# Patent

## Title
Compositions and methods of increasing stress tolerance in plants

## Abstract

The present invention provides novel isolated FT polynucleotides and polypeptides encoded by the FT polynucleotides. Also provided are the antibodies that immunospecifically bind to a FT polypeptide or any derivative, variant, mutant or fragment of the FT polypeptide, polynucleotide or antibody. The invention additionally provides methods of constructing transgenic plants that have altered levels of FT polynucleotides and polypeptides.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. Ser. No. 10/160,764, filed May 31, 2002, now issued U.S. Pat. No. 7,172,881, which claims the benefit of U.S. Ser. No. 60/294,766, filed May 31, 2001 and U.S. Ser. No. 60/348,909 filed Oct. 22, 2001 each of which are incorporated herein by reference in their entireties.


FIELD OF THE INVENTION
The invention relates in part to novel plant farnesyl transferase alpha and beta subunit polynucleotides and polypeptides. Also included are transgenic plants expressing the novel polynucleotides and polypeptides. The invention also includes transgenic plant cells, tissues and plants having novel phenotypes resulting from the expression of these polynucleotides in either the sense or antisense orientation.
BACKGROUND OF THE INVENTION
Most higher plants encounter at least transient decreases in relative water content at some stage of their life cycle and, as a result, have evolved a number of desiccation protection mechanisms. If however, the change in water deficit is prolonged the effects on the plants growth and development can be profound. Decreased water content due to drought, cold or salt stress can irreparably damage plant cells which in turn limits plant growth and crop productivity in agriculture.
Plants respond to adverse conditions of drought, salinity and cold with a variety of morphological and physiological changes. Although our understanding of plant tolerance mechanisms to these stresses is incomplete, the plant hormone abscisic acid (ABA) is believed to be an essential mediator between environmental stimulus and plant responses. ABA levels increase in response to water deficits and exogenously applied ABA mimics many of the responses induced by water-stress. Once ABA is synthesized it causes the closure of the leaf stomata thereby decreasing water loss through transpiration.
The identification of genes that transduce ABA into a cellular response opens the possibility of exploiting these regulators to enhance desiccation tolerance in crop species. In principle, these ABA signaling genes can be coupled with the appropriate controlling elements to allow optimal plant growth, development and productivity. Thus, not only would these genes allow the genetic tailoring of crops to withstand transitory environmental stresses, but they should also broaden the environments where traditional crops can be grown.
The recent isolation of an Arabidopsis thaliana mutant, era1, is hypersensitive to ABA and has been shown to also be tolerant to conditions of water deprivation. ERA1 has been identified as a β subunit of farnesyl transferase. Farnesyl transferase is a heterodimeric enzyme that provides the specific addition of a farnesyl pyrophosphate moiety onto the substrate target sequence. The target sequence is defined as a sequence of four amino acids which are present at the carboxy terminus of the protein and is referred to as a CaaX motif in which the “C” is cysteine, “a” is any aliphatic amino acid and “X” is any amino acid. The α subunit is common with a second prenylation enzyme, geranylgeranyl transferase, that has a different β subunit and adds a geranylgeranyl isoprenyl pyrophosphate moiety to the target sequence.
Prenylation is a multistep pathway which includes prenylation of the cysteine residue of the CaaX site, cleavage of the -aaX tripeptide and methylation of the prenyl-cysteine residue. Potentially, each of these steps could represent a target for genetic manipulation of the prenylation process to generate a desired phenotype such as stress tolerance.
In plants, prenylation has been linked to cell cycle control, meristem development, and phytohormone signal transduction, however, few details of the role of prenylation, the substrate proteins or the extent to which the plant system will be analogous to the mammalian and yeast systems are known. The most characterized substrates for CaaX modification are the Ras and a-factor proteins of yeast. Although there are three steps to complete protein maturation, abolition or modification of any one step does not necessarily result in cessation of target biological activities. Ras function is attenuated if the -aaX tripeptide is not cleaved but not abolished and some proteins retain the -aaX tripeptide after farnesylation. These observations may be substrate specific as, in contrast, there are examples indicating some proteins are fully functional only after being properly prenylated such as in regulating processes such as mitogen response in mammals and mating pheromone in yeast.
In Arabidopsis thaliana, more than 600 proteins contain a CaaX motif, suggesting a role for the post-translational modification by prenylation in numerous cellular processes. In Arabidopsis thaliana, it has been demonstrated that the loss-of-function of the β-subunit of farnesyl transferase will result in a ABA-hypersensitive phenotype. Although it is still not clear why plants lacking the functional β-subunit of farnesyl transferase become more sensitive to ABA, it clearly suggests that protein prenylation is involved in regulation of the homeostasis of ABA sensitivity. The balance of ABA cellular responses, whether more sensitive or less sensitive to ABA, is possibly regulated by the relative activities of prenylated proteins.
This invention is directed at the manipulation of the farnesyl transferase (FT) subunits, either α or β (FTA, FTB) to alter farnesyl transferase enzyme expression and activity. Farnesyl transferase catalyses the first step of farnesylation in which a 15-carbon farnesyl moiety is added to the cysteine residue of the target sequence CaaX. Included in this invention are vector constructs containing FTA or FTB sequences under the control of appropriate regulatory sequences to produce phenotypes such as, but not limited to, water-stress tolerance, increased biomass accumulation, increased yield or delayed senescence. Manipulation of the FTA subunit may also affect the activity of geranylgeranyl transferase and the phenoytypes associated with this manipulation are encompassed by this invention.
SUMMARY OF THE INVENTION
The present invention is based in part upon the discovery of novel farnesyl transferase nucleic acid sequences and polypeptides from Arabidopsis thaliana, Brassica napus, Glycine max and Zea maize. The nucleic acids, polynucleotides, proteins and polypeptides, or fragments thereof described herein are collectively referred to as FT nucleic acids and polypeptides.
Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or a fragment, homolog, analog or derivative thereof. The nucleic acid can include, e.g., a nucleic acid sequence encoding a polypeptide at least 99% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36, or SEQ ID NO:37, a nucleic acid sequence encoding a polypeptide at least 85% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 or a nucleic acid sequence encoding a polypeptide at least 99% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:33, SEQ ID NO:36, or SEQ ID NO:39 The nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
The invention also includes the nucleic acid sequences of SEQ ID NO: 2, 3, 4, 29, 30, 32, 35, 38, 40-57 or 58. Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein. In some aspects the FT nucleic acid is operably linked to a promoter. Examples of promoter includes a constitutive promoter (e.g., 35S CaMV, MuA), an ABA inducible promoter (e.g., RD29A), tissue specific promoters (e.g., CUT1) or a guard cell-specific promoter (e.g., 35S, MuA and RD29A)
The invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described herein.
The invention is also directed to plants and cells transformed with a FT nucleic acid or a vector comprising a FT nucleic acid. Also included in the invention is the seed, and progeny of the transformed plants or cells.
The invention is also further directed to the use of plants and cells transformed with a FT nucleic acid or a vector comprising a FT nucleic acid in generation of mutant libraries and genetic screening protocols.
In a further aspect, the invention includes a substantially purified FT polypeptide, e.g., any of the FT polypeptides encoded by an FT nucleic acid, and fragments, homologs, analogs, and derivatives thereof.
In still a further aspect, the invention provides an antibody that binds specifically to an FT polypeptide. The antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof. The invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
The invention also includes a method of producing a transgenic plant which has increased stress resistance such as, but not limited to, water deficit, or increased biomass, increased yield; delayed senescence or increases ABA sensitivity by introducing into one or more cells of a plant a compound that alters FT expression or activity in the plant. In one aspect the compound is a FT nucleic acid. The nucleic acid can be for example a inhibitor or farnesylation or genanylgerylation. Alternatively, the compound is a FT double stranded RNA-inhibition hair-pin nucleic acid or FT antisense nucleic acid.
The invention further provides a method for producing a FT polypeptide by providing a cell containing an FT nucleic acid, e.g., a vector that includes a FT nucleic acid, and culturing the cell under conditions sufficient to express the FT polypeptide encoded by the nucleic acid. The expressed FT polypeptide is then recovered from the cell. Preferably, the cell produces little or no endogenous FT polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
The invention is also directed to methods of identifying a FT polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
The invention further provides methods of identifying a compound that modulates the activity of a FT polypeptide by contacting a FT polypeptide with a compound and determining whether the FT polypeptide activity is modified.
The invention is also directed to compounds that modulate FT polypeptide activity identified by contacting a FT polypeptide with the compound and determining whether the compound modifies activity of the FT polypeptide, binds to the FT polypeptide, or binds to a nucleic acid molecule encoding a FT polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration depicting the pBI121 antisense FTA vector construct.
FIG. 2 is an illustration of genomic Southern hybridization analysis of anti-FTA transgenic Arabidopsis thaliana. 
FIG. 3 is an illustration of Northern analysis of five 35S-anti-FTA Arabidopsis thaliana lines (T3 plants).
FIG. 4 shows a Western expression analysis using anti-FTA antibodies to detect the FTA polypeptides.
FIG. 5 is a set of photographs showing ABA effects on seedling growth and development. FTA Antisense transgenic seedlings exhibit enhanced ABA sensitivity.
FIG. 6 shows the effect of ABA on seedling growth and development.
FIG. 7 shows photographs of wild type Columbia (A) and four antisense FTA transgenic lines (B, C, D, E) of Arabidopsis thaliana after 8 days without watering.
FIG. 8 is an illustration of the homology among FTA nucleic acid (A) and amino acid (B) sequences from various plant species based on ClustalW analysis (percent identity shown).
FIG. 9 is an illustration of the homology among FTB nucleic acid and amino acid sequences from various plant species based on ClustalW analysis (percent identity shown).
FIG. 10 is an illustration of transgenic performance during water stress.
FIG. 11 is an illustration of shoot fresh weight, or biomass accumulation, after 6 days of water stress treatment and 6 days recovery time.
FIG. 12 is an illustration of seed yield (grams) obtained under optimal conditions or following a 6 day water stress treatment.
FIG. 13 is an illustration of vegetative growth under optimal conditions, shown is shoot fresh weight 6 days after the first flower opened.
FIG. 14 is an illustration of the effect of a biotic stress coupled with drought stress treatment on seed yield.
FIG. 15 is a representative illustration of gel electrophoresis analysis of PCR products in an assay to detect transgenic lines of Brassica napus. 



DETAILED DESCRIPTION OF INVENTION
The present invention provides a novel farnesyl transferase (FT) nucleic acid sequences (SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37) and their encoded polypeptides (SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39) isolated from Brassica napus (Bn), Arabidopsis thaliana (At), Glycine max (Gm) and Zea maize (Zm). The sequences are collectively referred to as “FT nucleic acids” or FT polynucleotides” and the corresponding encoded polypeptide is referred to as a “FT polypeptide” or “FT protein”. Farnesyl transferase subunits, Alpha (α) and Beta (β) are referred to as FTA and FTB, respectively. Glycine max is also referred to as soy or soybean throughout the specification. Zea maize is also referred to as Zea mays or corn throughout the specification. These terms are interchangeable. Unless indicated otherwise, “FT” is meant to refer to any of the novel sequences disclosed herein.
Table A provides a summary of the FT nucleic acids and their encoded polypeptides.







TABLE A







Sequences and Corresponding SEQ ID Numbers










FT





Assign-

SEQ ID NO
SEQ ID NO


ment
Identification
(nucleic acid)
(polypeptide)













1
Arabidopsis thaliana farnesyl
1
5



transerase alpha subunit


2
Brassica napus farnesyl
6
7



transerase alpha subunit


3
Brassica napus farnesyl
8
9



transerase beta subunit


4
Glycine max alpha subunit
31
33


5
Glycine max beta subunit
34
36


6
Zea maize beta subunit
37
39









Also included in the invention are nucleic acids that are complementary to the disclosed FT nucleic acid sequences. For example, SEQ ID NO: 2, 3, 29, 30, 32, 35 or 38. Further provide by the invention are constructs comprising FT antisense nucleic acid molecules as disclosed in for example SEQ ID NO:4, 40-58.
Based on their structural and functional relatedness to known farnesyl transferase proteins, the FT proteins are novel members of the farnesyl transferase family of proteins. (See, Example 3) FT nucleic acids, and their encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, the nucleic acids can be used produce transgenic plants that have an increase resistance to biotic and abiotic stresses, e.g., chilling stress, salt stress, heat stress, water stress, wound healing, pathogen challenge, or herbicides.
This invention includes methods to up-regulate the FT enzyme activity in transgenic plants, cells and tissue cultures by using an over-expression vector construct and methods to down-regulate the FT enzyme activity in transgenic plants, cells and tissue cultures by using a double stranded RNA-inhibition, hairpin vector construct. These methods are by way of example to produce the up-regulation or down-regulation effects and are not meant to be limiting as to the method of achieving this outcome.
Additionally, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, FT activity. Alternatively, the FT nucleic acids and polypeptides can be used to identify proteins that are members of the farnesyl transferase family of associated proteins.
Further, the modulation or inhibition of FT activity maybe achieved by modifications to the nucleic acid sequences of FTA or FTB by the actions of chemical mutagens or irradiation. Expression of FT nucleic acids which encode enzymatically non-functional FT polypeptides can be used to evoke a dominant-negative inhibitory effect on FT activity.
Additional utilities for FT nucleic acids and polypeptides according to the invention are disclosed herein.
FT Nucleic Acids
The nucleic acids of the invention include those that encode a FT polypeptide or protein. As used herein, the terms polypeptide and protein are interchangeable.
In some embodiments, a FT nucleic acid encodes a mature FT polypeptide. As used herein, a “mature” form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein. The product “mature” form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell in which the gene product arises. Examples of such processing steps leading to a “mature” form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a “mature” form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
Among the FT nucleic acids is the nucleic acid whose sequence is provided in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 or a fragment thereof. Additionally, the invention includes mutant or variant nucleic acids of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or a fragment thereof, any of whose bases may be changed from the corresponding base shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, while still encoding a protein that maintains at least one of its FT-like activities and physiological functions. The invention further includes the complement of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, including fragments, derivatives, analogs and homologs thereof. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
One aspect of the invention pertains to isolated nucleic acid molecules that encode FT proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify FT-encoding nucleic acids (e.g., FT mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of FT nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
“Probes” refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated FT nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or a complement of any one of the nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 as a hybridization probe, FT nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to FT nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or a complement thereof. Oligonucleotides may be chemically synthesized and may be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37. For example, a complimentary nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:35 or SEQ ID NO:38. In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or a portion of this nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, thereby forming a stable duplex.
As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of FT. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein by reference in its entirety).
A “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a FT polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39, as well as a polypeptide having FT activity, e.g. substrate binding.
The nucleotide sequence determined from the cloning of the Arabidopsis thaliana FT gene allows for the generation of probes and primers designed for use in identifying and/or cloning FT homologues in other cell types, e.g., from other tissues, as well as FT homologues from other plants. The probe/primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37; or an anti-sense strand nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37; or of a naturally occurring mutant of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37.
Probes based on the Arabidopsis thaliana FT nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a FT protein, such as by measuring a level of a FT-encoding nucleic acid in a sample of cells from a subject e.g., detecting FT mRNA levels or determining whether a genomic FT gene has been mutated or deleted.
A “polypeptide having a biologically active portion of FT” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a “biologically active portion of FT” can be prepared by isolating a portion of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 that encodes a polypeptide having a FT biological activity (biological activities of the FT proteins are described below), expressing the encoded portion of FT protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of FT. In another embodiment, a nucleic acid fragment encoding a biologically active portion of FT includes one or more regions.
FT Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 due to the degeneracy of the genetic code. These nucleic acids thus encode the same FT protein as that encoded by the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, e.g., the polypeptide of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39.
In addition to the Arabidopsis thaliana FT nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of FT may exist within a population (e.g., the plant). Such genetic polymorphism in the FT gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a FT protein, preferably a plant FT protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the FT gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in FT that are the result of natural allelic variation and that do not alter the functional activity of FT are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding FT proteins from other species, and thus that have a nucleotide sequence that differs from the sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the FT cDNAs of the invention can be isolated based on their homology to the Arabidopsis thaliana FT nucleic acids disclosed herein using the cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding FT proteins derived from species other than Arabidopsis thaliana) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C. This hybridization is followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of the FT sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, thereby leading to changes in the amino acid sequence of the encoded FT protein, without altering the functional ability of the FT protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of FT without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the FT proteins of the present invention, are predicted to be particularly unamenable to alteration.
Another aspect of the invention pertains to nucleic acid molecules encoding FT proteins that contain changes in amino acid residues that are not essential for activity. Such FT proteins differ in amino acid sequence from SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39. Preferably, the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39.
An isolated nucleic acid molecule encoding a FT protein homologous to the protein of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in FT is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a FT coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for FT biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
In one embodiment, a mutant FT protein can be assayed for (1) the ability to form protein:protein interactions with other FT proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant FT protein and a FT receptor; (3) the ability of a mutant FT protein to bind to an intracellular target protein or biologically active portion thereof, (e.g., avidin proteins); (4) the ability to bind FT protein; or (5) the ability to specifically bind an anti-FT protein antibody.
Antisense FT Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire FT coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a FT protein of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39, or antisense nucleic acids complementary to a FT nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37 are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding FT (e.g. SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37). The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of Arabidopsis thaliana FT corresponds to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding FT (e.g. SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37). The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).
In various embodiments the anti-sense FT nucleic acid molecule includes the sequences of SEQ ID NO: 2, 3, 29, 30, 32, 35 or 38.
Given the coding strand sequences encoding FT disclosed herein (e.g., SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of FT mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of FT mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of FT mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a FT protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in applications.
Double Stranded RNA Inhibition (RNAi) by Hairpin Nucleic Acids
Another aspect of the invention pertains to the use of post transcriptional gene silencing (PTGS) to repress gene expression. Double stranded RNA can initiate the sequence specific repression of gene expression in plants and animals. Double stranded RNA is processed to short duplex oligomers of 21-23 nucleotides in length. These small interfering RNA's suppress the expression of endogenous and heterologous genes in a sequence specific manner (Fire et al. Nature 391:806-811, Carthew, Curr. Opin. in Cell Biol., 13:244-248, Elbashir et al., Nature 411:494-498). A RNAi suppressing construct can be designed in a number of ways, for example, transcription of a inverted repeat which can form a long hair pin molecule, inverted repeats separated by a spacer sequence that could be an unrelated sequence such as GUS or an intron sequence. Transcription of sense and antisense strands by opposing promoters or cotranscription of sense and antisense genes.
FT Ribozymes and PNA Moieties
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave FT mRNA transcripts to thereby inhibit translation of FT mRNA. A ribozyme having specificity for a FT-encoding nucleic acid can be designed based upon the nucleotide sequence of a FT DNA disclosed herein (i.e., SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a FT-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, FT mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, FT gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the FT (e.g., the FT promoter and/or enhancers) to form triple helical structures that prevent transcription of the FT gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of FT can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of FT can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of FT can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
In another embodiment, PNAs of FT can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of FT can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
FT Polypeptides
A FT polypeptide of the invention includes the protein whose sequence is provided in SEQ ID NO:5, SEQ ID NO:7, OR SEQ ID NO:9. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39 while still encoding a protein that maintains its FT-like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more of the residues may be so changed in the mutant or variant protein. In some embodiments, the FT polypeptide according to the invention is a mature polypeptide.
In general, a FT-like variant that preserves FT-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated FT proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-FT antibodies. In one embodiment, native FT proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, FT proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a FT protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the FT protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of FT protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of FT protein having less than about 30% (by dry weight) of non-FT protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-FT protein, still more preferably less than about 10% of non-FT protein, and most preferably less than about 5% non-FT protein. When the FT protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language “substantially free of chemical precursors or other chemicals” includes preparations of FT protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of FT protein having less than about 30% (by dry weight) of chemical precursors or non-FT chemicals, more preferably less than about 20% chemical precursors or non-FT chemicals, still more preferably less than about 10% chemical precursors or non-FT chemicals, and most preferably less than about 5% chemical precursors or non-FT chemicals.
Biologically active portions of a FT protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the FT protein, e.g., the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39 that include fewer amino acids than the full length FT proteins, and exhibit at least one activity of a FT protein, e.g. substrate binding. Typically, biologically active portions comprise a domain or motif with at least one activity of the FT protein. A biologically active portion of a FT protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a FT protein of the present invention may contain at least one of the above-identified domains conserved between the FT proteins. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native FT protein.
A biologically active portion or a FT protein can be the N-terminal domain of the FT polypeptide. Alternatively, a biologically active portion or a FT protein can be the C-terminal domain of the FT polypeptide. Preferably, the biologically active portion comprises at least 75 amino acids of the C-terminal domain. More preferably, the biologically active portion comprises at least 25 amino acids of the C-terminal domain. Most preferably, the biologically active portion comprises at least 10 amino acids of the C-terminal.
In an embodiment, the FT protein has an amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39. In other embodiments, the FT protein is substantially homologous to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39 and retains the functional activity of the protein of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the FT protein is a protein that comprises an amino acid sequence at least 45% homologous to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39 and retains the functional activity of the FT proteins of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:36 or SEQ ID NO:39.
Determining Homology Between Two or More Sequence
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either of the sequences being compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”).
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37.
The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region. The term “percentage of positive residues” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of positive residues.
Chimeric and Fusion Proteins
The invention also provides FT chimeric or fusion proteins. As used herein, a FT “chimeric protein” or “fusion protein” comprises a FT polypeptide operatively linked to a non-FT polypeptide. An “FT polypeptide” refers to a polypeptide having an amino acid sequence corresponding to FT, whereas a “non-FT polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the FT protein, e.g., a protein that is different from the FT protein and that is derived from the same or a different organism. Within a FT fusion protein the FT polypeptide can correspond to all or a portion of a FT protein. In one embodiment, a FT fusion protein comprises at least one biologically active portion of a FT protein. In another embodiment, a FT fusion protein comprises at least two biologically active portions of a FT protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the FT polypeptide and the non-FT polypeptide are fused in-frame to each other. The non-FT polypeptide can be fused to the N-terminus or C-terminus of the FT polypeptide.
A FT chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide, a 6×His-tag). A FT-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the FT protein.
FT Agonists and Antagonists
The present invention also pertains to variants of the FT proteins that function as either FT agonists (mimetics) or as FT antagonists. An agonist can be for example an antisense nucleic acid molecule. Variants of the FT protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the FT protein. An agonist of the FT protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the FT protein. An antagonist of the FT protein can inhibit one or more of the activities of the naturally occurring form of the FT protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the FT protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function.
Variants of the FT protein that function as either FT agonists (mimetics) or as FT antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the FT protein for FT protein agonist or antagonist activity. In one embodiment, a variegated library of FT variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of FT variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential FT sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of FT sequences therein. There are a variety of methods which can be used to produce libraries of potential FT variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential FT sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
Polypeptide Libraries
In addition, libraries of fragments of the FT protein coding sequence can be used to generate a variegated population of FT fragments for screening and subsequent selection of variants of a FT protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a FT coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the FT protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of FT proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify FT variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
FT Antibodies
FT polypeptides, including chimeric polypeptides, or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens to generate antibodies that immunospecifically-bind these peptide components. Such antibodies include, e.g., polyclonal, monoclonal, chimeric, single chain, Fab fragments and a Fab expression library. In a specific embodiment, fragments of the FT polypeptides are used as immunogens for antibody production. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to a FT polypeptides, or derivative, fragment, analog or homolog thereof.
For the production of polyclonal antibodies, various host animals may be immunized by injection with the native peptide, or a synthetic variant thereof, or a derivative of the foregoing. Various adjuvants may be used to increase the immunological response and include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.) and human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum. 
For preparation of monoclonal antibodies directed towards a FT polypeptides, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, Kohler and Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see, Kozbor, et al., 1983. Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see, Cole, et al., 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by the use of human hybridomas (see, Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see, Cole, et al., 1985. In: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., pp. 77-96).
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a FT polypeptides (see, e.g., U.S. Pat. No. 4,946,778). In addition, methodologies can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a FT polypeptides or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a FT polypeptides may be produced by techniques known in the art including, e.g., (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
In one embodiment, methodologies for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of a FT polypeptides is facilitated by generation of hybridomas that bind to the fragment of a FT polypeptides possessing such a domain. Antibodies that are specific for a domain within a FT polypeptides, or derivative, fragments, analogs or homologs thereof, are also provided herein. The anti-FT polypeptide antibodies may be used in methods known within the art relating to the localization and/or quantitation of a FT polypeptide (e.g., for use in measuring levels of the peptide within appropriate physiological samples, for use in diagnostic methods, for use in imaging the peptide, and the like).
FT Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a FT protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., FT proteins, mutant forms of FT proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of FT proteins in prokaryotic or eukaryotic cells. For example, FT proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells, plant cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein, however carboxy terminus fusions are also common. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the FT expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, FT can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In yet another embodiment, a nucleic acid of the invention is expressed in plants cells using a plant expression vector. Examples of plant expression vectors systems include tumor inducing (Ti) plasmid or portion thereof found in Agrobacterium, cauliflower mosaic virus (CAMV) DNA and vectors such as pBI121.
For expression in plants, the recombinant expression cassette will contain in addition to the FT nucleic acids, a plant promoter region, a transcription initiation site (if the coding sequence to transcribed lacks one), and a transcription termination/polyadenylation sequence. The termination/polyadenylation region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
Examples of suitable promotors include promoters from plant viruses such as the 35S promoter from cauliflower mosaic virus (CaMV). Odell, et al., Nature, 313: 810-812 (1985). and promoters from genes such as rice actin (McElroy, et al., Plant Cell, 163-171 (1990)); ubiquitin (Christensen, et al., Plant Mol. Biol., 12: 619-632 (1992); and Christensen, et al., Plant Mol. Biol., 18: 675-689 (1992)); pEMU (Last, et al., Theor. Appl. Genet., 81: 581-588 (1991)); MAS (Velten, et al., EMBO J., 3: 2723-2730 (1984)); maize H3 histone (Lepetit, et al., Mol. Gen. Genet., 231: 276-285 (1992); and Atanassvoa, et al., Plant Journal, 2(3): 291-300 (1992)), the 5′- or 3′-promoter derived from T-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the GRP1-8 promoter, ALS promoter, (WO 96/30530), a synthetic promoter, such as, Rsyn7, SCP and UCP promoters, ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heat shock promoters, seed-specific promoters and other transcription initiation regions from various plant genes, for example, include the various opine initiation regions, such as for example, octopine, mannopine, and nopaline.

Additional regulatory elements that may be connected to a FT encoding nucleic acid sequence for expression in plant cells include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell. Such regulatory elements and methods for adding or exchanging these elements with the regulatory elements FT gene are known, and include, but are not limited to, 3′ termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al., Nucl. Acids Res., 12: 369-385 (1983)); the potato proteinase inhibitor II (PINII) gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650 (1986) and hereby incorporated by reference); and An, et al., Plant Cell, 1: 115-122 (1989)); and the CaMV 19S gene (Mogen, et al., Plant Cell, 2: 1261-1272 (1990)).

Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264: 4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene (DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuka, et al., Proc. Nat'l Acad. Sci. (USA), 88: 834 (1991)) and the barley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), or signals which cause proteins to be secreted such as that of PRIb (Lind, et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which target proteins to the plastids such as that of rapeseed enoyl-ACP reductase (Verwaert, et al., Plant Mol. Biol., 26: 189-202 (1994)) are useful in the invention.
In another embodiment, the recombinant expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Especially useful in connection with the nucleic acids of the present invention are expression systems which are operable in plants. These include systems which are under control of a tissue-specific promoter, as well as those which involve promoters that are operable in all plant tissues.
Organ-specific promoters are also well known. For example, the patatin class I promoter is transcriptionally activated only in the potato tuber and can be used to target gene expression in the tuber (Bevan, M., 1986, Nucleic Acids Research 14:4625-4636). Another potato-specific promoter is the granule-bound starch synthase (GBSS) promoter (Visser, R. G. R, et al., 1991, Plant Molecular Biology 17:691-699).
Other organ-specific promoters appropriate for a desired target organ can be isolated using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, P., 1986, Trans. R. Soc. London B314:343).
For in situ production of the antisense mRNA of GST, those regions of the GST gene which are transcribed into GST mRNA, including the untranslated regions thereof, are inserted into the expression vector under control of the promoter system in a reverse orientation. The resulting transcribed mRNA is then complementary to that normally produced by the plant.
The resulting expression system or cassette is ligated into or otherwise constructed to be included in a recombinant vector which is appropriate for plant transformation. The vector may also contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. After transforming the plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, in the case of Agrobacterium transformations, T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention encoded in a an open reading frame of a polynucleotide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
A number of types of cells may act as suitable host cells for expression of a polypeptide encoded by an open reading frame in a polynucleotide of the invention. Plant host cells include, for example, plant cells that could function as suitable hosts for the expression of a polynucleotide of the invention include epidermal cells, mesophyll and other ground tissues, and vascular tissues in leaves, stems, floral organs, and roots from a variety of plant species, such as Arabidopsis thaliana, Nicotiana tabacum, Brassica napus, Zea mays, and Glycine max. 
Alternatively, it may be possible to produce a polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional polypeptide, if the polypeptide is of sufficient length and conformation to have activity. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
A polypeptide may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed polypeptide or protein may then be purified from such culture (e.g., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the polypeptide or protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, Heparin-Toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, a polypeptide or protein may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein containing a six-residue histidine tag. The histidine-tagged protein will then bind to a Ni-affinity column. After elution of all other proteins, the histidine-tagged protein can be eluted to achieve rapid and efficient purification. One or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide. The protein or polypeptide thus purified is substantially free of other plant proteins or polypeptides and is defined in accordance with the present invention as “isolated.”
Transformed Plants Cells and Transgenic Plants
The invention includes protoplast, plants cells, plant tissue and plants (e.g., monocots and dicots transformed with a FT nucleic acid, a vector containing a FT nucleic acid or an expression vector containing a FT nucleic acid. Examples of nucleic acids suitable for transforming plant cells and plants include those nucleic acid sequences of SEQ ID NO: 4, 40-57 or 58. As used herein, “plant” is meant to include not only a whole plant but also a portion thereof (i.e., cells, and tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds).
The plant can be any plant type including, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Caco, and Populus. 
In some aspects of the invention, the transformed plant is resistant to biotic and abiotic stresses, e.g., chilling stress, salt stress, heat stress, water stress, disease, grazing pests and wound healing. Additionally, the invention also includes a transgenic plant that is resistant to pathogens such as for example fungi, bacteria, nematodes, viruses and parasitic weeds. Alternatively, the transgenic plant is resistant to herbicides. By resistant is meant the plant grows under stress conditions (e.g., high salt, decreased water, low temperatures) or under conditions that normally inhibit, to some degree, the growth of an untransformed plant. Methodologies to determine plant growth or response to stress include for example, height measurements, weight measurements, leaf area, ability to flower, water use, transpiration rates and yield.
The invention also includes cells, tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds and the progeny derived from the transformed plant.
Numerous methods for introducing foreign genes into plants are known and can be used to insert a gene into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al., (1993) “Procedure for Introducing Foreign DNA into Plants”, In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88 and Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, polyethylene glycol (PEG) transformation, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)), electroporation, protoplast transformation, micro-injection, flower dipping and particle or non-particle biolistic bombardment.
Agrobacterium-mediated Transformation
The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genes responsible for genetic transformation of plants. See, for example, Kado, Crit. Rev. Plant Sci., 10:1-32 (1991). Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided in Gruber et al., supra; and Moloney, et al, Plant Cell Reports, 8: 238-242 (1989).
Transgenic Arabidopsis plants can be produced easily by the method of dipping flowering plants into an Agrobacterium culture, based on the method of Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Wild type plants are grown until the plant has both developing flowers and open flowers. The plant are inverted for 1 minutes into a solution of Agrobacterium culture carrying the appropriate gene construct. Plants are then left horizontal in a tray and kept covered for two days to maintain humidity and then righted and bagged to continue growth and seed development. Mature seed was bulk harvested.
Direct Gene Transfer
A generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 mu.m. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate the plant cell walls and membranes. (Sanford, et al., Part. Sci. Technol., 5: 27-37 (1987); Sanford, Trends Biotech, 6: 299-302 (1988); Sanford, Physiol. Plant, 79: 206-209 (1990); Klein, et al., Biotechnology, 10: 286-291 (1992)).
Another method for physical delivery of DNA to plants is sonication of target cells as described in Zang, et al., BioTechnology, 9: 996-996 (1991). Alternatively, liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, for example, Deshayes, et al., EMBO J., 4: 2731-2737 (1985); and Christou, et al., Proc. Nat'l. Acad. Sci. (USA), 84: 3962-3966 (1987). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. See, for example, Hain, et al., Mol. Gen. Genet., 199: 161 (1985); and Draper, et al., Plant Cell Physiol., 23: 451-458 (1982).
Electroporation of protoplasts and whole cells and tissues has also been described. See, for example, Donn, et al., (1990) In: Abstracts of the VIIth Int;l. Congress on Plant Cell and Tissue Culture IAPTC, A2-38, page 53; D'Halluin et al., Plant Cell, 4: 1495-1505 (1992); and Spencer et al., Plant Mol. Biol., 24: 51-61 (1994).
Plants may also be transformed using the method of Held et al. (U.S. Application 20010026941). The method utilizes an accelerated aerosol beam of droplets which carries the desired molecules, DNA, into the target cells. The size of droplets produced by this method are reported to be sufficiently small as to transform bacterial cells of 1 to 2 microns in length.
Particle Wounding/Agrobacterium Delivery
Another useful basic transformation protocol involves a combination of wounding by particle bombardment, followed by use of Agrobacterium for DNA delivery, as described by Bidney, et al., Plant Mol. Biol., 18: 301-31 (1992). Useful plasmids for plant transformation include Bin 19. See Bevan, Nucleic Acids Research, 12: 8711-8721 (1984), and hereby incorporated by reference.
In general, the intact meristem transformation method involves imbibing seed for 24 hours in the dark, removing the cotyledons and root radical, followed by culturing of the meristem explants. Twenty-four hours later, the primary leaves are removed to expose the apical meristem. The explants are placed apical dome side up and bombarded, e.g., twice with particles, followed by co-cultivation with Agrobacterium. To start the co-cultivation for intact meristems, Agrobacterium is placed on the meristem. After about a 3-day co-cultivation period the meristems are transferred to culture medium with cefotaxime plus kanamycin for the NPTII selection.
The split meristem method involves imbibing seed, breaking of the cotyledons to produce a clean fracture at the plane of the embryonic axis, excising the root tip and then bisecting the explants longitudinally between the primordial leaves. The two halves are placed cut surface up on the medium then bombarded twice with particles, followed by co-cultivation with Agrobacterium. For split meristems, after bombardment, the meristems are placed in an Agrobacterium suspension for 30 minutes. They are then removed from the suspension onto solid culture medium for three day co-cultivation. After this period, the meristems are transferred to fresh medium with cefotaxime plus kanamycin for selection.
Transfer by Plant Breeding
Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conventional plant breeding methods can be used to transfer the gene and associated regulatory sequences via crossing and backcrossing. Such intermediate methods will comprise the further steps of: (1) sexually crossing the disease-resistant plant with a plant from the disease susceptible taxon; (2) recovering reproductive material from the progeny of the cross; and (3) growing disease-resistant plants from the reproductive material. Where desirable or necessary, the agronomic characteristics of the susceptible taxon can be substantially preserved by expanding this method to include the further steps of repetitively: (1) backcrossing the disease-resistant progeny with disease-susceptible plants from the susceptible taxon; and (2) selecting for expression of a hydrogen peroxide producing enzyme activity (or an associated marker gene) among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with the gene or genes imparting oxalic acid degrading and/or hydrogen peroxide enzyme activity.
By the term “taxon” herein is meant a unit of botanical classification. It thus includes, genus, species, cultivars, varieties, variants and other minor taxonomic groups which lack a consistent nomenclature.
Regeneration of Transformants
The development or regeneration of plants from either single plant protoplasts or various explants is well known in the art (Weissbach and Weissbach, 1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al., 1983). In particular, U.S. Pat. No. 5,349,124 (specification incorporated herein by reference) details the creation of genetically transformed lettuce cells and plants resulting therefrom which express hybrid crystal proteins conferring insecticidal activity against Lepidopteran larvae to such plants.
This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.
Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, or pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
A preferred transgenic plant is an independent segregant and can transmit the FT gene and its activity to its progeny. A more preferred transgenic plant is homozygous for the gene, and transmits that gene to all of its offspring on sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for increased expression of the FT transgene.
Method of Producing Transgenic Plants
Included in the invention are methods of producing a transgenic plant that has increased stress resistance, delayed senescence or increased sensitivity to ABA. The method includes introducing into one or more plant cells a compound that alters farnesyl transferase expression (i.e. farnesyl transferase alpha or beta) or activity in the plant. The compound can be, e.g., (i) a farnesyl transferase polypeptide inhibitor; (ii) a nucleic acid encoding a farnesyl transferase polypeptide inhibitor; (iii) a nucleic acid that decreases expression of a nucleic acid that encodes a farnesyl transferase polypeptide and, derivatives, fragments, analogs and homologs thereof, (iv) an antisense farnesyl transferase nucleic acid. A nucleic acid that decreases expression of a nucleic acid that encodes a farnesyl transferase polypeptide includes, e.g., antisense nucleic acids or RNA inhibitory nucleic acids. The nucleic acid can be either endogenous or exogenous. Preferably the compound is a farnesyl transferase polypeptide or a nucleic acid encoding a farnesyl transferase polypeptide. For example the compound is the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37. More preferably the compound is a nucleic acid complementarity to a nucleic acid encoding a farnesyl transferase polypeptide. For example an anti-sense nucleic acid molecule. Exemplary compounds include SEQ ID NO:1, 3, 4, 29, 30, 32, 35, 38, 40-57 and 58.
Also included in the invention is a plant where a mutation has been introduced in the gene encoding farnesyl transferase (i.e. alpha or beta) which results in a plant that has decreased farnesyl transferase activity and increased tolerance to stress as compared to a wild type plant. The mutation may be introduced by chemical or mechanical means.
Examples of stresses include, for example, chilling stress, heat stress, salt stress, water stress, nutrient limitation stress, disease, grazing pests, wound healing, pathogens such as for example fungi, bacteria, nematodes, viruses or parasitic weed and herbicides.
Increases stress resistance is meant that the transgenic plant can grows under stress conditions (e.g., high salt, decreased water, low temperatures) or under conditions that normally inhibit the growth of an untransformed plant. Methodologies to determine plant growth or response to stress include for example, height measurements, weight measurements, leaf area, ability to flower, water use, transpiration rates and yield
Sensitivity to ABA can be assessed using a concentration curve of ABA and germinating seeds on plates as described in Example 11. Often germination is assessed and used to determine sensitivity. However, sensitivity can be observed at more developmental stages than simply germination. For example, increased sensitivity may be observed at the stage of cotyledon expansion, expansion of the first true leaf, or developmental arrest in the seedling stage.
The concentration of ABA at which sensitivity is observed varies in a species dependent manner. For example, transgenic Arabidopsis thaliana will demonstrate sensitivity at a lower concentration than observed in Brassica or soybean.
By increased ABA sensitivity it is meant that the transgenic plant is seen to display a phenotype at a lower concentration of ABA than that used to observe the same phenotype in a wild type plant. Methodologies to determine ABA sensitivity include for example, plant germination, growth or development.
The plant can be any plant type including, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Caco, and Populus. 
Screening Methods
The isolated nucleic acid molecules of the invention (e.g., SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:37) can be used to express FT protein (e.g., via a recombinant expression vector in a host cell), to detect FT mRNA (e.g., in a biological sample) or a genetic lesion in a FT gene, and to modulate FT activity, as described further, below. In addition, the FT proteins can be used to screen compounds that modulate the FT protein activity or expression. In addition, the anti-FT antibodies of the invention can be used to detect and isolate FT proteins and modulate FT activity.
The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to FT proteins or have a stimulatory or inhibitory effect on, e.g., FT protein expression or FT protein activity. The invention also includes compounds identified in the screening assays described herein. The invention also includes methods of identifying related genes using the transgenic plants of this invention in screening protocols utilizing mutagenesis, gene tagging, insertional gene tagging, activation tagging or other such methods of gene or phenotype identification.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to a FT protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.
A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a FT protein, or a biologically-active portion thereof, is contacted with a test compound and the ability of the test compound to bind to a FT protein determined. The cell, for example, can be of mammalian origin, plant cell or a yeast cell. Determining the ability of the test compound to bind to the FT protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the FT protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a FT protein, or a biologically-active portion thereof, with a known compound which binds FT to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FT protein, wherein determining the ability of the test compound to interact with a FT protein comprises determining the ability of the test compound to preferentially bind to FT protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a FT protein, or a biologically-active portion thereof, with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the FT protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of FT or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the FT protein to bind to or interact with a FT target molecule. As used herein, a “target molecule” is a molecule with which a FT protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a FT interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A FT target molecule can be a non-FT molecule or a FT protein or polypeptide of the invention In one embodiment, a FT target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with FT.
Determining the ability of the FT protein to bind to or interact with a FT target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the FT protein to bind to or interact with a FT target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a FT-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a FT protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the FT protein or biologically-active portion thereof. Binding of the test compound to the FT protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the FT protein or biologically-active portion thereof with a known compound which binds FT to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FT protein, wherein determining the ability of the test compound to interact with a FT protein comprises determining the ability of the test compound to preferentially bind to FT or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting FT protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the FT protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of FT can be accomplished, for example, by determining the ability of the FT protein to bind to a FT target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of FT protein can be accomplished by determining the ability of the FT protein further modulate a FT target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described above.
In yet another embodiment, the cell-free assay comprises contacting the FT protein or biologically-active portion thereof with a known compound which binds FT protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FT protein, wherein determining the ability of the test compound to interact with a FT protein comprises determining the ability of the FT protein to preferentially bind to or modulate the activity of a FT target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of FT protein. In the case of cell-free assays comprising the membrane-bound form of FT protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of FT protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either FT protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to FT protein, or interaction of FT protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-FT fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or FT protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of FT protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the FT protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated FT protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with FT protein or target molecules, but which do not interfere with binding of the FT protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or FT protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the FT protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the FT protein or target molecule.
In another embodiment, modulators of FT protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of FT mRNA or protein in the cell is determined. The level of expression of FT mRNA or protein in the presence of the candidate compound is compared to the level of expression of FT mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of FT mRNA or protein expression based upon this comparison. For example, when expression of FT mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of FT mRNA or protein expression. Alternatively, when expression of FT mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of FT mRNA or protein expression. The level of FT mRNA or protein expression in the cells can be determined by methods described herein for detecting FT mRNA or protein.
In yet another aspect of the invention, the FT proteins can be used as “bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with FT (“FT-binding proteins” or “FT-bp”) and modulate FT activity. Such FT-binding proteins are also likely to be involved in the propagation of signals by the FT proteins as, for example, upstream or downstream elements of the FT pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for FT is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a FT-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with FT.
In yet another aspect of the invention are methods which utilize the transgenic plants of the invention to identify FT-interacting components via genetic screening protocols. These components can be for example, regulatory elements which modify FT-gene expression, interacting proteins which directly modify FT activity or interacting proteins which modify components of the same signal transduction pathway and thereby exert an effect on the expression or activity of FT. Briefly, genetic screening protocols are applied to the transgenic plants of the invention and in so doing identify related genes which are not identified using a wild type background for the screen. For example an activation tagged library (Weigel, et al., 2000. Plant Physiol. 122: 1003-1013), can be produced using the transgenic plants of the invention as the genetic background. Plants are then screened for altered phenotypes from that displayed by the parent plants. Alternative methods of generating libraries from the transgenic plants of the invention can be used, for example, chemical or irradiation induced mutations, insertional inactivation or activation methods.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Cloning of Arabidopsis thaliana FTA and Construction of Transformation Vector
The Arabidopsis thaliana FTA sequence was obtained by RT-PCR from total RNA isolated from leaf tissue using primers corresponding to SEQ ID NO:11 and SEQ ID NO:12. The resulting fragment was digested with BamHI and SmaI and cloned into the plasmid pCR2.1 The Clonetech vector pBI121 was used as the backbone for the antisense construct. The GUS gene was removed by BamHI and Eco1CRI digestion and replaced with the FTA insert that was cut from pCR2.1-FTA using SmaI and BamHI and ligated into the vector SEQ ID NO:4.









TABLE 1







SEQ ID NO: 11:
5′-AAAGGATCCTCAAATTGCTGCCACTGTAAT-3′



 


SEQ ID NO: 12:
5′-AAACCCGGGATGAATTTCGACGAGAACGTG-3′









Example 2
Cloning of Non-Full Length Brassica napus FTA and FTB Nucleic Acid Sequences
RNA was isolated from leaf and root tissue using the Qiagen RNeasy kit. RT-PCR was performed by known techniques using the primers shown in Table 2. The FTA sequence was obtained using the primer pair SEQ ID NO:19 and SEQ ID NO:20. The FTB sequence was obtained using the primer pair SEQ ID NO:21 and SEQ ID NO:22.









TABLE 2







SEQ ID NO: 19:
5′-GGATCCATGGATTACTTCCGTGCGATTTACTTCTCC-3′



 


SEQ ID NO: 20:
5′-AAAAAGCTTCCATGCCCAATAGTTAGCTCTTATTGGATC-3′


 


SEQ ID NO: 21:
5′-AAAAAGCTTTGGCTTTGTTACTGGATTCTTCATTCAAT-3′


 


SEQ ID NO: 22:
5′-AAATCTAGAAGCTTCATAATACCGATCCAAGACAATGTT-3′









PCR products were separated from the RT-PCR reaction mixture using the Qiagen PCR column spin kit and ligated into the cloning vector pBluescript KS+. The vector was digested with EcoRV and treated with Taq polymerase in the presence of dTTP to produce a 3′ overhang for ligation with the PCR products. The ligation products were transformed into E. coli DH5α cells, positive colonies were selected and the resulting inserts sequenced.
Example 3
Cloning of Non-Full Length FTA and FTB Nucleic Acid Sequences from Glycine max and Zea maize 
RNA was isolated from leaf and root tissue using the Qiagen RNeasy kit. RT-PCR was performed by known techniques using the primers shown in Table 3. The Glycine max FTA sequence was obtained using the primer pair SEQ ID NO:23 and SEQ ID NO:24. The Glycine max FTB sequence was obtained using the primer pair SEQ ID NO:25 and SEQ ID NO:26. The Zea maize FTB sequence was obtained using the primer pair SEQ ID NO:27 and SEQ ID NO:28.









TABLE 3







SEQ ID NO: 23:
5′-AAAGGATCCATGGAATCTGGGTCTAGCGA-3′



 


SEQ ID NO: 24:
5′-AAATCTAGAAGGAAGTCTGCTCTTGCGC-3′


 


SEQ ID NO: 25:
5′-AAATCTAGAGCCACCATTCCTCGCAACG-3′


 


SEQ ID NO: 26:
5′-AAAGAGCTCGTGGTGGAGAATCTGGGTGC-3′


 


SEQ ID NO: 27:
5′-GGCGGATCCCGACCTACCGAGG-3′


 


SEQ ID NO: 28:
5′-AAAGAGCTCGTGGATGGATTGGCTCCAGC-3′









PCR products were separated from the RT-PCR reaction mixture using the Qiagen PCR column spin kit and ligated into the cloning vector pBluescript KS+. The vector was digested with EcoRV and treated with Taq polymerase in the presence of dTTP to produce a 3′ overhang for ligation with the PCR products. The ligation products were transformed into E. coli DH5α cells, positive colonies were selected and the resulting inserts sequenced.
Example 4
Sequence Analysis
Arabidopsis thaliana FTA
A disclosed nucleic acid of 999 nucleotides (also referred to as FT1) is shown in Table 4A. The primers used in the PCR are depicted in bold.







TABLE 4A





FT1 Nucleotide Sequence (SEQ ID NO: 1).
















aaacccgggatgaatttcgacgagaccgtgccactgagccaacgattggagtggtcagacgtggt



 


cccattgactcaggacgatggtccgaatccagtggtgccaattgcctacaaggaagagttccgcg


 


agactatggattacttccgtgcgatttacttttccgacgagcgatctcctcgcgcactacgactc


 


acggaagaaaccctcctcttaaactccggcaactacacagtgtggcatttcaggcgcctagtact


 


cgaggcccttaatcacgacttgtttgaagaactcgagttcatcgaacgcattgctgaggataact


 


ctaagaactaccaactgtggcatcatcggcgatgggttgcagagaaactgggtcctgatgttgca


 


gggagagaacttgaatttacccgtagagtactttcacttgatgccaaacattatcatgcttggtc


 


acataggcagtggacactacgggcattaggaggatgggaagatgagctcgattactgtcacgagc


 


tccttgaagctgacgtctttaacaattccgcctggaatcagaggtattatgtcatcacccaatct


 


cctttgttgggaggcctagaagccatgagagaatctgaagtaagctacacaatcaaagccatttt


 


aaccaatcctgcaaacgagagctcatggcgatacctaaaagcgctttacaaagacgacaaagaat


 


cctggattagtgatccaagtgtttcctcagtctgtttgaatgttctatcccgcacagattgcttc


 


catggattcgctctgagcacccttttggatcttctatgtgatggactgagaccaaccaacgagca


 


taaagactcagtgagagctctagctaatgaagaaccagagactaacttggccaatttggtgtgta


 


ctattcttggtcgtgtagatcctataagagctaactattgggcatggaggaagagcaagattaca


 


gtggcagcaatttgaggatccttt









A disclosed FT1 polypeptide (SEQ ID NO:5) encoded by SEQ ID NO:1 has 326 amino acid residues and is presented in Table 4B using the one-letter amino acid code.







TABLE 4B





Encoded FT1 protein sequence (SEQ ID NO: 5).
















MNFDETVPLSQRLEWSDVVPLTQDDGPNPVVPIAYKEEFRETMDYFRAIYFSDERSPRALRLTE



 


ETLLLNSGNYTVWHFRRLVLEALNHDLFEELEFIERIAEDNSKNYQLWHHRRWVAEKLGPDVAG


 


RELEFTRRVLSLDAKHYHAWSHRQWTLRALGGWEDELDYCHELLEADVFNNSAWNQRYYVITQS


 


PLLGGLEAMRESEVSYTIKAILTNPANESSWRYLKALYKDDKESWISDPSVSSVCLNVLSRTDC


 


FHGFALSTLLDLLCDGLRPTNEHKDSVRALANEEPETNLANLVCTILGRVDPIRANYWAWRKSK


 


ITVAAI









Due to the nature of the cloning strategy the sequence presented does not contain any 5′ or 3′ non-translated sequence. Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques. The percent identity of the Arabidopsis thaliana nucleotide sequence and its encoded amino acid sequence to that of published sequences is shown in FIG. 8.
The present invention also includes a nucleic acid sequence complimentary to the Arabidopsis thaliana farnesyl transferase alpha subunit of SEQ ID NO:1. The disclosed complimentary sequence is shown as SEQ ID NO:2. The nucleic acid sequence of SEQ ID NO:3 shows the nucleic acid sequence of SEQ ID NO:2 that has been prepared for ligation into an expression vector.








SEQ ID NO: 2








aaaggatcctcaaattgctgccactgtaatcttgctcttcctccatgccc


 


aatagttagctcttataggatctacacgaccaagaatagtacacaccaaa


 


ttggccaagttagtctctggttcttcattagctagagctctcactgagtc


 


tttatgctcgttggttggtctcagtccatcacatagaagatccaaaaggg


 


tgctcagagcgaatccatggaagcaatctgtgcgggatagaacattcaaa


 


cagactgaggaaacacttggatcactaatccaggattctttgtcgtcttt


 


gtaaagcgcttttaggtatcgccatgagctctcgtttgcaggattggtta


 


aaatggctttgattgtgtagcttacttcagattctctcatggcttctagg


 


cctcccaacaaaggagattgggtgatgacataatacctctgattccaggc


 


ggaattgttaaagacgtcagcttcaaggagctcgtgacagtaatcgagct


 


catcttcccatcctcctaatgcccgtagtgtccactgcctatgtgaccaa


 


gcatgataatgtttggcatcaagtgaaagtactctacgggtaaattcaag


 


ttctctccctgcaacatcaggacccagtttctctgcaacccatcgccgat


 


gatgccacagttggtagttcttagagttatcctcagcaatgcgttcgatg


 


aactcgagttcttcaaacaagtcgtgattaagggcctcgagtactaggcg


 


cctgaaatgccacactgtgtagttgccggagtttaagaggagggtttctt


 


ccgtgagtcgtagtgcgcgaggagatcgctcgtcggaaaagtaaatcgca


 


cggaagtaatccatagtctcgcggaactcttccttgtaggcaattggcac


 


cactggattcggaccatcgtcctgagtcaatgggaccacgtctgaccact


 


ccaatcgttggctcagtggcacggtctcgtcgaaattcatcccgggttt


 







SEQ ID NO: 3







 tcaaattgctgccactgtaatcttgctcttcctccatgcccaata


 


gttagctcttataggatctacacgaccaagaatagtacacaccaaattgg


 


ccaagttagtctctggttcttcattagctagagctctcactgagtcttta


 


tgctcgttggttggtctcagtccatcacatagaagatccaaaagggtgct


 


cagagcgaatccatggaagcaatctgtgcgggatagaacattcaaacaga


 


ctgaggaaacacttggatcactaatccaggattctttgtcgtctttgtaa


 


agcgcttttaggtatcgccatgagctctcgtttgcaggattggttaaaat


 


ggctttgattgtgtagcttacttcagattctctcatggcttctaggcctc


 


ccaacaaaggagattgggtgatgacataatacctctgattccaggcggaa


 


ttgttaaagacgtcagcttcaaggagctcgtgacagtaatcgagctcatc


 


ttcccatcctcctaatgcccgtagtgtccactgcctatgtgaccaagcat


 


gataatgtttggcatcaagtgaaagtactctacgggtaaattcaagttct


 


ctccctgcaacatcaggacccagtttctctgcaacccatcgccgatgatg


 


ccacagttggtagttcttagagttatcctcagcaatgcgttcgatgaact


 


cgagttcttcaaacaagtcgtgattaagggcctcgagtactaggcgcctg


 


aaatgccacactgtgtagttgccggagtttaagaggagggtttcttccgt


 


gagtcgtagtgcgcgaggagatcgctcgtcggaaaagtaaatcgcacgga


 


agtaatccatagtctcgcggaactcttccttgtaggcaattggcaccact


 


ggattcggaccatcgtcctgagtcaatgggaccacgtctgaccactccaa


 


tcgttggctcagtggcacggtctcgtcgaaattcat 






Brassica napus FTA

A disclosed nucleic acid of 822 nucleotides (also referred to as FT2) is shown in Table 5A.







TABLE 5A





FT2 Nucleotide Sequence (SEQ ID NO: 6).
















ATGGATTACTTCCGTGCGATTTACTTCTCCGACGAGCGTTCTGCTCGCGC



 


GCTGCGACTCACGGAAGAAGCTCTCCGCTTAAACTCGGGCAACTACACCG


 


TGTGGCACTTCGGGCGCTTAGTACTCGAGGAGCTTAATAACGACTTGTAT


 


GAAGAGCTCAAGTTCATCGAAAGCATTGCTGAGGATAACTCTAAGAACTA


 


CCAGTTGTGGCATCATCGACGATGGGTCGCAGAGAAACTGGGTCCTGATG


 


TTGCAGGAAAGGAACTTGAGTTTACTCGGAGGGTACTATCACTTGATGCC


 


AAGCATTATCATGCTTGGTCACATAGGCAGTGGGCGCTACAAGCATTAGG


 


AGGATGGGAAAATGAGCTTAACTACTGCCACGAGCTCCTTGAAGCTGACG


 


TCTTTAACAACTCTGCATGGAATCAGAGGTATTACGTTATAACTAGATCA


 


CCTTCGTTGGGAGGCCTAGAAGCCATGAGAGAATCTGAAGTAAGCTACAC


 


AGTCAAAGCCATTTTAGCAAATCCCGGGAACGAGAGCTCTTGGAGGTACC


 


TGAAAGCCCTTTACAAAGACGACACAGAGTCTTGGATTAGTGATCCAAGT


 


GTTTCCTCAGTCTGTTTGAAAGTTCTCTCACGCGCGGACTGCTTCCATGG


 


ATTCGCTCTGAGCACCCTTTTGGATCTTCTGTGCGATGGGTTGAGACCAA


 


CCAACGAGCATAGAGACTCGGTGAAAGCTCTAGCTAATGAAGAACCAGAG


 


ACTAACTTGGCCAATTTGGTGTGTACCATTCTGTGTCGTGTTGATCCAAT


 


AAGAGCTAACTATTGGGCATGG









A disclosed FT2 polypeptide (SEQ ID NO:7) encoded by SEQ ID NO:6 has 274 amino acid residues and is presented in Table 5B using the one-letter amino acid code.







TABLE 5B





Encoded FT2 protein sequence (SEQ ID NO: 7).
















MDYFRAIYFSDERSARALRLTEEALRLNSGNYTVWHFGRLVLEELNNDLY



 


EELKFIESIAEDNSKNYQLWHHRRWVAEKLGPDVAGLEKEFTRRVLSLDA


 


KHYHAWSHRQWALQALGGWENELNYCHELLEADVFNNSAWNQRYYVITRS


 


PSLGGLEAMRESEVSYTVKAILANPGNESSWRYLKALYKDDTESWISDPS


 


VSSVCLKVLSRADCFHGFALSTLLDLLCDGLRPTNEHRDSVKALANEEPE


 


TNLANLVCTILCRVDPIRANYWAWKL









Due to the nature of the cloning strategy the sequence presented is not full length. Compared to the Arabidopsis thaliana sequence there are 42 amino acids missing from the amino terminus and 10 amino acids from the carboxy terminus. The percent identity of the Brassica napus nucleotide sequence and its encoded amino acid sequence to that of published sequences is shown in FIG. 8.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
The present invention also includes a nucleic acid sequence complimentary to the Brassica napus farnesyl transferase alpha subunit of SEQ ID NO:6. The disclosed complimentary sequence is shown as SEQ ID NO:29.








SEQ ID NO: 29








CCATGCCCAATAGTTAGCTCTTATTGGATCAACACGACACAGAATGGTAC


 


ACACCAAATTGGCCAAGTTAGTCTCTGGTTCTTCATTAGCTAGAGCTTTC


 


ACCGAGTCTCTATGCTCGTTGGTTGGTCTCAACCCATCGCACAGAAGATC


 


CAAAAGGGTGCTCAGAGCGAATCCATGGAAGCAGTCCGCGCGTGAGAGAA


 


CTTTCAAACAGACTGAGGAAACACTTGGATCACTAATCCAAGACTCTGTG


 


TCGTCTTTGTAAAGGGCTTTCAGGTACCTCCAAGAGCTCTCGTTCCCGGG


 


ATTTGCTAAAATGGCTTTGACTGTGTAGCTTACTTCAGATTCTCTCATGG


 


CTTCTAGGCCTCCCAACGAAGGTGATCTAGTTATAACGTAATACCTCTGA


 


TTCCATGCAGAGTTGTTAAAGACGTCAGCTTCAAGGAGCTCGTGGCAGTA


 


GTTAAGCTCATTTTCCCATCCTCCTAATGCTTGTAGCGCCCACTGCCTAT


 


GTGACCAAGCATGATAATGCTTGGCATCAAGTGATAGTACCCTCCGAGTA


 


AACTCAAGTTCCTTTCCTGCAACATCAGGACCCAGTTTCTCTGCGACCCA


 


TCGTCGATGATGCCACAACTGGTAGTTCTTAGAGTTATCCTCAGCAATGC


 


TTTCGATGAACTTGAGCTCTTCATACAAGTCGTTATTAAGCTCCTCGAGT


 


ACTAAGCGCCCGAAGTGCCACACGGTGTAGTTGCCCGAGTTTAAGCGGAG


 


AGCTTCTTCCGTGAGTCGCAGCGCGCGAGCAGAACGCTCGTCGGAGAAGT


 


AAATCGCACGGAAGTAATCCAT






Brassica napus FTB

A disclosed nucleic acid of 1110 nucleotides (also referred to as FT3) is shown in Table 6A.







TABLE 6A





FT3 Nucleotide Sequence (SEQ ID NO: 8).
















TGGCTTTGTTACTGGATTCTTCATTCAATTGCTTTGCTTGGGGAGTCTGT



 


GGATGATGACTTAGAAAACAATGCAATCGATTTTCTTGGACGTTGCCAGG


 


GTTCTGATGGTGGATATGGTGGTGGTCCTGGCCAACTTCCACATCTTGCA


 


ACAAGTTATGCTGCAGTGAATACACTTGTTACTTTAGGAGGTGAGAAAGC


 


CTTCTCTTCAATTAACAGAGAACAAATGGCTTGTTTCTTAAGACGAATGA


 


AGGATACAAATGGAGGTTTCAGGATGCATAATATGGGAGAAATAGATGTG


 


CGAGCGTGCTACACTGCGATTTTGATTGCAAGCATCCTGAACATTGTGGA


 


TGATGAACTCACCCGCGGCTTAGGAGATTACATTTTGAGTTGCCAAACTT


 


ATGAAGGTGGCATTGGAGGGGAACCTGGCTCCGAAGCTCATGGTGGGTAC


 


ACGTACTGTGGGTTGGCTACTATGATTTTAATCAATGAAGTCGACCGCTT


 


GAATTTGGATTCGTTAATGAATTGGGTTGTACATCGACAAGGAGTAGAAA


 


TGGGATTCCAAGGTAGGACGAACAAATTGGTCGACGGTTGCTACACGTTT


 


TGGCAGGCAGCCCCCTGTGTTCTACTACAGCGATTTTTTTCATCCCAGGA


 


TATGGCACCTCATGGATCATCATCACATATGTCACAAGGGACAGATGAAG


 


ATCACGAGGAACATGGTCATGATGAAGATGATCCTGAAGACAGTGATGAA


 


GATGATTCTGATGAGGATAGCGATGAAGATTCAGGGAATGGTCACCAAGT


 


TCATCATACGTCTACCTACATTGACAGGAGAATTCAACCTGTTTTTGATA


 


GCCTCGGCTTGCAAAGATATGTGCTCTTGTGCTCTCAGGTTGCTGATGGT


 


GGATTCAGAGACAAGCTGAGGAAACCCCGTGACTTCTACCACACATGTTA


 


CTGCCTAAGCGGTCTTTCCGTGGCTCAACACGCTTGGTCAAAAGACGAGG


 


ACACTCCTCCTTTGACTCGTGACATTTTGGGTGGCTACGCAAACCACCTT


 


GAACCTGTTCACCTCCTCCACAACATTGTCTTGGATCGGTATTATGAAGC


 


TTCTAGATTT









A disclosed FT3 polypeptide (SEQ ID NO:9) encoded by SEQ ID NO:7 has 370 amino acid residues and is presented in Table 6B using the one-letter amino acid code.







TABLE 6B





Encoded FT3 protein sequence (SEQ ID NO: 9).
















WLCYWILHSIALLGESVDDDLENNAIDFLGRCQGSDGGYGGGPGQLPHLA



 


TSYAAVNTLVTLGGEKAFSSINREQMACFLRRMKDTNGGFRMHNMGEIDV


 


RACYTAILIASILNIVDDELTRGLGDYILSCQTYEGGIGGEPGSEAHGGY


 


TYCGLATMILINEVDRLNLDSLMNWVVHRQGVEMGFQGRTNKLVDGCYTF


 


WQAAPCVLLQRFFSSQDMAPHGSSSHMSQGTDEDHEEHGHDEDDPEDSDE


 


DDSDEDSDEDSGNGHQVHHTSTYIDRRIQPVFDSLGLQRYVLLCSQVADG


 


GFRDKLRKPRDFYHTCYCLSGLSVAQHAWSKDEDTPPLTRDILGGYANHL


 


EPVHLLHNILVDRYYEASRF









Due to the nature of the cloning strategy the sequence presented is not full length. Compared to the Arabidopsis thaliana sequence there are 31 amino acids missing from the amino terminus and 5 amino acids from the carboxy terminus. The percent identity of the Brassica napus nucleotide sequence and its encoded amino acid sequence to that of published sequences is shown in FIG. 9.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques. Sequence comparisons have been performed and percent identities are shown in FIG. 8 and FIG. 9.
The present invention also includes a nucleic acid sequence complimentary to the Brassica napus farnesyl transferase beta subunit of SEQ ID NO:8. The disclosed complimentary sequence is shown as SEQ ID NO:30.








SEQ ID NO: 30








AAATCTAGAAGCTTCATAATACCGATCCAAGACAATGTTGTGGAGGAGGT


 


GAACAGGTTCAAGGTGGTTTGCGTAGCCACCCAAAATGTCACGAGTCAAA


 


GGAGGAGTGTCCTCGTCTTTTGACCAAGCGTGTTGAGCCACGGAAAGACC


 


GCTTAGGCAGTAACATGTGTGGTAGAAGTCACGGGGTTTCCTCAGCTTGT


 


CTCTGAATCCACCATCAGCAACCTGAGAGCACAAGAGCACATATCTTTGC


 


AAGCCGAGGCTATCAAAAACAGGTTGAATTCTCCTGTCAATGTAGGTAGA


 


CGTATGATGAACTTGGTGACCATTCCCTGAATCTTCATCGCTATCCTCAT


 


CAGAATCATCTTCATCACTGTCTTCAGGATCATCTTCATCATGACCATGT


 


TCCTCGTGATCTTCATCTGTCCCTTGTGACATATGTGATGATGATCCATG


 


AGGTGCCATATCCTGGGATGAAAAAAATCGCTGTAGTAGAACACAGGGGG


 


CTGCCTGCCAAAACGTGTAGCAACCGTCGACCAATTTGTTCGTCCTACCT


 


TGGAATCCCATTTCTACTCCTTGTCGATGTACAACCCAATTCATTAACGA


 


ATCCAAATTCAAGCGGTCGACTTCATTGATTAAAATCATAGTAGCCAACC


 


CACAGTACGTGTACCCACCATGAGCTTCGGAGCCAGGTTCCCCTCCAATG


 


CCACCTTCATAAGTTTGGCAACTCAAAATGTAATCTCCTAAGCCGCGGGT


 


GAGTTCATCATCCACAATGTTCAGGATGCTTGCAATCAAAATCGCAGTGT


 


AGCACGCTCGCACATCTATTTCTCCCATATTATGCATCCTGAAACCTCCA


 


TTTGTATCCTTCATTCGTCTTAAGAAACAAGCCATTTGTTCTCTGTTAAT


 


TGAAGAGAAGGCTTTCTCACCTCCTAAAGTAACAAGTGTATTCACTGCAG


 


CATAACTTGTTGCAAGATGTGGAAGTTGGCCAGGACCACCACCATATCCA


 


CCATCAGAACCCTGGCAACGTCCAAGAAAATCGATTGCATTGTTTTCTAA


 


GTCATCATCCACAGACTCCCCAAGCAAAGCAATTGAATGAAGAATCCAGT


 


AACAAAGCCA






Glycine max FTA

A disclosed nucleic acid of 1041 nucleotides (also referred to as FT4) is shown in Table 7A.







TABLE 7A





FT4 Nucleotide Sequence (SEQ ID NO: 31).
















ATGGAATCTGGGTCTAGCGAAGGAGAAGAGGTGCAGCAACGCGTGCCGTT



 


GAGGGAGAGAGTGGAGTGGTCAGATGTTACTCCGGTTCCTCAAAACGACG


 


GCCCTAACCCTGTCGTTCCGATCCAGTACACTGAAGAGTTTTCCGAAGTT


 


ATGGATTACTTTCGCGCCGTTTACCTCACCGATGAACGCTCCCCTCGCGC


 


CCTCGCTCTCACAGCCGAAGCCGTTCAATTCAACTCCGGCAACTACACTG


 


TGTGGCATTTCCGACGGTTGTTACTTGAGTCGCTAAAAGTCGACTTGAAC


 


GATGAACTGGAGTTTGTGGAGCGTATGGCCGCTGGAAATTCTAAAAATTA


 


TCAGATGTGnATGTTCTGTAGGCATCCTAGACGATGGGTTGCCGAGAAGT


 


TAGGTCCTGAAGCTAGAAACAATGAGCTCGAGTTCACCAAAAAGATACTG


 


TCCGTTGATGCCAAACATTATCATGCATGGTCTCATAGACAGTGGGCTCT


 


TCAAACACTAGGAGGATGGGAAGATGAACTTAATTATTGCACAGAACTAC


 


TTAAAGAAGACATTTTTAACAATTCTGCTTGGAATCAGAGATATTTTGTC


 


ATAACAAGGTCTCCTTTCTTGGGGGGCCTAAAAGCTATGAGAGAGTCTGA


 


AGTGCTTTACACCATCGAAGCCATTATAGCCTACCCTGAAAATGAAAGCT


 


CGTGGAGATATCTACGAGGACTTTATAAAGGTGAAACTACTTCATGGGTA


 


AATGATCCTCAAGTTTCTTCAGTATGCTTAAAGATTTTGAGAACTAAGAG


 


CAACTACGTGTTTGCTCTTAGCACTATTTTAGATCTTATATGCTTTGGTT


 


ATCAACCAAATGAAGACATTAGAGATGCCATTGACGCCTTAAAGACCGCA


 


GATATGGATAAACAAGATTTAGATGATGATGAGAAAGGGGAACAACAAAA


 


TTTAAATATAGCACGAAATATTTGTTCTATCCTAAAACAAGTTGATCCAA


 


TTAGAACCAACTATTGGATTTGGCGCAAGAGCAGACTTCCT









A disclosed FT4 polypeptide (SEQ ID NO:33) encoded by SEQ ID NO:31 has 347 amino acid residues and is presented in Table 7B using the one-letter amino acid code.







TABLE 7B





Encoded FT4 protein sequence (SEQ ID NO: 33).
















MESGSSEGEEVQQRVPLRERVEWSDVTPVPQNDGPNPVVPIQYTEEFSEV



 


MDYFRAVYLTDERSPRALALTAEAVQFNSGNYTVWHFRRLLLESLKVDLN


 


DELEFVERMAAGNSKNYQMXMFCRHPRRWVAEKLGPEARNNELEFTKKIL


 


SVDAKHYHAWSHRQWALQTLGGWEDELNYCTELLKEDIFNNSAWNQRYFV


 


ITRSPFLGGLKAMRESEVLYTIEAIIAYPENESSWRYLRGLYKGETTSWV


 


NDPQVSSVCLKILRTKSNYVFALSTILDLICFGYQPNEDIRDAIDALKTA


 


DMDKQDLDDDEKGEQQNLNIARNICSILKQVDPIRTNYWIWRKSRLP









Due to the nature of the cloning strategy the sequence presented is not full length. The percent identity of the Glycine max nucleotide sequence and its encoded amino acid sequence to that of other sequences is shown in FIG. 8.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
The present invention also includes a nucleic acid sequence complimentary to the Glycine max alpha subunit of SEQ ID NO:31. The disclosed complimentary sequence is shown as SEQ ID NO:32.








SEQ ID NO: 32








AGGAAGTCTGCTCTTGCGCCAAATCCAATAGTTGGTTCTAATTGGATCAA


 


CTTGTTTTAGGATAGAACAAATATTTCGTGCTATATTTAAATTTTGTTGT


 


TCCCCTTTCTCATCATCATCTAAATCTTGTTTATCCATATCTGCGGTCTT


 


TAAGGCGTCAATGGCATCTCTAATGTCTTCATTTGGTTGATAACCAAAGC


 


ATATAAGATCTAAAATAGTGCTAAGAGCAAACACGTAGTTGCTCTTAGTT


 


CTCAAAATCTTTAAGCATACTGAAGAAACTTGAGGATCATTTACCCATGA


 


AGTAGTTTCACCTTTATAAAGTCCTCGTAGATATCTCCACGAGCTTTCAT


 


TTTCAGGGTAGGCTATAATGGCTTCGATGGTGTAAAGCACTTCAGACTCT


 


CTCATAGCTTTTAGGCCCCCCAAGAAAGGAGACCTTGTTATGACAAAATA


 


TCTCTGATTCCAAGCAGAATTGTTAAAAATGTCTTCTTTAAGTAGTTCTG


 


TGCAATAATTAAGTTCATCTTCCCATCCTCCTAGTGTTTGAAGAGCCCAC


 


TGTCTATGAGACCATGCATGATAATGTTTGGCATCAACGGACAGTATCTT


 


TTTGGTGAACTCGAGCTCATTGTTTCTAGCTTCAGGACCTAACTTCTCGG


 


CAACCCATCGTCTAGGATGCCTACAGAACATNCACATCTGATAATTTTTA


 


GAATTTCCAGCGGCCATACGCTCCACAAACTCCAGTTCATCGTTCAAGTC


 


GACTTTTAGCGACTCAAGTAACAACCGTCGGAAATGCCACACAGTGTAGT


 


TGCCGGAGTTGAATTGAACGGCTTCGGCTGTGAGAGCGAGGGCGCGAGGG


 


GAGCGTTCATCGGTGAGGTAAACGGCGCGAAAGTAATCCATAACTTCGGA


 


AAACTCTTCAGTGTACTGGATCGGAACGACAGGGTTAGGGCCGTCGTTTT


 


GAGGAACCGGAGTAACATCTGACCACTCCACTCTCTCCCTCAACGGCACG


 


CGTTGCTGCACCTCTTCTCCTTCGCTAGACCCAGATTCCAT






Glycine max FTB

A disclosed nucleic acid of 1035 nucleotides (also referred to as FT5) is shown in Table 8A.







TABLE 8A





FT5 Nucleotide Sequence (SEQ ID NO: 34).
















GCCACCATTCCTCGCAACGCCCAAACCCTCATGTTGGAGCTTCAACGCGA



 


TAATCACATGCAGTATGTCTCCAAAGGCCTTCGCCATCTCAGTTCCGCAT


 


TTTCCGTTTTGGACGCTAATCGACCCTGGCTCTGCTACTGGATCTTCCAC


 


TCCATTGCTTTGTTGGGAGAATCCGTCGATGATGAACTCGAAGATAACGC


 


TATCGATTTTCTTAACCGTTGCCAGGATCCGAATGGTGGATATGCCGGGG


 


GACCAGGCCAGATGCCTCATATTGCCACAACTTATGCTGCTGTTAATTCA


 


CTTATTACTTTGGGTGGTGAGAAATCCCTGGCATCAATTAATAGAGATAA


 


ACTGTATGGGTTTCTGCGGCGGATGAAGCAACCAAATGGTGGATTCAGGA


 


TGCATGATGAAGGTGAAATTGATGTTCGAGCTTGCTACACTGCCATTTCT


 


GTTGCAAGTGTTTTGAACATTTTGGATGATGAGCTGATCCAGAATGTTGG


 


AGACTACATTATAAGCTGTCAAACATATGAGGGTGGCATTGCTGGTGAGC


 


CTGGTTCTGAGGCTCATGGTGGGTACACCTTTTGTGGATTAGCTACAATG


 


ATTCTGATTGGTGAGGTTAATCACTTGGATCTGCCTCGATTAGTTGACTG


 


GGTGGTATTCCGACAAGGTAAGGAATGTGGATTCCAGGGGAGAACAAATA


 


AACTGGTGGATGGATGCTATTCCTTTTGGCAGGGAGGTGCTGTTGCTCTA


 


TTGCAAAGATTATCTTCTATTATCAACAAACAGATGGAAGAGACATCACA


 


GATTTTTGCGGTATCTTATGTATCTGAAGCAAAAGAAAGTTTGGATGGAA


 


CCTCTAGTCATGCAACATGCCGTGGTGAGCATGAAGGCACCAGTGAATCC


 


AGTTCATCTGATTTTAAAAATATTGCCTATAAATTTATTAATGAGTGGAG


 


AGCACAAGAACCACTTTTTCACAGTATTGCTTTACAGCAATATATTCTCT


 


TATGTGCACAGGAGCAAGAGGGTGGACTGAGAGACAAACCGGGTAAACGT


 


AGAGATCATTATCACACATGTTACTGTTTAAGTGGACTCTCATTGTGCCA


 


GTATAGTTGGTCAAAGCACCCAGATTCTCCACCAC









A disclosed FT5 polypeptide (SEQ ID NO:36) encoded by SEQ ID NO:34 has 378 amino acid residues and is presented in Table 8B using the one-letter amino acid code.







TABLE 8B





Encoded FT5 protein sequence (SEQ ID NO: 36).
















ATIPRNAQTLMLELQRDNHMQYVSKGLRHLSSAFSVLDANRPWLCYWIFH



 


SIALLGESVDDELEDNAIDFLNRCQDPNGGYAGGPGQMPHIATTYAAVNS


 


LITLGGEKSLASINRDKLYGFLRRMKQPNGGFRMHDEGEIDVRACYTAIS


 


VASVLNILDDELIQNVGDYIISCQTYEGGIAGEPGSEAHGGYTFCGLATM


 


ILIGEVNHLDLPRLVDWVVFRQGKECGFQGRTNKLVDGCYSFWQGGAVAL


 


LQRLSSIINKQMEETSQIFAVSYVSEAKESLDGTSSHATCRGEHEGTSES


 


SSSDFKNIAYKFINEWRAQEPLFHSIALQQYILLCAQEQEGGLRDKPGKR


 


RDHYHTCYCLSGLSLCQYSWSKHPDSPP









Due to the nature of the cloning strategy the sequence presented is not full length. The percent identity of the Glycine max nucleotide sequence and its encoded amino acid sequence to that of other sequences is shown in FIG. 8.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
The present invention also includes a nucleic acid sequence complimentary to the Glycine max beta subunit of SEQ ID NO:34. The disclosed complimentary sequence is shown as SEQ ID NO:35.








SEQ ID NO: 35








GTGGTGGAGAATCTGGGTGCTTTGACCAACTATACTGGCACAATGAGAGT


 


CCACTTAAACAGTAACATGTGTGATAATGATCTCTACGTTTACCCGGTTT


 


GTCTCTCAGTCCACCCTCTTGCTCCTGTGCACATAAGAGAATATATTGCT


 


GTAAAGCAATACTGTGAAAAAGTGGTTCTTGTGCTCTCCACTCATTAATA


 


AATTTATAGGCAATATTTTTAAAATCAGATGAACTGGATTCACTGGTGCC


 


TTCATGCTCACCACGGCATGTTGCATGACTAGAGGTTCCATCCAAACTTT


 


CTTTTGCTTCAGATACATAAGATACCGCAAAAATCTGTGATGTCTCTTCC


 


ATCTGTTTGTTGATAATAGAAGATAATCTTTGCAATAGAGCAACAGCACC


 


TCCCTGCCAAAAGGAATAGCATCCATCCACCAGTTTATTTGTTCTCCCCT


 


GGAATCCACATTCCTTACCTTGTCGGAATACCACCCAGTCAACTAATCGA


 


GGCAGATCCAAGTGATTAACCTCACCAATCAGAATCATTGTAGCTAATCC


 


ACAAAAGGTGTACCCACCATGAGCCTCAGAACCAGGCTCACCAGCAATGC


 


CACCCTCATATGTTTGACAGCTTATAATGTAGTCTCCAACATTCTGGATC


 


AGCTCATCATCCAAAATGTTCAAAACACTTGCAACAGAAATGGCAGTGTA


 


GCAAGCTCGAACATCAATTTCACCTTCATCATGCATCCTGAATCCACCAT


 


TTGGTTGCTTCATCCGCCGCAGAAACCCATACAGTTTATCTCTATTAATT


 


GATGCCAGGGATTTCTCACCACCCAAAGTAATAAGTGAATTAACAGCAGC


 


ATAAGTTGTGGCAATATGAGGCATCTGGCCTGGTCCCCCGGCATATCCAC


 


CATTCGGATCCTGGCAACGGTTAAGAAAATCGATAGCGTTATCTTCGAGT


 


TCATCATCGACGGATTCTCCCAACAAAGCAATGGAGTGGAAGATCCAGTA


 


GCAGAGCCAGGGTCGATTAGCGTCCAAAACGGAAAATGCGGAACTGAGAT


 


GGCGAAGGCCTTTGGAGACATACTGCATGTGATTATCGCGTTGAAGCTCC


 


AACATGAGGGTTTGGGCGTTGCGAGGAATGGTGGC






Zea maize FTB

A disclosed nucleic acid of 1235 nucleotides (also referred to as FT6) is shown in Table 9A.







TABLE 9A





FT6 Nucleotide Sequence (SEQ ID NO: 37).
















GGCGGATCCCGACCTACCGAGGCTCACGGTGACGCAGGTGGAGCAGATGA



 


AGGTGGAGGCCAGGGTTGGCGACATCTACCGCTCCCTCTTCGGGGCCGCG


 


CCCAACACGAAATCCATCATGCTAGAGCTGTGGCGTGATCAGCATATCGA


 


GTATCTGACGCCTGGGCTGAGGCATATGGGACCAGCCTTTCATGTTCTAG


 


ATGCCAATCGCCCTTGGCTATGCTACTGGATGGTTCATCCACTTGCTTTG


 


CTGGATGAAGCACTTGATGATGATCTTGAGAATGATATCATAGACTTCTT


 


AGCTCGATGTCAGGATAAAGATGGTGGATATAGTGGTGGACCTGGACAGT


 


TGCCTCACCTAGCTACGACTTATGCTGCTGTAAATACACTTGTGACAATA


 


GGGAGCGAAAGAGCATTGTCATCAATCAATAGGGGCAACCTGTACAATTT


 


TATGCTGCAGATGAAAGATGTATCAGGTGCTTTCAGAATGCATGATGGTG


 


GCGAAATTGATGTCCGTGCTTCCTACACCGCTATATCGGTTGCCAGCCTT


 


GTGAATATTCTTGATTTTAAACTGGCAAAAGGTGTAGGCGACTACATAGC


 


AAGATGTCAAACTTATGAAGGTGGTATTGCTGGGGAGCCTTATGCTGAAG


 


CACATGGTGGGTATACATTCTGTGGATTGGCTGCTTTGATCCTGCTTAAT


 


GAGGCAGAGAAAGTTGACTTGCCTAGTTTGATTGGCTGGGTGGCTTTTCG


 


TCAAGGAGTGGAATGCGGATTTCAAGGACGAACTAATAAATTGGTTGATG


 


GTTGCTACTCCTTTTGGCAGGGAGCTGCCATTGCTTTCACACAAAAGTTA


 


ATTACGATTGTTGATAAGCAATTGAGGTCCTCGTATTCCTGCAAAAGGCC


 


ATCAGGAGAGGATGCCTGCAGCACCAGTTCATATGGGTGCACCGCGAATA


 


AGTCTTCCTCTGCTGTGGACTATGCGAAGTTTGGATTTGATTTTATACAA


 


CAGAGCAACCAAATTGGCCCACTCTTCCATAACATTGCCCTGCAACAATA


 


CATCCTACTTTGTTCTCAGGTACTAGAGGGAGGCTTGAGGGATAAGCCTG


 


GAAAGAACAGAGATCACTATCATTCATGCTACTGCCTCAGTGGCCTCGCA


 


GTTAGCCAGTACAGTGCCATGACTGATACTGGTTCGTGCCCATTACCTCA


 


GCATGTGCTTGGACCGTACTCTAATTTGCTGGAGCCAATCCATCC









A disclosed FT6 polypeptide (SEQ ID NO:39) encoded by SEQ ID NO:37 has 414 amino acid residues and is presented in Table 9B using the one-letter amino acid code.







TABLE 9B





Encoded FT6 protein sequence (SEQ ID NO: 39).
















ADPDLPRLTVTQVEQMKVEARVGDIYRSLFGAAPNTKSIMLELWRDQHIE



 


YLTPGLRHMGPAFHVLDANRPWLCYWMVHPLALLDEALDDDLENDIIDFL


 


ARCQDKDGGYSGGPGQLPHLATTYAAVNTLVTIGSERALSSINRGNLYNF


 


MLQMKDVSGAFRMHDGGEIDVRASYTAISVASLVNILDFKLAKGVGDYIA


 


RCQTYEGGIAGEPYAEAHGGYTFCGLAALILLNEAEKVDLPSLIGWVAFR


 


QGVECGFQGRTNKLVDGCYSFWQGAAIAFTQKLITIVDKQLRSSYSCKRP


 


SGEDACSTSSYGCTANKSSSAVDYAKFGFDFIQQSNQIGPLFHNIALQQY


 


ILLCSQVLEGGLRDKPGKNRDHYHSCYCLSGLAVSQYSAMTDTGSCPLPQ


 


HVLGPYSNLLEPIH









Due to the nature of the cloning strategy the sequence presented is not full length. The percent identity of the Glycine max nucleotide sequence and its encoded amino acid sequence to that of other sequences is shown in FIG. 8.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
The present invention also includes a nucleic acid sequence complimentary to the Zea maize beta subunit of SEQ ID NO:37. The disclosed complimentary sequence is shown as SEQ ID NO:38.








SEQ ID NO: 38








GGATGGATTGGCTCCAGCAAATTAGAGTACGGTCCAAGCACATGCTGAGG


 


TAATGGGCACGAACCAGTATCAGTCATGGCACTGTACTGGCTAACTGCGA


 


GGCCACTGAGGCAGTAGCATGAATGATAGTGATCTCTGTTCTTTCCAGGC


 


TTATCCCTCAAGCCTCCCTCTAGTACCTGAGAACAAAGTAGGATGTATTG


 


TTGCAGGGCAATGTTATGGAAGAGTGGGCCAATTTGGTTGCTCTGTTGTA


 


TAAAATCAAATCCAAACTTCGCATAGTCCACAGCAGAGGAAGACTTATTC


 


GCGGTGCACCCATATGAACTGGTGCTGCAGGCATCCTCTCCTGATGGCCT


 


TTTGCAGGAATACGAGGACCTCAATTGCTTATCAACAATCGTAATTAACT


 


TTTGTGTGAAAGCAATGGCAGCTCCCTGCCAAAAGGAGTAGCAACCATCA


 


ACCAATTTATTAGTTCGTCCTTGAAATCCGCATTCCACTCCTTGACGAAA


 


AGCCACCCAGCCAATCAAACTAGGCAAGTCAACTTTCTCTGCCTCATTAA


 


GCAGGATCAAAGCAGCCAATCCACAGAATGTATACCCACCATGTGCTTCA


 


GCATAAGGCTCCCCAGCAATACCACCTTCATAAGTTTGACATCTTGCTAT


 


GTAGTCGCCTACACCTTTTGCCAGTTTAAAATCAAGAATATTCACAAGGC


 


TGGCAACCGATATAGCGGTGTAGGAAGCACGGACATCAATTTCGCCACCA


 


TCATGCATTCTGAAAGCACCTGATACATCTTTCATCTGCAGCATAAAATT


 


GTACAGGTTGCCCCTATTGATTGATGACAATGCTCTTTCGCTCCCTATTG


 


TCACAAGTGTATTTACAGCAGCATAAGTCGTAGCTAGGTGAGGCAACTGT


 


CCAGGTCCACCACTATATCCACCATCTTTATCCTGACATCGAGCTAAGAA


 


GTCTATGATATCATTCTCAAGATCATCATCAAGTGCTTCATCCAGCAAAG


 


CAAGTGGATGAACCATCCAGTAGCATAGCCAAGGGCGATTGGCATCTAGA


 


ACATGAAAGGCTGGTCCCATATGCCTCAGCCCAGGCGTCAGATACTCGAT


 


ATGCTGATCACGCCACAGCTCTAGCATGATGGATTTCGTGTTGGGCGCGG


 


CCCCGAAGAGGGAGCGGTAGATGTCGCCAACCCTGGCCTCCACCTTCATC


 


TGCTCCACCTGCGTCACCGTGAGCCTCGGTAGGTCGGGATCCGCC






The FTA and FTB nucleic acids and amino acids disclosed above have homology to other members of the FT protein family (GenBank ID NOs: U63298, U83707, and U73203; WO 00/14207; Cutler et al., Science 273(5279):1239-41, 1996; Ziegelhoffer et al., Proc Natl Acad Sci USA. 97(13):7633-8, 2000). The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Tables 10A-10D. In the ClustalW alignment, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.







TABLE 10A





ClustalW Nucleic Acid Analysis of FT Alpha Subunits















 1) BNA-12; FT2 (SEQ ID NO: 6)


 2) At-FT-A; FT1 (SEQ ID NO: 1)


 3) PPI-Soy-FTA; FT4 (SEQ ID NO: 31)


 4) Pea-FT-A (SEQ ID NO: 59)


 5) Tomato-FTA (SEQ ID NO: 60)


 6) Rice-FT-A (SEQ ID NO: 61)


 7) Zea mays-FT-A (SEQ ID NO: 62)


 8) Soy1-FT-A (SEQ ID NO: 63)


 9) Soy2-FT-A (SEQ ID NO: 64)


10) Triticum-FT-A (SEQ ID NO: 65)


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 10B





ClustalW Amino Acid Analysis of FT Alpha Subunits















1) BNA-12; FT2 (SEQ ID NO: 7)


2) At-FT-A; FT1 (SEQ ID NO: 5)


3) PPI-Soy-FTA; FT4 (SEQ ID NO: 33)


4) Pea-FT-A (SEQ ID NO: 66)


5) Tomato-FTA (SEQ ID NO: 67)


6) Rice-FT-A (SEQ ID NO: 68)


7) Zea mays-FT-A (SEQ ID NO: 69)


8) Soy1-FT-A (SEQ ID NO: 70)


9) Soy2-FT-A (SEQ ID NO: 71)


10) Triticum-FT-A (SEQ ID NO: 72)


 








 








 








 








 








 






















TABLE 10C





ClustalW Nucleic Acid Analysis of FT Beta Subunits















1) PPI-BnFTb; FT3 (SEQ ID NO: 8)


2) era1 (SEQ ID NO: 73)


3) Wiggum (SEQ ID NO: 74)


4) PPI-Soy-FTB; FT5 (SEQ ID NO: 34)


5) DuP-Soy-FTB (SEQ ID NO: 75)


6) PPI-Corn-FTB; FT6 (SEQ ID NO: 37)


7) DuP-Corn-FTB (SEQ ID NO: 76)


8) Pea-FT-B (SEQ ID NO: 77)


9) Tomato (SEQ ID NO: 78)


10) Tobacco (SEQ ID NO: 79)


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 10D





ClustalW Amino Acid Analysis of FT Beta Subunits















1) PPI-BnFTB; FT3 (SEQ ID NO: 9)


2) era1 (SEQ ID NO: 80)


3) Wiggum (SEQ ID NO: 81)


4) PPI-Soy-FTB; FT5 (SEQ ID NO: 36)


5) DuP-Soy-FTB (SEQ ID NO: 82)


6) PPI-Corn-FTB; FT6 (SEQ ID NO: 39)


7) DuP-Corn-FTB (SEQ ID NO: 83)


8) Pea-FT-B (SEQ ID NO: 84)


9) Tomato (SEQ ID NO: 85)


10) Tobacco (SEQ ID NO: 86)


 








 








 








 








 








 








 








 















Also included in the invention is the farnesyl transferase alpha consensus sequence of SEQ ID NO:87 and the farnesyl transferase beta consensus sequence of SEQ ID NO:88 To generate the consensus sequence, the farnesyl transferase alpha and farnesyl transferase beta sequences of the invention were aligned using the program BioEdit. The homology between the farnesyl transferase alpha (FTA) polypeptide sequences of the invention is shown graphically in the ClustalW analysis shown in Table 10E. The homology between the farnesyl transferase beta (FTB) polypeptide sequences of the invention is shown graphically in the ClustalW analysis shown in Table 10F.







TABLE 10E





ClustalW Amino Acid Analysis of FT Alphs





















 








 








 








 






















TABLE 10F





ClustalW Amino Acid Analysis of FT Beta





















 








 








 








 








 








 















Also included in the invention is the farnesyl transferase alpha consensus sequence of SEQ ID NO:89 and the farnesyl transferase beta consensus sequence of SEQ ID NO:90. To generate the consensus sequence, the farnesyl transferase alpha and farnesyl transferase beta sequences of the invention were aligned using the program BioEdit. The homology between the farnesyl transferase alpha (FTA) nucleic acid sequences of the invention is shown graphically in the ClustalW analysis shown in Table 10G. The homology between the farnesyl transferase beta (FTB) nucleic acid sequences of the invention is shown graphically in the ClustalW analysis shown in Table 10H.







TABLE 10G





ClustalW Nucleic Acid Analysis of FT Alpha





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 10H





ClustalW Nucleic Acid Analysis of FT Beta





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















Example 5
Vector Constructs for Transformation
The FTA or FTB sequences have be used to produce constructs suitable for transformation into plants and under the control of appropriate regulatory sequences. The gene sequences were in either the sense orientation for over-expression or the antisense orientation for down-regulation. Portions of these sequences have been used to construct a double-stranded-RNA-inhibition (dsRNAi) construct. A sequence of preferably not less than 21 nt was cloned as an inverse repeat separated by a linker that when expressed results in down-regulation of the target gene. Double antisense (DA) vectors have been created in which a direct repeat of an antisense sequence is separated by a spacer sequence such as GUS. Promoters have been used for constitutive expression such as the 35S CaMV promoter, the MuA Zea maize promoter or inducible by specific environmental or cellular cues such as the ABA levels or drought conditions which induce expression of the RD29A promoter. Alternatively, tissue or organelle specific promoters such as the HIC or CUT1 promoter can be used. Such constructs have been transformed into Arabidopsis thaliana, Brassica, Zea maize, Glycine max. Other species can be transformed as desired. Each species to be transformed may make use of specific regulatory sequences as appropriate for those particular species. Transformed plants have be selected and their phenotypic properties analyzed. The transgenic plants were assessed for characteristics such as increased tolerance to drought, altered biomass accumulation, yield, nutritional requirements such as minerals or micro-nutrients, biotic stress such as fungal, bacterial, or other such pathogen infection or attack or any other such physical or biochemical characteristic.
Example 6
Plant Transformation
Arabidopsis thaliana transgenic plants were made by flower dipping method into an Agrobacterium culture. Wild type plants were grown under standard conditions until they began flowering. The plant was inverted for 2 min into a solution of Agrobacterium culture. Plants were then bagged for two days to maintain humidity and then uncovered to continue growth and seed development. Mature seed was bulk harvested.
Transformed T1 plants were selected by germination and growth on MS plates containing 50 μg/ml kanamycin. Green, kanamycin resistant seedlings were identified after 2 weeks growth and transplanted to soil. Plants were bagged to ensure self fertilization and the T2 seed of each plant harvested separately. During growth of T1 plants leaf samples were harvested, DNA extracted and Southern analysis performed.
T2 seeds were analyzed for KanR segregation. From those lines that showed a 3:1 resistant phenotype surviving T2 plants were grown, bagged during seed set, and T3 seed harvested from each line. T3 seed was again used for KanR segregation analysis and those lines showing 100% KanR phenotype were selected as homozygous lines. Further analysis was done using T3 seed.
Transgenic Brassica napus plants were produced using Agrobacterium mediated transformation of cotyledon petiole tissue. Seeds were sterilized as follows. Seeds were wetted with 95% ethanol for a short period of time such as 15 seconds. Approximately 30 ml of sterilizing solution I was added (70% Javex, 100 μl Tween20) and left for approximately 15 minutes. Solution I was removed and replaced with 30 ml of solution II (0.25% mercuric chloride, 100 μl Tween20) and incubated for about 10 minutes. Seeds were rinsed with at least 500 ml double distilled sterile water and stored in a sterile dish. Seeds were germinated on plates of 1⁄2 MS medium, pH 5.8, supplemented with 1% sucrose and 0.7% agar. Fully expanded cotyledons were harvested and placed on Medium I (Murashige minimal organics (MMO), 3% sucrose, 4.5 mg/L benzyl adenine (BA), 0.7% phytoagar, pH5.8). An Agrobacterium culture containing the nucleic acid construct of interest was grown for 2 days in AB Minimal media. The cotyledon explants were dipped such that only the cut portion of the petiole is contacted by the Agrobacterium solution. The explants were then embedded in Medium I and maintained for 5 days at 24° C., with 16, 8 hr light dark cycles. Explants were transferred to Medium II (Medium 1,300 mg/L timentin,) for a further 7 days and then to Medium III (Medium II, 20 mg/L kanamycin). Any root or shoot tissue which had developed at this time was dissected away. Transfer explants to fresh plates of Medium III after 14-21 days. When regenerated shoot tissue developed the regenerated tissue was transferred to Medium IV (MMO, 3% sucrose, 1.0% phytoagar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin). Once healthy shoot tissue developed shoot tissue dissected from any callus tissue was dipped in 10×IBA and transferred to Medium V (Murashige and Skooge (MS), 3% sucrose, 0.2 mg/L indole butyric acid (IBA), 0.7% agar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin) for rooting. Healthy plantlets were transferred to soil.
Transgenic Glycine max, Zea maize and cotton can be produced using Agrobacterium-based methods which are known to one of skill in the art. Alternatively one can use a particle or non-particle biolistic bombardment transformation method. An example of non-particle biolistic transformation is given in U.S. Patent Application 20010026941. Viable plants are propagated and homozygous lines are generated. Plants are tested for the presence of drought tolerance, physiological and biochemical phenotypes as described elsewhere.
The following table identifies the constructs and the species which they have been transformed.









TABLE 11





SEQ ID NO:
SEQ
Species Transformed




















SEQ ID NO: 4
pBI121-35S-anti-AtFTA
Arabidopsis







thaliana


SEQ ID NO: 40
pBI121-35S-AtFTA
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 41
pBI121-rd29A-anti-AtFTA
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 42
pBI121-35S-DA-AtFTA
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 43
pBI121-RD29A-DA-AtFTA
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 44
MuA-anti-GmFTA



Glycine







max


SEQ ID NO: 45
RD29A-anti-GmFTA



Glycine







max


SEQ ID NO: 46
MuA-HP-GmFTA-Nos-Term



Glycine







max


SEQ ID NO: 47
RD29AP-HP-GmFTA-Nos-Term



Glycine







max


SEQ ID NO: 48
pBI121-35S-Anti-AtFTB
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 49
pBI121-RD29AP-Anti-AtFTB
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 50
pBI121-35S-HP-AtFTB
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 51
pBI121-RD29AP-HP-AtFTB
Arabidopsis
Brassica




thaliana
napus


SEQ ID NO: 52
pBI121-35S-AtFTB
Arabidopsis




thaliana


SEQ ID NO: 53
MuA-anti-GmFTB-Nos-Term



Glycine







max


SEQ ID NO: 54
RD29AP-anti-GmFTB-Nos-Term



Glycine







max


SEQ ID NO: 55
MuA-HP-GmFTB-Nos-Term



Glycine







max


SEQ ID NO: 56
RD29AP-HP-GmFTB-Nos-Term



Glycine







max


SEQ ID NO: 57
MuA-anti-Zea maizeFTB-Nos-Term


Zea






maize


SEQ ID NO: 58
MuA-HP-Zea maizeFTB-Nos-Term


Zea






maize









Non-limiting examples of vector constructs suitable for plant transformation are given in SEQ ID NO: 4, 40-58.








SEQ ID NO: 4








 aaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagcccacagatggttagagaggcttacgcagcaggtct


 


catcaagacgatctacccgagcaataatctccaggaaatcaaataccttc


 


ccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaag


 


aacacagagaaagatatatttctcaagatcagaagtactattccagtatg


 


gacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggag


 


tctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagatt


 


caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcat


 


acagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatgg


 


tggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca


 


gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaa


 


cctcctcggattccattgcccagctatctgtcactttattgtgaagatag


 


tggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaag


 


gccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccccc


 


acccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaa


 


agcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaa


 


tcccactatccttcgcaagacccttcctctatataaggaagttcatttca


 


tttggagagaacacgggggactctagaggatcctcaaattgctgccactg


 


taatcttgctcttcctccatgcccaatagttagctcttataggatctaca


 


cgaccaagaatagtacacaccaaattggccaagttagtctctggttcttc


 


attagctagagctctcactgagtctttatgctcgttggttggtctcagtc


 


catcacatagaagatccaaaagggtgctcagagcgaatccatggaagcaa


 


tctgtgcgggatagaacattcaaacagactgaggaaacacttggatcact


 


aatccaggattctttgtcgtctttgtaaagcgcttttaggtatcgccatg


 


agctctcgtttgcaggattggttaaaatggctttgattgtgtagcttact


 


tcagattctctcatggcttctaggcctcccaacaaaggagattgggtgat


 


gacataatacctctgattccaggcggaattgttaaagacgtcagcttcaa


 


ggagctcgtgacagtaatcgagctcatcttcccatcctcctaatgcccgt


 


agtgtccactgcctatgtgaccaagcatgataatgtttggcatcaagtga


 


aagtactctacgggtaaattcaagttctctccctgcaacatcaggaccca


 


gtttctctgcaacccatcgccgatgatgccacagttggtagttcttagag


 


ttatcctcagcaatgcgttcgatgaactcgagttcttcaaacaagtcgtg


 


attaagggcctcgagtactaggcgcctgaaatgccacactgtgtagttgc


 


cggagtttaagaggagggtttcttccgtgagtcgtagtgcgcgaggagat


 


cgctcgtcggaaaagtaaatcgcacggaagtaatccatagtctcgcggaa


 


ctcttccttgtaggcaattggcaccactggattcggaccatcgtcctgag


 


tcaatgggaccacgtctgaccactccaatcgttggctcagtggcacggtc


 


tcgtcgaaattcatcccctcgaatttccccgatcgttcaaacatttggca


 


ataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatca


 


tataatttctgttgaattacgttaagcatgtaataattaacatgtaatgc


 


atgacgttatttatgagatgggtttttatgattagagtcccgcaattata


 


catttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaat


 


tatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgt


 


cgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatc


 


gccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcc


 


cgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgcccgctc


 


ctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgt


 


caagctctaaatcgggggctccctttagggttccgatttagtgctttacg


 


gcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggc


 


catcgccctgatagacggtttttcgccctttgacgttggagtccacgttc


 


tttaatagtggactcttgttccaaactggaacaacactcaaccctatctc


 


gggctattcttttgatttataagggattttgccgatttcggaaccaccat


 


caaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgc


 


aactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctca


 


ctggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgt


 


tattaagttgtctaagcgtcaattt 


 


 






SEQ ID NO:4 is the nucleic acid sequence of pBI121-antisense-FTA vector construct used to transform Arabidopsis plants. Italicized sequences are the right and left border repeats (1-24, 5226-5230). Underlined sequence is the 35S promoter (2515-3318). Bold sequence is the anti-sense Farnesyl transferase alpha sequence (3334-4317).








SEQ ID NO: 40








gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagcccacagatggttagagaggcttacgcagcaggtct


 


catcaagacgatctacccgagcaataatctccaggaaatcaaataccttc


 


ccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaag


 


aacacagagaaagatatatttctcaagatcagaagtactattccagtatg


 


gacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggag


 


tctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagatt


 


caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcat


 


acagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatgg


 


tggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca


 


gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaa


 


cctcctcggattccattgcccagctatctgtcactttattgtgaagatag


 


tggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaag


 


gccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccccc


 


acccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaa


 


agcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaa


 


tcccactatccttcgcaagacccttcctctatataaggaagttcatttca


 


tttggagagaacacgggggactctagaggatccatgaatttcgacgagac


 


cgtgccactgagccaacgattggagtggtcagacgtggtcccattgactc


 


aggacgatggtccgaatccagtggtgccaattgcctacaaggaagagttc


 


cgcgagactatggattacttccgtgcgatttacttttccgacgagcgatc


 


tcctcgcgcactacgactcacggaagaaaccctcctcttaaactccggca


 


actacacagtgtggcatttcaggcgcctagtactcgaggcccttaatcac


 


gacttgtttgaagaactcgagttcatcgaacgcattgctgaggataactc


 


taagaactaccaactgtggcatcatcggcgatgggttgcagagaaactgg


 


gtcctgatgttgcagggagagaacttgaatttacccgtagagtactttca


 


cttgatgccaaacattatcatgcttggtcacataggcagtggacactacg


 


ggcattaggaggatgggaagatgagctcgattactgtcacgagctccttg


 


aagctgacgtctttaacaattccgcctggaatcagaggtattatgtcatc


 


acccaatctcctttgttgggaggcctagaagccatgagagaatctgaagt


 


aagctacacaatcaaagccattttaaccaatcctgcaaacgagagctcat


 


ggcgatacctaaaagctctttacaaagacgacaaagaatcctggattagt


 


gatccaagtgtttcctcagtctgtttgaatgttctatcccgcacagattg


 


cttccatggattcgctctgagcacccttttggatcttctatgtgatggac


 


tgagaccaaccaacgagcataaagactcagtgagagctctagctaatgaa


 


gaaccagagactaacttggccaatttggtgtgtactattcttggtcgtgt


 


agatcctgtaagagctaactattgggcatggaggaagagcaagattacag


 


tggcagcaatttgactcgaatttccccgatcgttcaaacatttggcaata


 


aagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatat


 


aatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatg


 


acgttatttatgagatgggtttttatgattagagtcccgcaattatacat


 


ttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattat


 


cgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgt


 


tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc


 


ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgc


 


accgatcgcccttcccaacagttgcgcagcctgaatggcgcccgctcctt


 


tcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaa


 


gctctaaatcgggggctccctttagggttccgatttagtgctttacggca


 


cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccat


 


cgccctgatagacggtttttcgccctttgacgttggagtccacgttcttt


 


aatagtggactcttgttccaaactggaacaacactcaaccctatctcggg


 


ctattcttttgatttataagggattttgccgatttcggaaccaccatcaa


 


acaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaac


 


tctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactg


 


gtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgttat


 


taagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca


(Underlined Seq: 35S promoter; Bold: AtFTA)


 







SEQ ID NO: 41







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagggagccatagatgcaattcaatcaaactgaaatttc


 


tgcaagaatctcaaacacggagatctcaaagtttgaaagaaaatttattt


 


cttcgactcaaaacaaacttacgaaatttaggtagaacttatatacatta


 


tattgtaattttttgtaacaaaatgtttttattattattatagaatttta


 


ctggttaaattaaaaatgaatagaaaaggtgaattaagaggagagaggag


 


gtaaacattttcttctattttttcatattttcaggataaattattgtaaa


 


agtttacaagatttccatttgactagtgtaaatgaggaatattctctagt


 


aagatcattatttcatctacttcttttatcttctaccagtagaggaataa


 


acaatatttagctcctttgtaaatacaaattaattttccttcttgacatc


 


attcaattttaattttacgtataaaataaaagatcatacctattagaacg


 


attaaggagaaatacaattcgaatgagaaggatgtgccgtttgttataat


 


aaacagccacacgacgtaaacgtaaaatgaccacatgatgggccaataga


 


catggaccgactactaataatagtaagttacattttaggatggaataaat


 


atcataccgacatcagttttgaaagaaaagggaaaaaaagaaaaaataaa


 


taaaagatatactaccgacatgagttccaaaaagcaaaaaaaaagatcaa


 


gccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacg


 


aaaacagacgcttcatacgtgtccctttatctctctcagtctctctataa


 


acttagtgagaccctcctctgttttactcacaaatatgcaaactagaaaa


 


caatcatcaggaataaagggtttgattacttctattggaaagactctaga


 


ggatcctcaaattgctgccactgtaatcttgctcttcctccatgcccaat


 


agttagctcttataggatctacacgaccaagaatagtacacaccaaattg


 


gccaagttagtctctggttcttcattagctagagctctcactgagtcttt


 


atgctcgttggttggtctcagtccatcacatagaagatccaaaagggtgc


 


tcagagcgaatccatggaagcaatctgtgcgggatagaacattcaaacag


 


actgaggaaacacttggatcactaatccaggattctttgtcgtctttgta


 


aagcgcttttaggtatcgccatgagctctcgtttgcaggattggttaaaa


 


tggctttgattgtgtagcttacttcagattctctcatggcttctaggcct


 


cccaacaaaggagattgggtgatgacataatacctctgattccaggcgga


 


attgttaaagacgtcagcttcaaggagctcgtgacagtaatcgagctcat


 


cttcccatcctcctaatgcccgtagtgtccactgcctatgtgaccaagca


 


tgataatgtttggcatcaagtgaaagtactctacgggtaaattcaagttc


 


tctccctgcaacatcaggacccagtttctctgcaacccatcgccgatgat


 


gccacagttggtagttcttagagttatcctcagcaatgcgttcgatgaac


 


tcgagttcttcaaacaagtcgtgattaagggcctcgagtactaggcgcct


 


gaaatgccacactgtgtagttgccggagtttaagaggagggtttcttccg


 


tgagtcgtagtgcgcgaggagatcgctcgtcggaaaagtaaatcgcacgg


 


aagtaatccatagtctcgcggaactcttccttgtaggcaattggcaccac


 


tggattcggaccatcgtcctgagtcaatgggaccacgtctgaccactcca


 


atcgttggctcagtggcacggtctcgtcgaaattcatcccctcgaatttc


 


cccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgt


 


tgccggtcttgcgatgattatcatataatttctgttgaattacgttaagc


 


atgtaataattaacatgtaatgcatgacgttatttatgagatgggttttt


 


atgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaat


 


atagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttac


 


tagatcgggaattcactggccgtcgttttacaacgtcgtgactgggaaaa


 


ccctggcgttacccaacttaatcgccttgcagcacatccccctttcgcca


 


gctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttg


 


cgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcg


 


ccacgttcgccggctttccccgtcaagctctaaatcgggggctcccttta


 


gggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattt


 


gggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcc


 


ctttgacgttggagtccacgttctttaatagtggactcttgttccaaact


 


ggaacaacactcaaccctatctcgggctattcttttgatttataagggat


 


tttgccgatttcggaaccaccatcaaacaggattttcgcctgctggggca


 


aaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagg


 


gcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagta


 


cattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgt


 


ttacaccacaatatatcctgcca


(Underlined Seq: RD29A promoter; Bold: Anti-


sense-AtFTA)


 







SEQ ID NO: 42







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagcccacagatggttagagaggcttacgcagcaggtct


 


catcaagacgatctacccgagcaataatctccaggaaatcaaataccttc


 


ccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaag


 


aacacagagaaagatatatttctcaagatcagaagtactattccagtatg


 


gacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggag


 


tctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagatt


 


caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcat


 


acagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatgg


 


tggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca


 


gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaa


 


cctcctcggattccattgcccagctatctgtcactttattgtgaagatag


 


tggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaag


 


gccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccccc


 


acccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaa


 


agcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaa


 


tcccactatccttcgcaagacccttcctctatataaggaagttcatttca


 


tttggagagaacacgggggactctagaggatcctcGCTCTTCCTCCATGC


 


CCAATAGTTAGCTCTTACAGGATCTACACGACCAAGAATAGTACACACCA


 


AATTGGCCAAGTTAGTCTCTGGTTCTTCATTAGCTAGAGCTCTCACTGAG


 


TCTTTATGCTCGTTGGTTGGTCTCAGTCCATCACATAGAAGATCCAAAAG


 


GGTGCTCAGAGCGAATCCATGGAAGCAATCTGTGCGGGATAGAACATTCA


 


AACAGACTGAGGAAACACTTGGATCACTAATCCAGGATTCTTTGTCGTCT


 


TTGTAAAGAGCTTTTAGGTATCGCCATGAGCTCTCGTTTGCAGGATTGGT


 


TAAAATGGCTTTGATTGTGTAGCTTACTTCAGATTCTCTCATGGCTTCTA


 


GGCCTCCCAACAAAGGAGATTGGGTGATGACATAATACCTCTGATTCCAG


 


GCGGAATTGTTAAAGACGTCAGCTTCAAGGAGCTCGTGACAGTAATCGAG


 


CTCATCTTCCCATCCTCCTAATGCCCGgaggatccccATCTACCCGCTTC


 


GCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAAC


 


CACAAACCGTTCTACTTTACTGGCTTTGGTCGTCATGAAGATGCGGACTT


 


GCGTGGCAAAGGATTCGATAACGTGCTGATGGTGCACGACCACGCATTAA


 


TGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCATTACCCTTACGCT


 


GAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATTGATGA


 


AACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCGAAGCGG


 


GCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACT


 


CAGCAAGCGCACTTACAGGCGATTAAAGAGCTGATAGCGCGTGACAAAAA


 


CCACCCAAGCGTGGTGATGTGGAGTATTGCCAACGAACCGGATACCCGTC


 


CGCAAGGTGCACGGGAATATTTCGCGCCACTGGCGGAAGCAACGCGTAAA


 


CTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTCTGCGACGC


 


TCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAACCGTT


 


ATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTA


 


CTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGTACACCGACATGTG


 


GAGTGAAGAGTATCAGTGTGCATGGCTGGATATGTATCACCGCGTCTTTG


 


ATCGCGTCAGCGCCGTCGTCGGTGAACAGGTATGGAATTTCGCCGATTTT


 


GCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACAAGAAAGGGATCTT


 


CACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAACGCT


 


GGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAATGA


 


ATCAACAACTCTCCTGGCGCACCATCGTCGGCTACAGCCTCGGGAATTGC


 


TACCGAGCTCGCTCTTCCTCCATGCCCAATAGTTAGCTCTTACAGGATCT


 


ACACGACCAAGAATAGTACACACCAAATTGGCCAAGTTAGTCTCTGGTTC


 


TTCATTAGCTAGAGCTCTCACTGAGTCTTTATGCTCGTTGGTTGGTCTCA


 


GTCCATCACATAGAAGATCCAAAAGGGTGCTCAGAGCGAATCCATGGAAG


 


CAATCTGTGCGGGATAGAACATTCAAACAGACTGAGGAAACACTTGGATC


 


ACTAATCCAGGATTCTTTGTCGTCTTTGTAAAGAGCTTTTAGGTATCGCC


 


ATGAGCTCTCGTTTGCAGGATTGGTTAAAATGGCTTTGATTGTGTAGCTT


 


ACTTCAGATTCTCTCATGGCTTCTAGGCCTCCCAACAAAGGAGATTGGGT


 


GATGACATAATACCTCTGATTCCAGGCGGAATTGTTAAAGACGTCAGCTT


 


CAAGGAGCTCGTGACAGTAATCGAGCTCATCTTCCCATCCTCCTAATGCC


 


CGctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaag


 


attgaatcctgttgccggtcttgcgatgattatcatataatttctgttga


 


attacgttaagcatgtaataattaacatgtaatgcatgacgttatttatg


 


agatgggtttttatgattagagtcccgcaattatacatttaatacgcgat


 


agaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgt


 


catctatgttactagatcgggaattcactggccgtcgttttacaacgtcg


 


tgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatc


 


cccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct


 


tcccaacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcc


 


cttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgg


 


gggctccctttagggttccgatttagtgctttacggcacctcgaccccaa


 


aaaacttgatttgggtgatggttcacgtagtgggccatcgccctgataga


 


cggtttttcgccctttgacgttggagtccacgttctttaatagtggactc


 


ttgttccaaactggaacaacactcaaccctatctcgggctattcttttga


 


tttataagggattttgccgatttcggaaccaccatcaaacaggattttcg


 


cctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggcc


 


aggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaa


 


accaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaa


 


gcgtcaatttgtttacaccacaatatatcctgcca


(Underlined Seq: 35S promoter; Bold: AtFTA anti-


sense sequence separated by GUS Seq.)


 







SEQ ID NO: 43







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagggagccatagatgcaattcaatcaaactgaaatttc


 


tgcaagaatctcaaacacggagatctcaaagtttgaaagaaaatttattt


 


cttcgactcaaaacaaacttacgaaatttaggtagaacttatatacatta


 


tattgtaattttttgtaacaaaatgtttttattattattatagaatttta


 


ctggttaaattaaaaatgaatagaaaaggtgaattaagaggagagaggag


 


gtaaacattttcttctattttttcatattttcaggataaattattgtaaa


 


agtttacaagatttccatttgactagtgtaaatgaggaatattctctagt


 


aagatcattatttcatctacttcttttatcttctaccagtagaggaataa


 


acaatatttagctcctttgtaaatacaaattaattttccttcttgacatc


 


attcaattttaattttacgtataaaataaaagatcatacctattagaacg


 


attaaggagaaatacaattcgaatgagaaggatgtgccgtttgttataat


 


aaacagccacacgacgtaaacgtaaaatgaccacatgatgggccaataga


 


catggaccgactactaataatagtaagttacattttaggatggaataaat


 


atcataccgacatcagttttgaaagaaaagggaaaaaaagaaaaaataaa


 


taaaagatatactaccgacatgagttccaaaaagcaaaaaaaaagatcaa


 


gccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacg


 


aaaacagacgcttcatacgtgtccctttatctctctcagtctctctataa


 


acttagtgagaccctcctctgttttactcacaaatatgcaaactagaaaa


 


caatcatcaggaataaagggtttgattacttctattggaaaggactctag


 


aggatcctcGCTCTTCCTCCATGCCCAATAGTTAGCTCTTACAGGATCTA


 


CACGACCAAGAATAGTACACACCAAATTGGCCAAGTTAGTCTCTGGTTCT


 


TCATTAGCTAGAGCTCTCACTGAGTCTTTATGCTCGTTGGTTGGTCTCAG


 


TCCATCACATAGAAGATCCAAAAGGGTGCTCAGAGCGAATCCATGGAAGC


 


AATCTGTGCGGGATAGAACATTCAAACAGACTGAGGAAACACTTGGATCA


 


CTAATCCAGGATTCTTTGTCGTCTTTGTAAAGAGCTTTTAGGTATCGCCA


 


TGAGCTCTCGTTTGCAGGATTGGTTAAAATGGCTTTGATTGTGTAGCTTA


 


CTTCAGATTCTCTCATGGCTTCTAGGCCTCCCAACAAAGGAGATTGGGTG


 


ATGACATAATACCTCTGATTCCAGGCGGAATTGTTAAAGACGTCAGCTTC


 


AAGGAGCTCGTGACAGTAATCGAGCTCATCTTCCCATCCTCCTAATGCCC


 


GgaggatccccATCTACCCGCTTCGCGTCGGCATCCGGTCAGTGGCAGTG


 


AAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTACTTTACTGGCTT


 


TGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACGTGC


 


TGATGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTAC


 


CGTACCTCGCATTACCCTTACGCTGAAGAGATGCTCGACTGGGCAGATGA


 


ACATGGCATCGTGGTGATTGATGAAACTGCTGCTGTCGGCTTTTCGCTCT


 


CTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGC


 


GAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGGCGATTAA


 


AGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGGAGTA


 


TTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCG


 


CCACTGGCGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTG


 


CGTCAATGTAATGTTCTGCGACGCTCACACCGATACCATCAGCGATCTCT


 


TTGATGTGCTGTGCCTGAACCGTTATTACGGATGGTATGTCCAAAGCGGC


 


GATTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACTTCTGGCCTGGCA


 


GGAGAAACTGTACACCGACATGTGGAGTGAAGAGTATCAGTGTGCATGGC


 


TGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAA


 


CAGGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGT


 


TGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGG


 


CGGCTTTTCTGCTGCAAAAACGCTGGACTGGCATGAACTTCGGTGAAAAA


 


CCGCAGCAGGGAGGCAAACAATGAATCAACAACTCTCCTGGCGCACCATC


 


GTCGGCTACAGCCTCGGGAATTGCTACCGAGCTCGCTCTTCCTCCATGCC


 


CAATAGTTAGCTCTTACAGGATCTACACGACCAAGAATAGTACACACCAA


 


ATTGGCCAAGTTAGTCTCTGGTTCTTCATTAGCTAGAGCTCTCACTGAGT


 


CTTTATGCTCGTTGGTTGGTCTCAGTCCATCACATAGAAGATCCAAAAGG


 


GTGCTCAGAGCGAATCCATGGAAGCAATCTGTGCGGGATAGAACATTCAA


 


ACAGACTGAGGAAACACTTGGATCACTAATCCAGGATTCTTTGTCGTCTT


 


TGTAAAGAGCTTTTAGGTATCGCCATGAGCTCTCGTTTGCAGGATTGGTT


 


AAAATGGCTTTGATTGTGTAGCTTACTTCAGATTCTCTCATGGCTTCTAG


 


GCCTCCCAACAAAGGAGATTGGGTGATGACATAATACCTCTGATTCCAGG


 


CGGAATTGTTAAAGACGTCAGCTTCAAGGAGCTCGTGACAGTAATCGAGC


 


TCATCTTCCCATCCTCCTAATGCCCGctcgaatttccccgatcgttcaaa


 


catttggcaataaagtttcttaagattgaatcctgttgccggtcttgcga


 


tgattatcatataatttctgttgaattacgttaagcatgtaataattaac


 


atgtaatgcatgacgttatttatgagatgggtttttatgattagagtccc


 


gcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaact


 


aggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattc


 


actggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttaccc


 


aacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagc


 


gaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatgg


 


cgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggc


 


tttccccgtcaagctctaaatcgggggctccctttagggttccgatttag


 


tgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcac


 


gtagtgggccatcgccctgatagacggtttttcgccctttgacgttggag


 


tccacgttctttaatagtggactcttgttccaaactggaacaacactcaa


 


ccctatctcgggctattcttttgatttataagggattttgccgatttcgg


 


aaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggacc


 


gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttg


 


cccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtcc


 


gcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatat


 


atcctgcca


(Underlined Seq: RD29A promoter; Bold: AtFTA anti-


sense sequence, separated by GUS Seq.)


 







SEQ ID NO: 44







GAATTCAAATTTTTCGCCAGTTCTAAATATCCGGAAACCTCTTGGGATGC


 


CATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAGGT


 


GGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGA


 


TACCTCTGCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGC


 


ACCGTGGAGTAAGAAGACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTG


 


ATGTAGTATCTCCATTGACGTAAGGGATGACGCACAATCCAACTATCCAT


 


CGCAAGACCATTGCTCTATATAAGAAAGTTAATATCATTTCGAGTGGCCA


 


CGCTGAGCTCAGGAAGTCTGCTCTTGCGCCAAATCCAATAGTTGGTTCTA


 


ATTGGATCAACTTGTTTTAGGATAGAACAAATATTTCGTGCTATATTTAA


 


ATTTTGTTGTTCCCCTTTCTCATCATCATCTAAATCTTGTTTATCCATAT


 


CTGCGGTCTTTAAGGCGTCAATGGCATCTCTAATGTCTTCATTTGGTTGA


 


TAACCAAAGCATATAAGATCTAAAATAGTGCTAAGAGCAAACACGTAGTT


 


GCTCTTAGTTCTCAAAATCTTTAAGCATACTGAAGAAACTTGAGGATCAT


 


TTACCCATGAAGTAGTTTCACCTTTATAAAGTCCTCGTAGATATCTCCAC


 


GAGCTTTCATTTTCAGGGTAGGCTATAATGGCTTCGATGGTGTAAAGCAC


 


TTCAGACTCTCTCATAGCTTTTAGGCCCCCCAAGAAAGGAGACCTTGTTA


 


TGACAAAATATCTCTGATTCCAAGCAGAATTGTTAAAAATGTCTTCTTTA


 


AGTAGTTCTGTGCAATAATTAAGTTCATCTTCCCATCCTCCTAGTGTTTG


 


AAGAGCCCACTGTCTATGAGACCATGCATGATAATGTTTGGCATCAACGG


 


ACAGTATCTTTTTGGTGAACTCGAGCTgagctcgaatttccccgatcgtt


 


caaacatttggcaataaagtttcttaagattgaatcctgttgccggtctt


 


gcgatgattatcatataatttctgttgaattacgttaagcatgtaataat


 


taacatgtaatgcatgacgttatttatgagatgggtttttatgattagag


 


tcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgca


 


aactaggataaattatcgcgcgcggtgtcatctatgttactagatcggga


 


attc


(Underlined MuA Promoter; Bold: Glycine max


anti-FTA; lower case: NOS terminater Seq.)


 







SEQ ID NO: 45







GGAGCCATAGATGCAATTCAATCAAACTGAAATTTCTGCAAGAATCTCAA


 


ACACGGAGATCTCAAAGTTTGAAAGAAAATTTATTTCTTCGACTCAAAAC


 


AAACTTACGAAATTTAGGTAGAACTTATATACATTATATTGTAATTTTTT


 


GTAACAAAATGTTTTTATTATTATTATAGAATTTTACTGGTTAAATTAAA


 


AATGAATAGAAAAGGTGAATTAAGAGGAGAGAGGAGGTAAACATTTTCTT


 


CTATTTTTTCATATTTTCAGGATAAATTATTGTAAAAGTTTACAAGATTT


 


CCATTTGACTAGTGTAAATGAGGAATATTCTCTAGTAAGATCATTATTTC


 


ATCTACTTCTTTTATCTTCTACCAGTAGAGGAATAAACAATATTTAGCTC


 


CTTTGTAAATACAAATTAATTTTCCTTCTTGACATCATTCAATTTTAATT


 


TTACGTATAAAATAAAAGATCATACCTATTAGAACGATTAAGGAGAAATA


 


CAATTCGAATGAGAAGGATGTGCCGTTTGTTATAATAAACAGCCACACGA


 


CGTAAACGTAAAATGACCACATGATGGGCCAATAGACATGGACCGACTAC


 


TAATAATAGTAAGTTACATTTTAGGATGGAATAAATATCATACCGACATC


 


AGTTTTGAAAGAAAAGGGAAAAAAAGAAAAAATAAATAAAAGATATACTA


 


CCGACATGAGTTCCAAAAAGCAAAAAAAAAGATCAAGCCGACACAGACAC


 


GCGTAGAGAGCAAAATGACTTTGACGTCACACCACGAAAACAGACGCTTC


 


ATACGTGTCCCTTTATCTCTCTCAGTCTCTCTATAAACTTAGTGAGACCC


 


TCCTCTGTTTTACTCACAAATATGCAAACTAGAAAACAATCATCAGGAAT


 


AAAGGGTTTGATTACTTCTATTGGAAAGAGGAAGTCTGCTCTTGCGCCAA


 


ATCCAATAGTTGGTTCTAATTGGATCAACTTGTTTTAGGATAGAACAAAT


 


ATTTCGTGCTATATTTAAATTTTGTTGTTCCCCTTTCTCATCATCATCTA


 


AATCTTGTTTATCCATATCTGCGGTCTTTAAGGCGTCAATGGCATCTCTA


 


ATGTCTTCATTTGGTTGATAACCAAAGCATATAAGATCTAAAATAGTGCT


 


AAGAGCAAACACGTAGTTGCTCTTAGTTCTCAAAATCTTTAAGCATACTG


 


AAGAAACTTGAGGATCATTTACCCATGAAGTAGTTTCACCTTTATAAAGT


 


CCTCGTAGATATCTCCACGAGCTTTCATTTTCAGGGTAGGCTATAATGGC


 


TTCGATGGTGTAAAGCACTTCAGACTCTCTCATAGCTTTTAGGCCCCCCA


 


AGAAAGGAGACCTTGTTATGACAAATATCTCTGATTCCAAGCAGAATTGT


 


TAAAAATGTCTTCTTTAAGTAGTTCTGTGCAATAATTAAGTTCATCTTCC


 


CATCCTCCTAGTGTTTGAAGAGCCCACTGTCTATGAGACCATGCATGATA


 


ATGTTTGGCATCAACGGACAGTATCTTTTTGGTGAACTCGAGCTgagctc


 


gaatttccccgatcgttcaaacatttggcaataaagtttcttaagattga


 


atcctgttgccggtcttgcgatgattatcatataatttctgttgaattac


 


gttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatg


 


ggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaa


 


acaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatct


 


atgttactagatcgggaattc


(Underlined RD29A Promoter; Bold: Glycine max


anti-Glycine max FTA; lower case: NOS terminater


Seq.)


 







SEQ ID NO: 46







GAATTCAAATTTTTCGCCAGTTCTAAATATCCGGAAACCTCTTGGGATGC


 


CATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAGGT


 


GGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGA


 


TACCTCTGCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGC


 


ACCGTGGAGTAAGAAGACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTG


 


ATGTAGTATCTCCATTGACGTAAGGGATGACGCACAATCCAACTATCCAT


 


CGCAAGACCATTGCTCTATATAAGAAAGTTAATATCATTTCGAGTGGCCA


 


CGCTGAGCTCAGGAAGTCTGCTCTTGCGCCAAATCCAATAGTTGGTTCTA


 


ATTGGATCAACTTGTTTTAGGATAGAACAAATATTTCGTGCTATATTTAA


 


ATTTTGTTGTTCCCCTTTCTCATCATCATCTAAATCTTGTTTATCCATAT


 


CTGCGGTCTTTAAGGCGTCAATGGCATCTCTAATGTCTTCATTTGGTTGA


 


TAACCAAAGCATATAAGATCTAAAATAGTGCTAAGAGCAAACACGTAGTT


 


GCTCTTAGTTCTCAAAATCTTTAAGCATACTGAAGAAACTTGAGGATCAT


 


TTACCCATGAAGTAGTTTCACCTTTATAAAGTCCTCGTAGATATCTCCAC


 


GAGCTTTCATTTTCAGGGTAGGCTATAATGGCTTCGATGGTGTAAAGCAC


 


TTCAGACTCTCTCATAGCTTTTAGGCCCCCCAAGAAAGGAGACCTTGTTA


 


TGACAAAATATCTCTGATTCCAAGCAGAATTGTTAAAAATGTCTTCTTTA


 


AGTAGTTCTGTGCAATAATTAAGTTCATCTTCCCATCCTCCTAGTGTTTG


 


AAGAGCCCACTGTCTATGAGACCATGCATGATAATGTTTGGCATCAACGG


 


ACAGTATCTTTTTGGTGAACTCGAGCTTAAAGGTGAAACTACTTCATGGG


 


TAAATGATCCTCAAGTTTCTTCAGTATGCTTAAAGATTTTGAGAACTAAG


 


AGCAACTACGTGTTTGCTCTTAGCACTATTTTAGATCTTATATGCTTTGG


 


TTATCAACCAAATGAAGACATTAGAGATGCCATTGACGCCTTAAAGACCG


 


CAGATATGGATAAACAAGATTTAGATGATGATGAGAAAGGGGAACAACAA


 


AATTTAAATATACCACGAAATATTTGTTCTATCCTAAAACAAGTTGATCC


 


AATTAGAACCAACTATTGGATTTGGCGCAAGAGCAGACTTCCTgagctcg


 


aatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaa


 


tcctgttgccggtcttgcgatgattatcatataatttctgttgaattacg


 


ttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgg


 


gtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaa


 


caaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatcta


 


tgttactagatcgggaattc


(Underlined: Glycine max FTA Anti-Sense section;


Bold: MuA Promoter; Italics: Glycine max FTA Sense


section; lower case: NOS terminater Seq.)


 







SEQ ID NO: 47







ggagccatagatgcaattcaatcaaactgaaatttctgcaagaatctcaa


 


acacggagatctcaaagtttgaaagaaaatttatttcttcgactcaaaac


 


aaacttacgaaatttaggtagaacttatatacattatattgtaatttttt


 


gtaacaaaatgtttttattattattatagaattttactggttaaattaaa


 


aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttctt


 


ctattttttcatattttcaggataaattattgtaaaagtttacaagattt


 


ccatttgactagtgtaaatgaggaatattctctagtaagatcattatttc


 


atctacttcttttatcttctaccagtagaggaataaacaatatttagctc


 


ctttgtaaatacaaattaattttccttcttgacatcattcaattttaatt


 


ttacgtataaaataaaagatcatacctattagaacgattaaggagaaata


 


caattcgaatgagaaggatgtgccgtttgttataataaacagccacacga


 


cgtaaacgtaaaatgaccacatgatgggccaatagacatggaccgactac


 


taataatagtaagttacattttaggatggaataaatatcataccgacatc


 


agttttgaaagaaaagggaaaaaaagaaaaaataaataaaagatatacta


 


ccgacatgagttccaaaaagcaaaaaaaaagatcaagccgacacagacac


 


gcgtagagagcaaaatgactttgacgtcacaccacgaaaacagacgcttc


 


atacgtgtccctttatctctctcagtctctctataaacttagtgagaccc


 


tcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaat


 


aaagggtttgattacttctattggaaagAGGAAGTCTGCTCTTGCGCCAA


 


ATCCAATAGTTGGTTCTAATTGGATCAACTTGTTTTAGGATAGAACAAAT


 


ATTTCGTGCTATATTTAAATTTTGTTGTTCCCCTTTCTCATCATCATCTA


 


AATCTTGTTTATCCATATCTGCGGTCTTTAAGGCGTCAATGGCATCTCTA


 


ATGTCTTCATTTGGTTGATAACCAAAGCATATAAGATCTAAAATAGTGCT


 


AAGAGCAAACACGTAGTTGCTCTTAGTTCTCAAAATCTTTAAGCATACTG


 


AAGAAACTTGAGGATCATTTACCCATGAAGTAGTTTCACCTTTATAAAGT


 


CCTCGTAGATATCTCCACGAGCTTTCATTTTCAGGGTAGGCTATAATGGC


 


TTCGATGGTGTAAAGCACTTCAGACTCTCTCATAGCTTTTAGGCCCCCCA


 


AGAAAGGAGACCTTGTTATGACAAAATATCTCTGATTCCAAGCAGAATTG


 


TTAAAAATGTCTTCTTTAAGTAGTTCTGTGCAATAATTAAGTTCATCTTC


 


CCATCCTCCTAGTGTTTGAAGAGCCCACTGTCTATGAGACCATGCATGAT


 


AATGTTTGGCATCAACGGACAGTATCTTTTTGGTGAACTCGAGCTTAAAG


 


GTGAAACTACTTCATGGGTAAATGATCCTCAAGTTTCTTCAGTATGCTTA


 


AAGATTTTGAGAACTAAGAGCAACTACGTGTTTGCTCTTAGCACTATTTT


 


AGATCTTATATGCTTTGGTTATCAACCAAATGAAGACATTAGAGATGCCA


 


TTGACGCCTTAAAGACCGCAGATATGGATAAACAAGATTTAGATGATGAT


 


GAGAAAGGGGAACAACAAAATTTAAATATAGCACGAAATATTTGTTCTAT


 


CCTAAAACAAGTTGATCCAATTAGAACCAACTATTGGATTTGGCGCAAGA


 


GCAGACTTCCTgagctcgaatttccccgatcgttcaaacatttggcaata


 


aagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatat


 


aatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatg


 


acgttatttatgagatgggtttttatgattagagtcccgcaattatacat


 


ttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattat


 


cgcgcgcggtgtcatctatgttactagatcgggaattc


(Bold lower case: RD29A Promoter; Underline, Upper


case: Antisense GmFTA; Upper case: Sense GmFTA;


lower case: NOS terminater)


 







SEQ ID NO: 48







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagcccacagatggttagagaggcttacgcagcaggtct


 


catcaagacgatctacccgagcaataatctccaggaaatcaaataccttc


 


ccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaag


 


aacacagagaaagatatatttctcaagatcagaagtactattccagtatg


 


gacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggag


 


tctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagatt


 


caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcat


 


acagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatgg


 


tggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca


 


gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaa


 


cctcctcggattccattgcccagctatctgtcactttattgtgaagatag


 


tggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaag


 


gccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccccc


 


acccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaa


 


agcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaa


 


tcccactatccttcgcaagacccttcctctatataaggaagttcatttca


 


tttggagagaacacgggggactctagaggatccgtccggaattcccgggt


 


cgacccacgcgtccgggagattcagcgagataagcaattggattatctga


 


tgaaaggcttaaggcagcttggtccgcagttttcttccttagatgctaat


 


cgaccttggctttgttactggattcttcattcaatagctttgcttgggga


 


gactgtggatgatgaattagaaagcaatgccattgacttccttggacgct


 


gccagggctctgaaggtggatacggtggtggtcctggccaacttccacat


 


cttgcaactacttatgctgcagtgaatgcacttgttactttaggaggtga


 


caaagccctttcttcaattaatagagaaaaaatgtcttgttttttaagac


 


ggatgaaggatacaagtggaggtttcaggatgcatgatatgggagaaatg


 


gatgttcgtgcatgctacactgcaatttcggttgcaagcatcctaaatat


 


tatggatgatgaactcacccagggcctaggagattacatcttgagttgcc


 


aaacttatgaaggtggcattggaggggaacctggctccgaagctcacggt


 


gggtatacctactgtggtttggctgctatgattttaatcaatgaggtcga


 


ccgtttgaatttggattcattaatgaattgggctgtacatcgacaaggag


 


tagaaatgggatttcaaggtaggacgaacaaattggtcgatggttgctac


 


acattttggcaggcagccccttgtgttctactacaaagattatattcaac


 


caatgatcatgacgttcatggatcatcacatatatcagaagggacaaatg


 


aagaacatcatgctcatgatgaagatgaccttgaagacagtgatgatgat


 


gatgattctgatgaggacaacgatgaagattcagtgaatggtcacagaat


 


ccatcatacatccacctacattaacaggagaatgcaactggtttttgata


 


gcctcggcttgcagagatatgtactcttgtgctctaagatccctgacggt


 


ggattcagagacaagccgaggaaaccccgtgacttctaccacacatgtta


 


ctgcctgagcggcttgtctgtggctcagcacgcttggttaaaagacgagg


 


acactcctcctttgactcgcgacattatgggtggctactcgaatctcctt


 


gaacctgttcaacttcttcacaacattgtcatggatcagtataatgaagc


 


tatcgagttcttctttaaagcagcatgactcgaatttccccgatcgttca


 


aacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgc


 


gatgattatcatataatttctgttgaattacgttaagcatgtaataatta


 


acatgtaatgcatgacgttatttatgagatgggtttttatgattagagtc


 


ccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaa


 


ctaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaat


 


tcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttac


 


ccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaata


 


gcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaat


 


ggcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccg


 


gctttccccgtcaagctctaaatcgggggctccctttagggttccgattt


 


agtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttc


 


acgtagtgggccatcgccctgatagacggtttttcgccctttgacgttgg


 


agtccacgttctttaatagtggactcttgttccaaactggaacaacactc


 


aaccctatctcgggctattcttttgatttataagggattttgccgatttc


 


ggaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtgga


 


ccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgt


 


tgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgt


 


ccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaat


 


atatcctgcca


(Underline: 35S promoter; Bold: anti-AtFTB)


 







SEQ ID NO: 49







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagggagccatagatgcaattcaatcaaactgaaatttc


 


tgcaagaatctcaaacacggagatctcaaagtttgaaagaaaatttattt


 


cttcgactcaaaacaaacttacgaaatttaggtagaacttatatacatta


 


tattgtaattttttgtaacaaaatgtttttattattattatagaatttta


 


ctggttaaattaaaaatgaatagaaaaggtgaattaagaggagagaggag


 


gtaaacattttcttctattttttcatattttcaggataaattattgtaaa


 


agtttacaagatttccatttgactagtgtaaatgaggaatattctctagt


 


aagatcattatttcatctacttcttttatcttctaccagtagaggaataa


 


acaatatttagctcctttgtaaatacaaattaattttccttcttgacatc


 


attcaattttaattttacgtataaaataaaagatcatacctattagaacg


 


attaaggagaaatacaattcgaatgagaaggatgtgccgtttgttataat


 


aaacagccacacgacgtaaacgtaaaatgaccacatgatgggccaataga


 


catggaccgactactaataatagtaagttacattttaggatggaataaat


 


atcataccgacatcagttttgaaagaaaagggaaaaaaagaaaaaataaa


 


taaaagatatactaccgacatgagttccaaaaagcaaaaaaaaagatcaa


 


gccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacg


 


aaaacagacgcttcatacgtgtccctttatctctctcagtctctctataa


 


acttagtgagaccctcctctgttttactcacaaatatgcaaactagaaaa


 


caatcatcaggaataaagggtttgattacttctattggaaaggactctag


 


aggatccgtccggaattcccgggtcgacccacgcgtccgggagattcagc


 


gagataagcaattggattatctgatgaaaggcttaaggcagcttggtccg


 


cagttttcttccttagatgctaatcgaccttggctttgttactggattct


 


tcattcaatagctttgcttggggagactgtggatgatgaattagaaagca


 


atgccattgacttccttggacgctgccagggctctgaaggtggatacggt


 


ggtggtcctggccaacttccacatcttgcaactacttatgctgcagtgaa


 


tgcacttgttactttaggaggtgacaaagccctttcttcaattaatagag


 


aaaaaatgtcttgttttttaagacggatgaaggatacaagtggaggtttc


 


aggatgcatgatatgggagaaatggatgttcgtgcatgctacactgcaat


 


ttcggttgcaagcatcctaaatattatggatgatgaactcacccagggcc


 


taggagattacatcttgagttgccaaacttatgaaggtggcattggaggg


 


gaacctggctccgaagctcacggtgggtatacctactgtggtttggctgc


 


tatgattttaatcaatgaggtcgaccgtttgaatttggattcattaatga


 


attgggctgtacatcgacaaggagtagaaatgggatttcaaggtaggacg


 


aacaaattggtcgatggttgctacacattttggcaggcagccccttgtgt


 


tctactacaaagattatattcaaccaatgatcatgacgttcatggatcat


 


cacatatatcagaagggacaaatgaagaacatcatgctcatgatgaagat


 


gaccttgaagacagtgatgatgatgatgattctgatgaggacaacgatga


 


agattcagtgaatggtcacagaatccatcatacatccacctacattaaca


 


ggagaatgcaactggtttttgatagcctcggcttgcagagatatgtactc


 


ttgtgctctaagatccctgacggtggattcagagacaagccgaggaaacc


 


ccgtgacttctaccacacatgttactgcctgagcggcttgtctgtggctc


 


agcacgcttggttaaaagacgaggacactcctcctttgactcgcgacatt


 


atgggtggctactcgaatctccttgaacctgttcaacttcttcacaacat


 


tgtcatggatcagtataatgaagctatcgagttcttctttaaagcagcat


 


gactcgaatttccccgatcgttcaaacatttggcaataaagtttcttaag


 


attgaatcctgttgccggtcttgcgatgattatcatataatttctgttga


 


attacgttaagcatgtaataattaacatgtaatgcatgacgttatttatg


 


agatgggtttttatgattagagtcccgcaattatacatttaatacgcgat


 


agaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgt


 


catctatgttactagatcgggaattcactggccgtcgttttacaacgtcg


 


tgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatc


 


cccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct


 


tcccaacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcc


 


cttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgg


 


gggctccctttagggttccgatttagtgctttacggcacctcgaccccaa


 


aaaacttgatttgggtgatggttcacgtagtgggccatcgccctgataga


 


cggtttttcgccctttgacgttggagtccacgttctttaatagtggactc


 


ttgttccaaactggaacaacactcaaccctatctcgggctattcttttga


 


tttataagggattttgccgatttcggaaccaccatcaaacaggattttcg


 


cctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggcc


 


aggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaa


 


accaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaa


 


gcgtcaatttgtttacaccacaatatatcctgcca


(Underline: RD29A Promoter; Bold: anti-AtFTB)


 







SEQ ID NO: 50







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagcccacagatggttagagaggcttacgcagcaggtct


 


catcaagacgatctacccgagcaataatctccaggaaatcaaataccttc


 


ccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaag


 


aacacagagaaagatatatttctcaagatcagaagtactattccagtatg


 


gacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggag


 


tctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagatt


 


caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcat


 


acagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatgg


 


tggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca


 


gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaa


 


cctcctcggattccattgcccagctatctgtcactttattgtgaagatag


 


tggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaag


 


gccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccccc


 


acccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaa


 


agcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaa


 


tcccactatccttcgcaagacccttcctctatataaggaagttcatttca


 


tttggagagaacacgggggactctagaggatcctcCTCCTAGGCCCTGGG


 


TGAGTTCATCATCCATAATATTTAGGATGCTTGCAACCGAAATTGCAGTG


 


TAGCATGCACGAACATCCATTTCTCCCATATCATGCATCCTGAAACCTCC


 


ACTTGTATCCTTCATCCGTCTTAAAAAACAAGACATTTTTTCTCTATTAA


 


TTGAAGAAAGGGCTTTGTCACCTCCTAAAGTAACAAGTGCATTCACTGCA


 


GCATAAGTAGTTGCAAGATGTGGAAGTTGGCCAGGACCACCACCGTATCC


 


ACCTTCAGAGCCCTGGCAGCGTCCAAGGAAGTCAATGGCATTGCTTTCTA


 


ATTCATCATCCACAGTCTCCCCAAGCAAAGCTATTGAATGAAGAATCCAG


 


TAACAAAGCCAAGGTCGATTAGCATCTAAGGAAGAAAACTGCGGACCAAG


 


CTGCCTTAAGCCTTTCATCAGATAATCCAATTGCTTATCTCGCTGAATCT


 


CCCGGACGCGTGGGTCGACCCGGGAATTCCGGACgaggatccccATCTAC


 


CCGCTTCGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCT


 


GATTAACCACAAACCGTTCTACTTTACTGGCTTTGGTCGTCATGAAGATG


 


CGGACTTGCGTGGCAAAGGATTCGATAACGTGCTGATGGTGCACGACCAC


 


GCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCATTACCC


 


TTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGA


 


TTGATGAAACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTC


 


GAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGG


 


GGAAACTCAGCAAGCGCACTTACAGGCGATTAAAGAGCTGATAGCGCGTG


 


ACAAAAACCACCCAAGCGTGGTGATGTGGAGTATTGCCAACGAACCGGAT


 


ACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACTGGCGGAAGCAAC


 


GCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTCT


 


GCGACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTG


 


AACCGTTATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGA


 


GAAGGTACTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGTACACCG


 


ACATGTGGAGTGAAGAGTATCAGTGTGCATGGCTGGATATGTATCACCGC


 


GTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAACAGGTATGGAATTTCGC


 


CGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACAAGAAAG


 


GGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAA


 


AAACGCTGGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAA


 


ACAATGAATCAACAACTCTCCTGGCGCACCATCGTCGGCTACAGCCTCGG


 


GAATTGCTACCGAGCTCgtccggaattcccgggtcgacccacgcgtccgg


 


gagattcagcgagataagcaattggattatctgatgaaaggcttaaggca


 


gcttggtccgcagttttcttccttagatgctaatcgaccttggctttgtt


 


actggattcttcattcaatagctttgcttggggagactgtggatgatgaa


 


ttagaaagcaatgccattgacttccttggacgctgccagggctctgaagg


 


tggatacggtggtggtcctggccaacttccacatcttgcaactacttatg


 


ctgcagtgaatgcacttgttactttaggaggtgacaaagccctttcttca


 


attaatagagaaaaaatgtcttgttttttaagacggatgaaggatacaag


 


tggaggtttcaggatgcatgatatgggagaaatggatgttcgtgcatgct


 


acactgcaatttcggttgcaagcatcctaaatattatggatgatgaactc


 


acccagggcctaggagctcgaatttccccgatcgttcaaacatttggcaa


 


taaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcat


 


ataatttctgttgaattacgttaagcatgtaataattaacatgtaatgca


 


tgacgttatttatgagatgggtttttatgattagagtcccgcaattatac


 


atttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaatt


 


atcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtc


 


gttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcg


 


ccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggccc


 


gcaccgatcgcccttcccaacagttgcgcagcctgaatggcgcccgctcc


 


tttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtc


 


aagctctaaatcgggggctccctttagggttccgatttagtgctttacgg


 


cacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggcc


 


atcgccctgatagacggtttttcgccctttgacgttggagtccacgttct


 


ttaatagtggactcttgttccaaactggaacaacactcaaccctatctcg


 


ggctattcttttgatttataagggattttgccgatttcggaaccaccatc


 


aaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgca


 


actctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac


 


tggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgtt


 


attaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca


(Underline: 35S promoter; Bold uppercase:


antisense AtFTB; Lower case Bold: sense AtFTB)


 







SEQ ID NO: 51







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggggtcgaaggtgccgatatcattacgacagcaa


 


cggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggccc


 


ggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgagat


 


gcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgccac


 


agacccggatgatccccgatcgttcaaacatttggcaataaagtttctta


 


agattgaatcctgttgccggtcttgcgatgattatcatataatttctgtt


 


gaattacgttaagcatgtaataattaacatgtaatgcatgacgttattta


 


tgagatgggtttttatgattagagtcccgcaattatacatttaatacgcg


 


atagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggt


 


gtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctct


 


ggtggtggttctggtggcggctctgagggtggtggctctgagggtggcgg


 


ttctgagggtggcggctctgagggaggcggttccggtggtggctctggtt


 


ccggtgattttgattatgaaaagatggcaaacgctaataagggggctatg


 


accgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaact


 


tgattctgtcgctactgattacggtgctgctatcgatggtttcattggtg


 


acgtttccggccttgctaatggtaatggtgctactggtgattttgctggc


 


tctaattcccaaatggctcaagtcggtgacggtgataattcacctttaat


 


gaataatttccgtcaatatttaccttccctccctcaatcggttgaatgtc


 


gcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg


 


ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg


 


cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccc


 


aggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagc


 


ggataacaatttcacacaggaaacagctatgaccatgattacgccaagct


 


tgcatgcctgcagggagccatagatgcaattcaatcaaactgaaatttct


 


gcaagaatctcaaacacggagatctcaaagtttgaaagaaaatttatttc


 


ttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattat


 


attgtaattttttgtaacaaaatgtttttattattattatagaattttac


 


tggttaaattaaaaatgaatagaaaaggtgaattaagaggagagaggagg


 


taaacattttcttctattttttcatattttcaggataaattattgtaaaa


 


gtttacaagatttccatttgactagtgtaaatgaggaatattctctagta


 


agatcattatttcatctacttcttttatcttctaccagtagaggaataaa


 


caatatttagctcctttgtaaatacaaattaattttccttcttgacatca


 


ttcaattttaattttacgtataaaataaaagatcatacctattagaacga


 


ttaaggagaaatacaattcgaatgagaaggatgtgccgtttgttataata


 


aacagccacacgacgtaaacgtaaaatgaccacatgatgggccaatagac


 


atggaccgactactaataatagtaagttacattttaggatggaataaata


 


tcataccgacatcagttttgaaagaaaagggaaaaaaagaaaaaataaat


 


aaaagatatactaccgacatgagttccaaaaagcaaaaaaaaagatcaag


 


ccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacga


 


aaacagacgcttcatacgtgtccctttatctctctcagtctctctataaa


 


cttagtgagaccctcctctgttttactcacaaatatgcaaactagaaaac


 


aatcatcaggaataaagggtttgattacttctattggaaaggactctaga


 


ggatcctcCTCCTAGGCCCTGGGTGAGTTCATCATCCATAATATTTAGGA


 


TGCTTGCAACCGAAATTGCAGTGTAGCATGCACGAACATCCATTTCTCCC


 


ATATCATGCATCCTGAAACCTCCACTTGTATCCTTCATCCGTCTTAAAAA


 


ACAAGACATTTTTTCTCTATTAATTGAAGAAAGGGCTTTGTCACCTCCTA


 


AAGTAACAAGTGCATTCACTGCAGCATAAGTAGTTGCAAGATGTGGAAGT


 


TGGCCAGGACCACCACCGTATCCACCTTCAGAGCCCTGGCAGCGTCAAGG


 


AAGTCAATGGCATTGCTTTCTAATTCATCATCCACAGTCTCCCCAAGCAA


 


AGCTATTGAATGAAGAATCCAGTAACAAAGCCAAGGTCGATTAGCATCTA


 


AGGAAGAAAACTGCGGACCAAGCTGCCTTAAGCCTTTCATCAGATAATCC


 


AATTGCTTATCTCGCTGAATCTCCCGGACGCGTGGGTCGACCCGGGAATT


 


CCGGACgaggatccccATCTACCCGCTTCGCGTCGGCATCCGGTCAGTGG


 


CAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTACTTTACT


 


GGCTTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAA


 


CGTGCTGATGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACT


 


CCTACCGTACCTCGCATTACCCTTACGCTGAAGAGATGCTCGACTGGGCA


 


GATGAACATGGCATCGTGGTGATTGATGAAACTGCTGCTGTCGGCTTTTC


 


GCTCTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGT


 


ACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGGCG


 


ATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTG


 


GAGTATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATT


 


TCGCGCCACTGGCGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATC


 


ACCTGCGTCAATGTAATGTTCTGCGACGCTCACACCGATACCATCAGCGA


 


TCTCTTTGATGTGCTGTGCCTGAACCGTTATTACGGATGGTATGTCCAAA


 


GCGGCGATTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACTTCTGGCC


 


TGGCAGGAGAAACTGTACACCGACATGTGGAGTGAAGAGTATCAGTGTGC


 


ATGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCG


 


GTGAACAGGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTG


 


CGCGTTGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAA


 


GTCGGCGGCTTTTCTGCTGCAAAAACGCTGGACTGGCATGAACTTCGGTG


 


AAAAACCGCAGCAGGGAGGCAAACAATGAATCAACAACTCTCCTGGCGCA


 


CCATCGTCGGCTACAGCCTCGGGAATTGCTACCGAGCTCgtccggaattc


 


ccgggtcgacccacgcgtccgggagattcagcgagataagcaattggatt


 


atctgatgaaaggcttaaggcagcttggtccgcagttttcttccttagat


 


gctaatcgaccttggctttgttactggattcttcattcaatagctttgct


 


tggggagactgtggatgatgaattagaaagcaatgccattgacttccttg


 


gacgctgccagggctctgaaggtggatacggtggtggtcctggccaactt


 


ccacatcttgcaactacttatgctgcagtgaatgcacttgttactttagg


 


aggtgacaaagccctttcttcaattaatagagaaaaaatgtcttgttttt


 


taagacggatgaaggatacaagtggaggtttcaggatgcatgatatggga


 


gaaatggatgttcgtgcatgctacactgcaatttcggttgcaagcatcct


 


aaatattatggatgatgaactcacccagggcctaggagctcgaatttccc


 


cgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttg


 


ccggtcttgcgatgattatcatataatttctgttgaattacgttaagcat


 


gtaataattaacatgtaatgcatgacgttatttatgagatgggtttttat


 


gattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatat


 


agcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttacta


 


gatcgggaattcactggccgtcgttttacaacgtcgtgactgggaaaacc


 


ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagc


 


tggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcg


 


cagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgcc


 


acgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg


 


gttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgg


 


gtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccct


 


ttgacgttggagtccacgttctttaatagtggactcttgttccaaactgg


 


aacaacactcaaccctatctcgggctattcttttgatttataagggattt


 


tgccgatttcggaaccaccatcaaacaggattttcgcctgctggggcaaa


 


ccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggc


 


aatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtaca


 


ttaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgttt


 


acaccacaatatatcctgcca


(Underline: RD29A promoter; Bold uppercase:


antisense AtFTB; Lower case Bold: sense AtFTB)


 







SEQ ID NO: 52







gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggc


 


gggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatg


 


acccccgccgatgacgcgggacaagccgttttacgtttggaactgacaga


 


accgcaacgttgaaggagccactcagccgcgggtttctggagtttaatga


 


gctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaa


 


ggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgt


 


tccataaattcccctcggtatccaattagagtctcatattcactctcaat


 


ccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg


 


gattgcacgcaggttctccggccgcttgggtggagaggctattcggctat


 


gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggct


 


gtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg


 


ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg


 


acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaag


 


ggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc


 


accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg


 


ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca


 


tcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg


 


atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc


 


aggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatgg


 


cgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat


 


tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg


 


ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccg


 


cttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct


 


tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa


 


tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccacc


 


gccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg


 


ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacg


 


ggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagca


 


acggccgacaagcacaacgccacgatcctgagcgacaatatgatcgggcc


 


cggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgaga


 


tgcaccgcgatatcttgctgcgttcggatattttcgtggagttcccgcca


 


cagacccggatgatccccgatcgttcaaacatttggcaataaagtttctt


 


aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgt


 


tgaattacgttaagcatgtaataattaacatgtaatgcatgacgttattt


 


atgagatgggtttttatgattagagtcccgcaattatacatttaatacgc


 


gatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg


 


tgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctc


 


tggtggtggttctggtggcggctctgagggtggtggctctgagggtggcg


 


gttctgagggtggcggctctgagggaggcggttccggtggtggctctggt


 


tccggtgattttgattatgaaaagatggcaaacgctaataagggggctat


 


gaccgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaac


 


ttgattctgtcgctactgattacggtgctgctatcgatggtttcattggt


 


gacgtttccggccttgctaatggtaatggtgctactggtgattttgctgg


 


ctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaa


 


tgaataatttccgtcaatatttaccttccctccctcaatcggttgaatgt


 


cgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttg


 


gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgg


 


gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc


 


caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag


 


cggataacaatttcacacaggaaacagctatgaccatgattacgccaagc


 


ttgcatgcctgcagcccacagatggttagagaggcttacgcagcaggtct


 


catcaagacgatctacccgagcaataatctccaggaaatcaaataccttc


 


ccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaag


 


aacacagagaaagatatatttctcaagatcagaagtactattccagtatg


 


gacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggag


 


tctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagatt


 


caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcat


 


acagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatgg


 


tggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca


 


gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaa


 


cctcctcggattccattgcccagctatctgtcactttattgtgaagatag


 


tggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaag


 


gccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccccc


 


acccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaa


 


agcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaa


 


tcccactatccttcgcaagacccttcctctatataaggaagttcatttca


 


tttggagagaacacgggggactctagaggatccatgccagtagtaacccg


 


cttgattcgtttgaagtgtgtagggctcagacttgaccggagtggactca


 


atcggcgaatctgtcacggaggacacggggaatcaacgcggcggagagtg


 


atggaagagctttcaagcctaaccgtgagtcagcgcgagcaatttctggt


 


ggagaacgatgtgttcgggatctataattacttcgacgccagcgacgttt


 


ctactcaaaaatacatgatggagattcagcgagataagcaattggattat


 


ctgatgaaaggcttaaggcagcttggtccgcagttttcttccttagatgc


 


taatcgaccttggctttgttactggattcttcattcaatagctttgcttg


 


gggagactgtggatgatgaattagaaagcaatgccattgacttccttgga


 


cgctgccagggctctgaaggtggatacggtggtggtcctggccaacttcc


 


acatcttgcaactacttatgctgcagtgaatgcacttgttactttaggag


 


gtgacaaagccctttcttcaattaatagagaaaaaatgtcttgtttttta


 


agacggatgaaggatacaagtggaggtttcaggatgcatgatatgggaga


 


aatggatgttcgtgcatgctacactgcaatttcggttgcaagcatcctaa


 


atattatggatgatgaactcacccagggcctaggagattacatcttgagt


 


tgccaaacttatgaaggtggcattggaggggaacctggctccgaagctca


 


cggtgggtatacctactgtggtttggctgctatgattttaatcaatgagg


 


tcgaccgtttgaatttggattcattaatgaattgggctgtacatcgacaa


 


ggagtagaaatgggatttcaaggtaggacgaacaaattggtcgatggttg


 


ctacacattttggcaggcagccccttgtgttctactacaaagattatatt


 


caaccaatgatcatgacgttcatggatcatcacatatatcagaagggaca


 


aatgaagaacatcatgctcatgatgaagatgaccttgaagacagtgatga


 


tgatgatgattctgatgaggacaacgatgaagattcagtgaatggtcaca


 


gaatccatcatacatccacctacattaacaggagaatgcaactggttttt


 


gatagcctcggcttgcagagatatgtactcttgtgctctaagatccctga


 


cggtggattcagagacaagccgaggaaaccccgtgacttctaccacacat


 


gttactgcctgagcggcttgtctgtggctcagcacgcttggttaaaagac


 


gaggacactcctcctttgactcgcgacattatgggtggctactcgaatct


 


ccttgaacctgttcaacttcttcacaacattgtcatggatcagtataatg


 


aagctatcgagttcttctttaaagcagcatgactcgaatttccccgatcg


 


ttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtc


 


ttgcgatgattatcatataatttctgttgaattacgttaagcatgtaata


 


attaacatgtaatgcatgacgttatttatgagatgggtttttatgattag


 


agtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcg


 


caaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgg


 


gaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcg


 


ttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgt


 


aatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcct


 


gaatggcgcccgctcctttcgctttcttcccttcctttctcgccacgttc


 


gccggctttccccgtcaagctctaaatcgggggctccctttagggttccg


 


atttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatg


 


gttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg


 


ttggagtccacgttctttaatagtggactcttgttccaaactggaacaac


 


actcaaccctatctcgggctattcttttgatttataagggattttgccga


 


tttcggaaccaccatcaaacaggattttcgcctgctggggcaaaccagcg


 


tggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcag


 


ctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaa


 


acgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacacca


 


caatatatcctgcca


(Underlined: 35S promoter; Bold: Sense AtFTB)


 







SEQ ID NO: 53







GAATTCAAATTTTTCGCCAGTTCTAAATATCCGGAAACCTCTTGGGATGC


 


CATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAGGT


 


GGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGA


 


TACCTCTGCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGC


 


ACCGTGGAGTAAGAAGACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTG


 


ATGTAGTATCTCCATTGACGTAAGGGATGACGCACAATCCAACTATCCAT


 


CGCAAGACCATTGCTCTATATAAGAAAGTTAATATCATTTCGAGTGGCCA


 


CGCTGAGCTCGTGGTGGAGAATCTGGGTGCTTTGACCAACTATACTGGCA


 


CAATGAGAGTCCACTTAAACAGTAACATGTGTGATAATGATCTCTACGTT


 


TACCCGGTTTGTCTCTCAGTCCACCCTCTTGCTCCTGTGCACATAAGAGA


 


ATATATTGCTGTAAAGCAATACTGTGAAAAAGTGGTTCTTGTGCTCTCCA


 


CTCATTAATAAATTTATAGGCAATATTTTTAAAATCAGATGAACTGGATT


 


CACTGGTGCCTTCATGCTCACCACGGCATGTTGCATGACTAGAGGTTCCA


 


TCCAAACTTTCTTTTGCTTCAGATACATAAGATACCGCAAAAATCTGTGA


 


TGTCTCTTCCATCTGTTTGTTGATAATAGAAGATAATCTTTGCAATAGAG


 


CAACAGCACCTCCCTGCCAAAAGGAATAGCATCCATCCACCAGTTTATTT


 


GTTCTCCCCTGGAATCCACATTCCTTACCTTGTCGGAATACCACCCAGTC


 


AACTAATCGAGGCAGATCCAAGTGATTAACCTCACCAATCAGAATCATTG


 


TAGCTAATCCACAAAAGGTGTACCCACCATGAGCCTCAGAACCAGGCTCA


 


CCAGCAATGCCACCCTCATATGTTTGACAGCTTATAATGTAGTCTCCAAC


 


ATTCTGGATCAGCTCATCATCCAAAATGTTCAAAACACTTGCAACAGAAA


 


TGGCAGTGTAGCAAGCTCGAACATCAATTTCACCTTCATCATGCATCCTG


 


AATCCACCATTTGGTTGCTTCATCCGCCGCAGAAACCCATACAGTTTATC


 


TCTATTAATTGATGCCAGGGATTTCTCACCACCCAAAGTAATAAGTGAAT


 


TAACAGCAGCATAAGTTGTGGCAATATGAGGCATCTGGCCTGGTCCCCCG


 


GCATATCCACCATTCGGATCCTGGCAACGGTTAAGAAAATCGATAGCGTT


 


ATCTTCGAGTTCATCATCGACGGATTCTCCCAACAAAGCAATGGAGTGGA


 


AGATCCAGTAGCAGAGCCAGGGTCGATTAGCGTCCAAAACGGAAAATGCG


 


GAACTGAGATGGCGAAGGCCTTTGGAGACATACTGCATGTGATTATCGCG


 


TTGAAGCTCCAACATGAGGGTTTGGGCGTTGCGAGGAATGGTGGCgagct


 


cgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattg


 


aatcctgttgccggtcttgcgatgattatcatataatttctgttgaatta


 


cgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagat


 


gggtttttatgattagagtcccgcaattatacatttaatacgcgatagaa


 


aacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatc


 


tatgttactagatcgggaattc


(Upper Case: MuA Promoter; Underlined: Antisense


GmFTB; Lower case: NOS terminater)


 







SEQ ID NO: 54







GGAGCCATAGATGCAATTCAATCAAACTGAAATTTCTGCAAGAATCTCAA


 


ACACGGAGATCTCAAAGTTTGAAAGAAAATTTATTTCTTCGACTCAAAAC


 


AAACTTACGAAATTTAGGTAGAACTTATATACATTATATTGTAATTTTTT


 


GTAACAAAATGTTTTTATTATTATTATAGAATTTTACTGGTTAAATTAAA


 


AATGAATAGAAAAGGTGAATTAAGAGGAGAGAGGAGGTAAACATTTTCTT


 


CTATTTTTTCATATTTTCAGGATAAATTATTGTAAAAGTTTACAAGATTT


 


CCATTTGACTAGTGTAAATGAGGAATATTCTCTAGTAAGATCATTATTTC


 


ATCTACTTCTTTTATCTTCTACCAGTAGAGGAATAAACAATATTTAGCTC


 


CTTTGTAAATACAAATTAATTTTCCTTCTTGACATCATTCAATTTTAATT


 


TTACGTATAAAATAAAAGATCATACCTATTAGAACGATTAAGGAGAAATA


 


CAATTCGAATGAGAAGGATGTGCCGTTTGTTATAATAAACAGCCACACGA


 


CGTAAACGTAAAATGACCACATGATGGGCCAATAGACATGGACCGACTAC


 


TAATAATAGTAAGTTACATTTTAGGATGGAATAAATATCATACCGACATC


 


AGTTTTGAAAGAAAAGGGAAAAAAAGAAAAAATAAATAAAAGATATACTA


 


CCGACATGAGTTCCAAAAAGCAAAAAAAAAGATCAAGCCGACACAGACAC


 


GCGTAGAGAGCAAAATGACTTTGACGTCACACCACGAAAACAGACGCTTC


 


ATACGTGTCCCTTTATCTCTCTCAGTCTCTCTATAAACTTAGTGAGACCC


 


TCCTCTGTTTTACTCACAAATATGCAAACTAGAAAACAATCATCAGGAAT


 


AAAGGGTTTGATTACTTCTATTGGAAAGGTGGTGGAGAATCTGGGTGCTT


 


TGACCAACTATACTGGCACAATGAGAGTCCACTTAAACAGTAACATGTGT


 


GATAATGATCTCTACGTTTACCCGGTTTGTCTCTCAGTCCACCCTCTTGC


 


TCCTGTGCACATAAGAGAATATATTGCTGTAAAGCAATACTGTGAAAAAG


 


TGGTTCTTGTGCTCTCCACTCATTAATAAATTTATAGGCAATATTTTTAA


 


AATCAGATGAACTGGATTCACTGGTGCCTTCATGCTCACCACGGCATGTT


 


GCATGACTAGAGGTTCCATCCAAACTTTCTTTTGCTTCAGATACATAAGA


 


TACCGCAAAAATCTGTGATGTCTCTTCCATCTGTTTGTTGATAATAGAAG


 


ATAATCTTTGCAATAGAGCAACAGCACCTCCCTGCCAAAAGGAATAGCAT


 


CCATCCACCAGTTTATTTGTTCTCCCCTGGAATCCACATTCCTTACCTTG


 


TCGGAATACCACCCAGTCAACTAATCGAGGCAGATCCAAGTGATTAACCT


 


CACCAATCAGAATCATTGTAGCTAATCCACAAAAGGTGTACCCACCATGA


 


GCCTCAGAACCAGGCTCACCAGCAATGCCACCCTCATATGTTTGACAGCT


 


TATAATGTAGTCTCCAACATTCTGGATCAGCTCATCATCCAAAATGTTCA


 


AAACACTTGCAACAGAAATGGCAGTGTAGCAAGCTCGAACATCAATTTCA


 


CCTTCATCATGCATCCTGAATCCACCATTTGGTTGCTTCATCCGCCGCAG


 


AAACCCATACAGTTTATCTCTATTAATTGATGCCAGGGATTTCTCACCAC


 


CCAAAGTAATAAGTGAATTAACAGCAGCATAAGTTGTGGCAATATGAGGC


 


ATCTGGCCTGGTCCCCCGGCATATCCACCATTCGGATCCTGGCAACGGTT


 


AAGAAAATCGATAGCGTTATCTTCGAGTTCATCATCGACGGATTCTCCCA


 


ACAAAGCAATGGAGTGGAAGATCCAGTAGCAGAGCCAGGGTCGATTAGCG


 


TCCAAAACGGAAAATGCGGAACTGAGATGGCGAAGGCCTTTGGAGACATA


 


CTGCATGTGATTATCGCGTTGAAGCTCCAACATGAGGGTTTGGGCGTTGC


 


GAGGAATGGTGGCgagctcgaatttccccgatcgttcaaacatttggcaa


 


taaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcat


 


ataatttctgttgaattacgttaagcatgtaataattaacatgtaatgca


 


tgacgttatttatgagatgggtttttatgattagagtcccgcaattatac


 


atttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaatt


 


atcgcgcgcggtgtcatctatgttactagatcgggaattc


(Upper Case: RD29A Promoter; Underlined: Antisense


GmFTB; Lower case: NOS) terminater


 







SEQ ID NO: 55







GAATTCAAATTTTTCGCCAGTTCTAAATATCCGGAAACCTCTTGGGATGC


 


CATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAGGT


 


GGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGA


 


TACCTCTGCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGC


 


ACCGTGGAGTAAGAAGACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTG


 


ATGTAGTATCTCCATTGACGTAAGGGATGACGCACAATCCAACTATCCAT


 


CGCAAGACCATTGCTCTATATAAGAAAGTTAATATCATTTCGAGTGGCCA


 


CGCTGAGCTCGTGGTGGAGAATCTGGGTGCTTTGACCAACTATACTGGCA


 


CAATGAGAGTCCACTTAAACAGTAACATGTGTGATAATGATCTCTACGTT


 


TACCCGGTTTGTCTCTCAGTCCACCCTCTTGCTCCTGTGCACATAAGAGA


 


ATATATTGCTGTAAAGCAATACTGTGAAAAAGTGGTTCTTGTGCTCTCCA


 


CTCATTAATAAATTTATAGGCAATATTTTTAAAATCAGATGAACTGGATT


 


CACTGGTGCCTTCATGCTCACCACGGCATGTTGCATGACTAGAGGTTCCA


 


TCCAAACTTTCTTTTGCTTCAGATACATAAGATACCGCAAAAATCTGTGA


 


TGTCTCTTCCATCTGTTTGTTGATAATAGAAGATAATCTTTGCAATAGAG


 


CAACAGCACCTCCCTGCCAAAAGGAATAGCATCCATCCACCAGTTTATTT


 


GTTCTCCCCTGGAATCCACATTCCTTACCTTGTCGGAATACCACCCAGTC


 


AACTAATCGAGGCAGATCCAAGTGATTAACCTCACCAATCAGAATCATTG


 


TAGCTAATCCACAAAAGGTGTACCCACCATGAGCCTCAGAACCAGGCTCA


 


CCAGCAATGCCACCCTCATATGTTTGACAGCTTATAATGTAGTCTCCAAC


 


ATTCTGGATCAGCTCATCATCCAAAATGTTCAAAACACTTGCAACAGAAA


 


TGGCAGTGTAGCAAGCTCGAACATCAATTTCACCTTCATCATGCATCCTG


 


AATCCACCATTTGGTTGCTTCATCCGCCGCAGAAACCCATACAGTTTATC


 


TCTATTAATTGATGCCAGGGATTTCTCACCACCCAAAGTAATAAGTGAAT


 


TAACAGCAGCATAAGTTGTGGCAATATGAGGCATCTGGCCTGGTCCCCCG


 


GCATATCCACCATTCGGATCCTGGCAACGGTTAAGAAAATCGATAGCGTT


 


ATCTTCGAGTTCATCATCGACGGATTCTCCCAACAAAGCAATGGAGTGGA


 


AGATCCAGTAGCAGAGCCAGGGTCGATTAGCGTCCAAAACGGAAAATGCG


 


GAACTGAGATGGCGAAGGCCTTTGGAGACATACTGCATGTGATTATCGCG


 


TTGAAGCTCCAACATGAGGGTTTGGGCGTTGCGAGGAATGGTGGCGGTGA


 


GGTTAATCACTTGGATCTGCCTCGATTAGTTGACTGGGTGGTATTCCGAC


 


AAGGTAAGGAATGTGGATTCCAGGGGAGAACAAATAAACTGGTGGATGGA


 


TGCTATTCCTTTTGGCAGGGAGGTGCTGTTGCTCTATTGCAAAGATTATC


 


TTCTATTATCAACAAACAGATGGAAGAGACATCACAGATTTTTGCGGTAT


 


CTTATGTATCTGAAGCAAAAGAAAGTTTGGATGGAACCTCTAGTCATGCA


 


ACATGCCGTGGTGAGCATGAAGGCACCAGTGAATCCAGTTCATCTGATTT


 


TAAAAATATTGCCTATAAATTTATTAATGAGTGGAGAGCACAAGAACCAC


 


TTTTTCACAGTATTGCTTTACAGCAATATATTCTCTTATGTGCACAGGAG


 


CAAGAGGGTGGACTGAGAGACAAACCGGGTAAACGTAGAGATCATTATCA


 


CACATGTTACTGTTTAAGTGGACTCTCATTGTGCCAGTATAGTTGGTCAA


 


AGCACCCAGATTCTCCACCACgagctcgaatttccccgatcgttcaaaca


 


tttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatg


 


attatcatataatttctgttgaattacgttaagcatgtaataattaacat


 


gtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgc


 


aattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactag


 


gataaattatcgcgcgcggtgtcatctatgttactagatcgggaattc


(Upper Case: MuA Promoter; Underlined: Antisense


GmFTB; Bold: Sense GmFTB; Lower case: NOS


terminater)


 







SEQ ID NO: 56







GGAGCCATAGATGCAATTCAATCAAACTGAAATTTCTGCAAGAATCTCAA


 


ACACGGAGATCTCAAAGTTTGAAAGAAAATTTATTTCTTCGACTCAAAAC


 


AAACTTACGAAATTTAGGTAGAACTTATATACATTATATTGTAATTTTTT


 


GTAACAAAATGTTTTTATTATTATTATAGAATTTTACTGGTTAAATTAAA


 


AATGAATAGAAAAGGTGAATTAAGAGGAGAGAGGAGGTAAACATTTTCTT


 


CTATTTTTTCATATTTTCAGGATAAATTATTGTAAAAGTTTACAAGATTT


 


CCATTTGACTAGTGTAAATGAGGAATATTCTCTAGTAAGATCATTATTTC


 


ATCTACTTCTTTTATCTTCTACCAGTAGAGGAATAAACAATATTTAGCTC


 


CTTTGTAAATACAAATTAATTTTCCTTCTTGACATCATTCAATTTTAATT


 


TTACGTATAAAATAAAAGATCATACCTATTAGAACGATTAAGGAGAAATA


 


CAATTCGAATGAGAAGGATGTGCCGTTTGTTATAATAAACAGCCACACGA


 


CGTAAACGTAAAATGACCACATGATGGGCCAATAGACATGGACCGACTAC


 


TAATAATAGTAAGTTACATTTTAGGATGGAATAAATATCATACCGACATC


 


AGTTTTGAAAAGAAGGGAAAAAAAGAAAAAAATAAATAAAAGATATACTA


 


CCGACATGAGTTCCAAAAAGCAAAAAAAAAGATCAAGCCGACACAGACAC


 


GCGTAGAGAGCAAAATGACTTTGACGTCACACCACGAAAACAGACGCTTC


 


ATACGTGTCCCTTTATCTCTCTCAGTCTCTCTATAAACTTAGTGAGACCC


 


TCCTCTGTTTTACTCACAAATATGCAAACTAGAAAACAATCATCAGGAAT


 


AAAGGGTTTGATTACTTCTATTGGAAAGGTGGTGGAGAATCTGGGTGCTT


 


TGACCAACTATACTGGCACAATGAGAGTCCACTTAAACAGTAACATGTGT


 


GATAATGATCTCTACGTTTACCCGGTTTGTCTCTCAGTCCACCCTCTTGC


 


TCCTGTGCACATAAGAGAATATATTGCTGTAAAGCAATACTGTGAAAAAG


 


TGGTTCTTGTGCTCTCCACTCATTAATAAATTTATAGGCAATATTTTTAA


 


AATCAGATGAACTGGATTCACTGGTGCCTTCATGCTCACCACGGCATGTT


 


GCATGACTAGAGGTTCCATCCAAACTTTCTTTTGCTTCAGATACATAAGA


 


TACCGCAAAAATCTGTGATGTCTCTTCCATCTGTTTGTTGATAATAGAAG


 


ATAATCTTTGCAATAGAGCAACAGCACCTCCCTGCCAAAAGGAATAGCAT


 


CCATCCACCAGTTTATTTGTTCTCCCCTGGAATCCACATTCCTTACCTTG


 


TCGGAATACCACCCAGTCAACTAATCGAGGCAGATCCAAGTGATTAACCT


 


CACCAATCAGAATCATTGTAGCTAATCCACAAAAGGTGTACCCACCATGA


 


GCCTCAGAACCAGGCTCACCAGCAATGCCACCCTCATATGTTTGACAGCT


 


TATAATGTAGTCTCCAACATTCTGGATCAGCTCATCATCCAAAATGTTCA


 


AAACACTTGCAACAGAAATGGCAGTGTAGCAAGCTCGAACATCAATTTCA


 


CCTTCATCATGCATCCTGAATCCACCATTTGGTTGCTTCATCCGCCGCAG


 


AAACCCATACAGTTTATCTCTATTAATTGATGCCAGGGATTTCTCACCAC


 


CCAAAGTAATAAGTGAATTAACAGCAGCATAAGTTGTGGCAATATGAGGC


 


ATCTGGCCTGGTCCCCCGGCATATCCACCATTCGGATCCTGGCAACGGTT


 


AAGAAAATCGATAGCGTTATCTTCGAGTTCATCATCGACGGATTCTCCCA


 


ACAAAGCAATGGAGTGGAAGATCCAGTAGCAGAGCCAGGGTCGATTAGCG


 


TCCAAAACGGAAAATGCGGAACTGAGATGGCGAAGGCCTTTGGAGACATA


 


CTGCATGTGATTATCGCGTTGAAGCTCCAACATGAGGGTTTGGGCGTTGC


 


GAGGAATGGTGGCGGTGAGGTTAATCACTTGGATCTGCCTCGATTAGTTG


 


ACTGGGTGGTATTCCGACAAGGTAAGGAATGTGGATTCCAGGGGAGAACA


 


AATAAACTGGTGGATGGATGCTATTCCTTTTGGCAGGGAGGTGCTGTTGC


 


TCTATTGCAAAGATTATCTTCTATTATCAACAAACAGATGGAAGAGACAT


 


CACAGATTTTTGCGGTATCTTATGTATCTGAAGCAAAAGAAAGTTTGGAT


 


GGAACCTCTAGTCATGCAACATGCCGTGGTGAGCATGAAGGCACCAGTGA


 


ATCCAGTTCATCTGATTTTAAAAATATTGCCTATAAATTTATTAATGAGT


 


GGAGAGCACAAGAACCACTTTTTCACAGTATTGCTTTACAGCAATATATT


 


CTCTTATGTGCACAGGAGCAAGAGGGTGGACTGAGAGACAAACCGGGTAA


 


ACGTAGAGATCATTATCACACATGTTACTGTTTAAGTGGACTCTCATTGT


 


GCCAGTATAGTTGGTCAAAGCACCCAGATTCTCCACCACgagctcgaatt


 


tccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcct


 


gttgccggtcttgcgatgattatcatataatttctgttgaattacgttaa


 


gcatgtaataattaacatgtaatgcatgacgttatttatgagatgggttt


 


ttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaa


 


atatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgtt


 


actagatcgggaattc


(Upper Case: RD29A Promoter; Underlined: Antisense


GmFTB; Bold: Sense GmFTB; Lower case: NOS


terminater)


 







SEQ ID NO: 57







GAATTCAAATTTTTCGCCAGTTCTAAATATCCGGAAACCTCTTGGGATGC


 


CATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAGGT


 


GGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGA


 


TACCTCTGCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGC


 


ACCGTGGAGTAAGAAGACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTG


 


ATGTAGTATCTCCATTGACGTAAGGGATGACGCACAATCCAACTATCCAT


 


CGCAAGACCATTGCTCTATATAAGAAAGTTAATATCATTTCGAGTGGCCA


 


CGCTGAGCTCGGATGGATTGGCTCCAGCAAATTAGAGTACGGTCCAAGCA


 


CATGCTGAGGTAATGGGCACGAACCAGTATCAGTCATGGCACTGTACTGG


 


CTAACTGCGAGGCCACTGAGGCAGTAGCATGAATGATAGTGATCTCTGTT


 


CTTTCCAGGCTTATCCCTCAAGCCTCCCTCTAGTACCTGAGAACAAAGTA


 


GGATGTATTGTTGCAGGGCAATGTTATGGAAGAGTGGGCCAATTTGGTTG


 


CTCTGTTGTATAAAATCAAATCCAAACTTCGCATAGTCCACAGCAGAGGA


 


AGACTTATTCGCGGTGCACCCATATGAACTGGTGCTGCAGGCATCCTCTC


 


CTGATGGCCTTTTGCAGGAATACGAGGACCTCAATTGCTTATCAACAATC


 


GTAATTAACTTTTGTGTGAAAGCAATGGCAGCTCCCTGCCAAAAGGAGTA


 


GCAACCATCAACCAATTTATTAGTTCGTCCTTGAAATCCGCATTCCACTC


 


CTTGACGAAAAGCCACCCAGCCAATCAAACTAGGCAAGTCAACTTTCTCT


 


GCCTCATTAAGCAGGATCAAAGCAGCCAATCCACAGAATGTATACCCACC


 


ATGTGCTTCAGCATAAGGCTCCCCAGCAATACCACCTTCATAAGTTTGAC


 


ATCTTGCTATGTAGTCGCCTACACCTTTTGCCAGTTTAAAATCAAGAATA


 


TTCACAAGGCTGGCAACCGATATAGCGGTGTAGGAAGCACGGACATCAAT


 


TTCGCCACCATCATGCATTCTGAAAGCACCTGATACATCTTTCATCTGCA


 


GCATAAAATTGTACAGGTTGCCCCTATTGATTGATGACAATGCTCTTTCG


 


CTCCCTATTGTCACAAGTGTATTTACAGCAGCATAAGTCGTAGCTAGGTG


 


AGGCAACTGTCCAGGTCCACCACTATATCCACCATCTTTATCCTGACATC


 


GAGCTAAGAAGTCTATGATATCATTCTCAAGATCATCATCAAGTGCTTCA


 


TCCAGCAAAGCAAGTGGATGAACCATCCAGTAGCATAGCCAAGGGCGATT


 


GGCATCTAGAACATGAAAGGCTGGTCCCATATGCCTCAGCCCAGGCGTCA


 


GATACTCGATATGCTGATCACGCCACAGCTCTAGCATGATGGATTTCGTG


 


TTGGGCGCGGCCCCGAAGAGGGAGCGGTAGATGTCGCCAACCCTGGCCTC


 


CACCTTCATCTGCTCCACCTGCGTCACCGTGAGCCTCGGTAGGTCGGGAT


 


CCGCCgagctcgaatttccccgatcgttcaaacatttggcaataaagttt


 


cttaagattgaatcctgttgccggtcttgcgatgattatcatataatttc


 


tgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgtta


 


tttatgagatgggtttttatgattagagtcccgcaattatacatttaata


 


cgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcg


 


cggtgtcatctatgttactagatcgggaattc


(Upper Case: MuA Promoter; Underlined: Antisense


Zea maize-FTB; Lower case: NOS terminater)


 







SEQ ID NO: 58







GAATTCAAATTTTTCGCCAGTTCTAAATATCCGGAAACCTCTTGGGATGC


 


CATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAGGT


 


GGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGA


 


TACCTCTGCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGC


 


ACCGTGGAGTAAGAAGACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTG


 


ATGTAGTATCTCCATTGACGTAAGGGATGACGCACAATCCAACTATCCAT


 


CGCAAGACCATTGCTCTATATAAGAAAGTTAATATCATTTCGAGTGGCCA


 


CGCTGAGCTCGGATGGATTGGCTCCAGCAAATTAGAGTACGGTCCAAGCA


 


CATGCTGAGGTAATGGGCACGAACCAGTATCAGTCATGGCACTGTACTGG


 


CTAACTGCGAGGCCACTGAGGCAGTAGCATGAATGATAGTGATCTCTGTT


 


CTTTCCAGGCTTATCCCTCAAGCCTCCCTCTAGTACCTGAGAACAAAGTA


 


GGATGTATTGTTGCAGGGCAATGTTATGGAAGAGTGGGCCAATTTGGTTG


 


CTCTGTTGTATAAAATCAAATCCAAACTTCGCATAGTCCACAGCAGAGGA


 


AGACTTATTCGCGGTGCACCCATATGAACTGGTGCTGCAGGCATCCTCTC


 


CTGATGGCCTTTTGCAGGAATACGAGGACCTCAATTGCTTATCAACAATC


 


GTAATTAACTTTTGTGTGAAAGCAATGGCAGCTCCCTGCCAAAAGGAGTA


 


GCAACCATCAACCAATTTATTAGTTCGTCCTTGAAATCCGCATTCCACTC


 


CTTGACGAAAAGCCACCCAGCCAATCAAACTAGGCAAGTCAACTTTCTCT


 


GCCTCATTAAGCAGGATCAAAGCAGCCAATCCACAGAATGTATACCCACC


 


ATGTGCTTCAGCATAAGGCTCCCCAGCAATACCACCTTCATAAGTTTGAC


 


ATCTTGCTATGTAGTCGCCTACACCTTTTGCCAGTTTAAAATCAAGAATA


 


TTCACAAGGCTGGCAACCGATATAGCGGTGTAGGAAGCACGGACATCAAT


 


TTCGCCACCATCATGCATTCTGAAAGCACCTGATACATCTTTCATCTGCA


 


GCATAAAATTGTACAGGTTGCCCCTATTGATTGATGACAATGCTCTTTCG


 


CTCCCTATTGTCACAAGTGTATTTACAGCAGCATAAGTCGTAGCTAGGTG


 


AGGCAACTGTCCAGGTCCACCACTATATCCACCATCTTTATCCTGACATC


 


GAGCTAAGAAGTCTATGATATCATTCTCAAGATCATCATCAAGTGCTTCA


 


TCCAGCAAAGCAAGTGGATGAACCATCCAGTAGCATAGCCAAGGGCGATT


 


GGCATCTAGAACATGAAAGGCTGGTCCCATATGCCTCAGCCCAGGCGTCA


 


GATACTCGATATGCTGATCACGCCACAGCTCTAGCATGATGGATTTCGTG


 


TTGGGCGCGGCCCCGAAGAGGGAGCGGTAGATGTCGCCAACCCTGGCCTC


 


CACCTTCATCTGCTCCACCTGCGTCACCGTGAGCCTCGGTAGGTCGGGAT


 


CCGCCggatccGCTGGGGAGCCTTATGCTGAAGCACATGGTGGGTATACA


 


TTCTGTGGATTGGCTGCTTTGATCCTGCTTAATGAGGCAGAGAAAGTTGA


 


CTTGCCTAGTTTGATTGGCTGGGTGGCTTTTCGTCAAGGAGTGGAATGCG


 


GATTTCAAGGACGAACTAATAAATTGGTTGATGGTTGCTACTCCTTTTGG


 


CAGGGAGCTGCCATTGCTTTCACACAAAAGTTAATTACGATTGTTGATAA


 


GCAATTGAGGTCCTCGTATTCCTGCAAAAGGCCATCAGGAGAGGATGCCT


 


GCAGCACCAGTTCATATGGGTGCACCGCGAATAAGTCTTCCTCTGCTGTG


 


GACTATGCGAAGTTTGGATTTGATTTTATACAACAGAGCAACCAAATTGG


 


CCCACTCTTCCATAACATTGCCCTGCAACAATACATCCTACTTTGTTCTC


 


AGGTACTAGAGGGAGGCTTGAGGGATAAGCCTGGAAAGAACAGAGATCAC


 


TATCATTCATGCTACTGCCTCAGTGGCCTCGCAGTTAGCCAGTACAGTGC


 


CATGACTGATACTGGTTCGTGCCCATTACCTCAGCATGTGCTTGGACCGT


 


ACTCTAATTTGCTGGAGCCAATCCATCCaagcttgaatttccccgatcgt


 


tcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtct


 


tgcgatgattatcatataatttctgttgaattacgttaagcatgtaataa


 


ttaacatgtaatgcatgacgttatttatgagatgggtttttatgattaga


 


gtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgc


 


aaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgga


 


agctt


(Upper Case: MuA Promoter; Underlined: Antisense


Zea maize-FTB; Bold: Sense Zea maize-FTB; Lower


case: NOS terminater)






Example 7
PCR Analysis of Putative Transgenic Plants
To verify that the putative transgenic plants carried the gene of interest PCR analysis was performed. Genomic DNA was isolated and PCR run according to standard protocols and conditions which are known to one of skill in the art. A typical reaction was performed in a volume of 25 μl and primer pairs used were dependent on the gene and promoter combination of the particular construct (Table 12).
Putative transgenic Brassica napus plants were screened using the primer combinations detailed in the table below. A representative gel showing PCR analysis results is shown in FIG. 15 which represents transgenic plants carrying the pRD29A-anti-FTA construct. Transformants were confirmed in an analogous manner for each species and construct transformation done.










TABLE 12





Construct





Name
Primer Name
Primer Sequence (5′-3′)







35S-antiFTA
SEQ ID NO: 10
GCCGACAGTGGTCCCAAAGATGG




SEQ ID NO: 11
AAAGGATCCTCAAATTGCTGCCAC




TGTAAT


 


rd29A-antiFTA
SEQ ID NO: 12
AAACCCGGGATGAATTTCGACGAG




AACGTG



SEQ ID NO: 13
GCAAGACCGGCAACAGGA


 


rd29B-antiFTA
SEQ ID NO: 14
TTTAAGCTTGACAGAAACAGTCAG




CGAGAC



SEQ ID NO: 11
AAACCCGGGATGAATTTCGACGAG




AACGTG


 


35S-DA-FTA
SEQ ID NO: 15
GCTCTTCCTCCATGCCCA



SEQ ID NO: 13
GCAAGACCGGCAACAGGA


 


rd29A-DA-FTA
SEQ ID NO: 16
TTTAAGCTTGGAGCCATAGATGCA




ATTCAA



SEQ ID NO: 17
CGGGCATTAGGAGGATGGGAA


 


35S-HP-FTB
SEQ ID NO: 10
GCCGACAGTGGTCCCAAAGATGG



SEQ ID NO: 18
GTCCGGAATTCCCGGGTC


 


rd29A-HP-FTB
SEQ ID NO: 16
TTTAAGCTTGGAGCCATAGATGCA




ATTCAA



SEQ ID NO: 18
GTCCGGAATTCCCGGGTC









Example 8
Southern Analysis
Genomic Southern analysis of anti-FTA transgenic Arabidopsis thaliana. The numbers indicate the line numbers. Five micrograms of genomic DNA of T1 plants was digested with HindIII (a unique site in the T-DNA plasmid) and separated in a 0.8% agarose gel. The NPTII coding region was used as the probe for radio-labeling. FIG. 2 shows a typical result from Southern analysis indicating the presence of the transgene.
Example 9
Northern Blots of Antisense FTA Lines
RNA was isolated from developing leaf tissue of five 35S-anti-FTA Arabidopsis thaliana lines (T3 plants). The blot was first probed with P32 labeled, single-stranded sense transcript of FTA (FIG. 3 panel A) which detects antisense transcript, then stripped and re-probed with the single-stranded anti-sense transcript of FTA (FIG. 3 panel B) that detects the sense transcript. FIG. 3 panel C shows the ethidium bromide stained gel for the blot. Approximately 5 μg of total RNA was loaded into each lane. FIG. 3 indicates the accumulation of the transgene anti-sense transcript and a reduction in the sense transcript in transgenic plants.
Example 10
Western Blot Antisense FTA Lines with Anti-FT-α Antibodies
The antibodies produced according to the methods of Example 19 were used to analyze protein extracts from transgenic plants on western blots. Lane 1 of FIG. 4 is a molecular weight standard, lane 2 purified FTA protein, lanes 3-10 are protein extracts from the ERA1 mutant, wild type, and 4 lines of transgenic Arabidopsis thaliana. FIG. 4 illustrates the reduction of detectable FTA protein in transgenic lines.
Example 11
ABA Sensitivity of Transgenic Seedlings
Seeds of wild type Columbia, era 1-2 and T3 homozygous seeds of two antisense, drought tolerant lines of 35S-antisense-FTA were plated on minimum medium (1⁄2 MS) supplemented with no ABA (A), 0.3 μM (B), 0.5 μM (C) or 1.0 μM ABA (D). Plates were chilled for 3 days in 4° C. in the dark, and incubated for 11 days at 22° C. with 24 hour continuous light. era1 and transgenic lines were more inhibited in germination than wild type plants. Results are shown in FIG. 5.
Twelve day old seedling phenotypes of wild type Columbia, era1-2 and two drought tolerant 35S-antisense-FTA lines (9.9 & 21.2) in minimum medium without (A) or with (B) 1 μM ABA. FIG. 6 shows the reduced root growth and development of era1 and transgenic lines relative to wild type plants. The 35S-antisense-FTA lines show reduced root growth, similar to the era1 mutant, in response to ABA.
A transgenic Brassica napus line carrying the 35S-antisense-FTA construct was assessed for ABA sensitivity. At about 10 μm an effect was observed showing reduced seedling development and vigor at the cotyledon and first leaf stage, thereby indicating an increased sensitivity to ABA
ABA sensitivity is assessed in all transgenic plants engineered to have reduced or increased FTA or FTB expression or activity by the methods above. The ABA concentration used varies depending upon the species under examination.
Example 12
Drought Experiment
To assess the response of plants under water stress or drought one can expose plants to various situations. For example, the plant can be removed from soil or media and placed on paper towel for a period of time, such as 4 hours, then returned to a plate to continue growth and development. Survival and vigor can be assessed.
Alternatively one can impose a water stress in such a way as to more closely resemble a field situation by withholding water for a period of time, such as up to 6 days. Plants were grown five plants per four inch pot, in a replicated water-stress experiment. All pots were filled with equal amounts of homogeneous premixed and wetted soil. Growth conditions were 16 hour daylight (150-200 μmol/m2/s) at 22° C. and 70% relative humidity. On the day that the first flower opened drought treatment was initiated first by equalizing the soil water content in each pot on a weight basis and then cessation of watering. At the end of the water stress treatment plants were typically either harvested for biomass data or re-watered to complete the life cycle and determination of biomass and yield data. Physiological parameters have been assessed under stressed and optimal conditions, for example, shoot and root biomass accumulation, soil water content, water loss alone or as a function of parameters such as biomass, seed yield, and leaf number and leaf area. FIG. 7 shows photographs of wild type Columbia (A) and four 35S-antisense-FTA transgenic Arabidopsis thaliana lines (B, C, D, E) after 8 days of water stress treatment. The control plant is visibly stressed and less healthy. This experiment has been conducted on transgenic lines containing vectors described by SEQ ID NO: 4, 40-58.
Drought or water stress tolerance is assessed in all transgenic plants engineered to have reduced or increased FTA or FTB expression or activity by the described methods.
Example 13
Analysis of Water Loss in Arabidopsis thaliana pRD29A-DA-FTA Lines During Drought Stress
Plants were grown 5 plants per 4 inch pot and 6 pots per line. When the plants had grown to the first flower stage drought treatment was initiated as described in Example 12. Pots were weighed daily and at the end of the 7 day drought treatment all plants were harvested for shoot fresh weight and dry weight determinations. FIG. 10 shows the water loss on a per shoot dry weight basis at 4 days of water stress treatment. Of the 31 lines examined in this experiment 25 showed lower water loss relative to the Columbia wild type, 22 of which were statistically significant. All lines had been assessed for ABA sensitivity as described in Example 6, increased ABA sensitivity (ABAS) also correlated with a decreased water loss during drought treatment. Those lines determined to have wild type ABA sensitivity (ABAWT) were the same 6 lines (lines 2, 36, 69, 29, 24, 21) that did not show a reduced water loss compared to wild type.
The above experiment was repeated using two ABAS lines, one ABAWT line and a Columbia control. Plants were harvested after 2, 4 and 6 days of water stress treatment for shoot dry weight determinations. ABAS transgenics had greater leaf and shoot biomass, greater soil water contents and lower water loss per shoot dry weight when compared to the ABAWT or Columbia controls. Results were consistent at all three harvest stages.
The data shown in this example was obtained using transgenic plants carrying the pRD29A-DA-FTA construct. The experiment has also been conducted on lines carrying variations of this construct such as 35S-DA-FTA, pRD29A-antisense-FTA or 35S-antisense-FTA, with similar water stress tolerant trends observed. Soil water loss is assessed in all transgenic plants engineered to have reduced or increased FTA or FTB expression or activity by the described methods.
Example 14
Analysis of Shoot Fresh Weight in Arabidopsis thaliana pRD29A-DA-FTA Lines During Drought Stress
Plants were grown 5 plants per 4 inch pot and 8 pots per line. When the plants had grown to the first flower stage drought treatment was initiated as described in Example 12. Plants were re-watered after 6 days drought treatment and allowed to recover for an additional 6 days. Plants were harvested and shoot fresh weights determined. FIG. 11 shows the shoot fresh weights. This experiment consisted of 25 transgenic lines, 2 of which are ABAWT (line 2 and 69) and a Columbia wild type control. All 23 ABAS transgenic lines had statistically significant greater shoot fresh weights, on average 44% greater.
The data shown in this example was obtained using transgenic plants carrying the pRD29A-DA-FTA construct. The experiment has been conducted on lines carrying variations of this construct such as 35S-DA-FTA, pRD29A-antisense-FTA or 35S-antisense-FTA, with similar trends observed.
Example 15
Analysis of Seed Yield in Arabidopsis thaliana pRD29A-DA-FTA Lines During Drought Stress and Under Optimal Conditions
Plants were grown 1 plant per 4 inch pot. When the plants had grown to the first flower stage drought treatment was initiated as described in Example 12. Plants were re-watered after 6 days drought treatment and allowed to grow to maturity. The optimal group was not exposed to the drought treatment.
Yield analysis indicates that although drought treatment results in decreased yields, the transgenics do not suffer as severely as controls and maintain a productivity advantage (FIG. 12) as shown previously in Experiment 14. Comparison of the yields produced by the ABAS transgenics versus the control plants show that a 15% greater yield was obtained under optimal conditions and a 20% increase under drought conditions. In the drought treatment group 8 of 9 transgenic lines showed greater yield than controls. Expression of yield of each line obtained under drought treatment as a percentage of its performance under optimum conditions indicates that 8 of 9 ABAS lines outperformed the control line while 4 of 9 out performed the ABAWT controls.
The data shown in this example was obtained using transgenic plants carrying the pRD29A-DA-FTA construct. The experiment has been conducted on lines carrying variations of this construct such as 35S-DA-FTA, pRD29A-antisense-FTA or 35S-antisense-FTA, with similar trends observed.
Example 16
Analysis of Vegetative Growth in Arabidopsis thaliana pRD29A-DA-FTA Lines Under Optimum Growth Conditions
Plants were grown 1 plant per 3 inch pot and 8 pots per line. Plants were harvested at three stages and fresh weights determined. Vegetative stage was defined as 14 day old seedlings, bolting stage as the appearance of first flower (19-21 day seedlings) and mid-flowering as 6 days from first flower. At each of the above stages respectively 7, 8 and 10 of the 10 ABAS transgenic lines tested showed statistically greater shoot fresh weight biomass than the control plants (FIG. 13). One Columbia line and an ABAWT (line 2) line were used as the control group. Additionally, there was a statistically significant trend for the transgenic lines to have an increased number of rosette leaves.
The data shown in this example was obtained using transgenic plants carrying the pRD29A-DA-FTA construct. The experiment has been conducted on lines carrying variations of this construct such as 35S-DA-FTA, pRD29A-antisense-FTA or 35S-antisense-FTA, with similar trends observed.
Example 17
Analysis of Arabidopsis thaliana pRD29A-DA-FTA Lines Under Drought Treatment and Biotic Stress
Plants were grown 1 plant per 4 inch pot and 8 pots. When the plants had grown to the first flower stage drought treatment was initiated as described in Example 12. Plants were re-watered after 7 days drought treatment and allowed to grow to maturity. One Columbian control line (col) and one transgenic line were evaluated. Analysis of seed yield indicated less than normal yields, approximately 12% of expected optimal yield. It was determined that the soil used contained a fungal contaminant that was responsible for the reduced yields as the biotic stress could be negated by sterilization of the soil prior to use. This biotic stress was less severe in the transgenic line compared to the control which had a yield 22% of the transgenic line. In the drought treatment groups of plants the biotic stress was reduced however, transgenics outperformed controls by nearly 4.5 fold (FIG. 14).
The data shown in this example was obtained using transgenic plants carrying the pRD29A-DA-FTA construct. The experiment has been conducted on lines carrying variations of this construct such as 35S-DA-FTA, pRD29A-antisense-FTA or 35S-antisense-FTA, with similar trends observed.
Example 18
Analysis of Arabidopsis thaliana pRD29A-DA-FTA Lines for Stomatal Number
The number of stomata on both the upper and lower surface of the leaf was assessed on two transgenic lines and a wild type Columbia control. Nail polish imprints were made of both upper and lower leaf surfaces of the fifth leaf, plants were at the early flowering stage. No differences in stoma density were observed.
The data shown in this example was obtained using transgenic plants carrying the pRD29A-DA-FTA construct. The experiment has been conducted on lines carrying variations of this construct such as 35S-DA-FTA, pRD29A-antisense-FTA or 35S-antisense-FTA, with similar trends observed.
Example 19
Production of Polyclonal Antibodies Against FT-A and FT-B
The isolated Arabidopsis thaliana FT sequences were cloned into the E. coli expression vector derived from pET11D. To generate the Histidine tagged FT-B construct the Arabidopsis thaliana FT-B clone and pET vector were digested with BamHI and ligated together. Restriction digests were performed to verify the orientation of the insert. To produce the FT-A construct the Arabidopsis thaliana FT-A clone and pET vector were digested with BamHI and EcoRI and subsequently ligated together. The resultant plasmids directed the expression of fusion proteins containing 6 consecutive histidine residues at the N-termini of AtFTA and AtFTB. The fusion proteins were expressed in the bacterial host BL21(DE3) and purified using Hi-Trap chelating chromatography as described by the manufacturer (Pharmacia). The soluble fraction of the crude bacterial extract containing the His-FT fusion proteins were loaded to a Hi-Trap column (1.5 cm×2.0 cm), and the proteins eluted with a 200 ml linear gradient of 0.0 to 0.3 M imidazole in column buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT). Fractions containing purified His-FT proteins were pooled, desalted and concentrated with a Centriprep-30 concentrator (Amicon). All purification steps were carried out at 4° C. To generate an antibody, the purified fusion protein was further separated by SDS/PAGE and the Coomassie stained band corresponding to the fusion protein was excised. Protein was eluted from the gel slice by electroelution and then emulsified in Ribi adjuvant (Ribi Immunochem) to a final volume of 1 ml. His-AtFTA or His-AtFTB (250 μg) were injected into a 3 kg New Zealand rabbit on day 1 and booster injections given on day 21 and day 35 with 200 μg of the protein. High-titer antisera were obtained one week after the final injection. These antibodies were used in the western analysis of example 10, FIG. 4.
Example 20
Screening for Related Genes
The transgenic plants of the invention can be used to identify genes which interact with the genes of the present invention. One can make use of the transgenic plants of the invention to screen for related genes, for example, suppressors, enhancers or modulators of gene expression or activity can be identified through genetic screening protocols. By way of example, a mutant library can be generated using the transgenic plants of the invention as the genetic background. Various methods are available and would be known to one of skill in the art. For example, chemical mutagens such as EMS can be used to induce point mutations in the genome, fast neutron irradiation of seeds can result in deletion mutations, T-DNA libraries can be produced that inactivate genes through insertional effects or activation tagging methods can be used to produce libraries with up-regulated genes. Analysis of these types of libraries can identify genes which rescue or modulate the phenotypes observed in the transgenic plants of the present invention.

## Claims

1. A method of producing a drought tolerant plant comprising:
a) providing a nucleic acid construct comprising a promoter operably linked to a farnesyltransferase alpha nucleic acid that inhibits farnesyltransferase alpha activity;
b) inserting said nucleic acid construct into a vector;
c) transforming a plant, tissue culture, or a plant with the vector to obtain a plant, tissue culture or a plant with decreased farnesyltransferase alpha activity;
d) growing said plant or regenerating a plant form said tissue culture or plant cells, wherein a drought tolerant plant is produced.

2. The method of claim 1, wherein said promoter is a constitutive promoter, an ABA inducible promoter, a tissue specific promoters or a guard cell specific promoter.

3. A drought tolerant transgenic plant produced by the method claim 1 or 2.

4. A transgenic seed produced by the transgenic plant of claim 3, wherein said seed produces a drought tolerant plant.