# Patent

## Title
Stress tolerant plants and methods thereof

## Abstract

The present invention provides a method and DNA molecules that when expressed in a plant produces transgenic plants with improved abiotic stress tolerance. The invention includes plant expression vectors comprising the DNA molecules, and plants containing such DNA molecules.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a division of U.S. application Ser. No. 11/007,819, filed Dec. 8, 2004, which claims benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application 60/528,540 filed on Dec. 10, 2003, which applications are herein incorporated in their entirety by reference.


INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Seq. Listing Copy 1 and Seq. Listing Copy 2) and a computer-readable form of the sequence listing, all on CD_ROMs, each containing the file named OsPK7Regular Filing.ST25.txt, which is 153,600 bytes (measured in MS-DOS) and was created on Dec. 7, 2004, are hereby incorporated by reference.
FIELD OF THE INVENTION
Described herein are inventions in the field of plant molecular biology and plant genetic engineering. In particular, DNA constructs encoding a polypeptide and transgenic plants containing the DNA constructs are provided. The transgenic plants are characterized by improved stress tolerance.
BACKGROUND OF THE INVENTION
One of the goals of plant genetic engineering is to produce plants with agronomically, horticulturally or economically important characteristics or traits. Traits of particular interest include high yield, improved quality and yield stability. The yield from a plant is greatly influenced by external environmental factors including water availability and heat, of which tolerance of extremes is in turn influenced by internal developmental factors. Enhancement of plant yield may be achieved by genetically modifying the plant to be tolerant to yield losses due to stressful environmental conditions, such as heat and drought stress.
Seed and fruit production are both limited inherently due to abiotic stress. Soybean (Glycine max), for instance, is a crop species that suffers from loss of seed germination during storage and fails to germinate when soil temperatures are cool (Zhang et al., Plant Soil 188: (1997)). This is also true in corn and other plants of agronomic importance. Improvement of abiotic stress tolerance in plants would be an agronomic advantage to growers allowing enhanced growth and/or germination in cold, drought, flood, heat, UV stress, ozone increases, acid rain, pollution, salt stress, heavy metals, mineralized soils, and other abiotic stresses.
Traditional breeding (crossing specific alleles of one genotype into another) has been used for centuries to increase abiotic stress tolerance and yield. Traditional breeding is limited inherently to the limited number of alleles present in the parental plants. This in turn limits the amount of genetic variability that can be added in this manner. Molecular biology has allowed the inventors of the instant invention to look far and wide for genes that will improve stress tolerance in plants. Protein phosphorylation is one of the major mechanisms controlling cellular functions in response to external signals in eukaryotes and kinases represent a large and diverse protein family. Protein kinases in plants have been shown to participate in a wide variety of developmental processes. Protein kinases also respond to environmental stresses
Members of the Snf1-related protein kinases play a major role in phosphorylation cascades involved in carbon assimilation in animals, fungi and plants. (Hardie D. G., Carling D. and Carlson M.; Ann. Rev. Biochem. 67: 821-855, 1998). Members of the AMP-activated/Snf1-related protein kinase subfamily are central components of highly conserved protein kinase cascades that now appear to be present in most, if not all, eukaryotic cells. Because the downstream targets of the action of these enzymes are many and varied, they have been discovered and rediscovered several times in different guises and by different approaches. Alderson and coworkers (Alderson A., et al. Proc. Natl. Acad. Sci. USA, 88: 8602-8605, 1991) cloned and sequenced a cDNA (RKIN1) encoding a Snf1 homolog from the higher plant rye. Transformation of an Snf1 mutant strain of yeast with a low-copy RKIN1 plasmid restored the ability to grow on nonfermentable carbon sources (Alderson A., et al. Proc. Natl. Acad. Sci. USA, 88: 8602-8605, 1991), showing that RKIN1 is functionally as well as structurally related to Snf1. Snf1 homologs were subsequently cloned from Arabidopsis thaliana (LeGuen L., Thomas M., Bianchi M., Halford N. G., and Kreis M., Gene 120: 249-254, 1992), barley, (Hannappel U., Vincente-Carbajosa J., Baker J. H. A., Shewery P. R., and Halford N. G., Plant Mol. Biol., 27: 1235-1240, 1995; Halford N. G., Vincente-Carbajosa J., Sabelli P. A., Shewery P. R., Hannappel U., and Kreis M., Plant J., 2: 791-797, 1992), tobacco (Muranaka T., Banno H., Machida Y., Mol. Cell. Biol. 14: 2958-2965, 1994) rice and maize (Ohba H. et al. Mo Genet., 263: 359-366, 2000). Two Snf1-related protein kinases from rice, OsPK4 and OsPK7, which are structurally very similar and share more than 75% homology with the wheat homolog WPK4, exhibit very different expression patterns as well as stress response in rice and maize plants (Ohba H. et al. Mo Genet., 263: 359-366, 2000). Based on yeast studies, Snf1 protein kinases including, OsPK4 and OsPK7, are expected to play a central role in energy metabolism to provide protection against environmental stress in the host organism. Very little or no changes were observed in the expression pattern of rice and maize OsPK7 genes in response to a variety of abiotic stresses such as light, nutrients, cold, drought, and salt. (Ohba H. et al. Mo Genet., 263: 359-366, 2000).
The current invention demonstrates and claims the utilization of the OsPK7 gene and its homologs to produce plants with enhanced abiotic stress tolerance, including response to suboptimal growth temperatures and amounts of water required for growth of natural plants.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a method of generating a transgenic plant with enhanced stress tolerance comprising the steps of transforming a plant cell with a DNA construct comprising a promoter that functions in the plant cell, operably linked to a DNA molecule that encodes a protein substantially homologous to a protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 and operably linked to a 3′termination region; and regenerating the plant cell into a fertile transgenic plant; and selecting said fertile transgenic plant containing the DNA construct; wherein the fertile transgenic plant exhibits enhanced stress tolerance compared to a plant of the same plant species not transformed to contain said DNA construct.
In one preferred embodiment of the invention a DNA construct is provided that contains a promoter that is a plant virus promoter. In another preferred embodiment of the invention a DNA construct is provided that contains a promoter that is a heterologous plant promoter. In another preferred embodiment of the invention the DNA construct contains a promoter that is a tissue specific or tissue enhanced promoter. In one aspect of the invention, the DNA construct contains a promoter that is a constitutive promoter. In another aspect of the invention the DNA construct contains a promoter that is a promoter that is found in association with the native gene in the genome.
In another preferred embodiment, the DNA molecule is substantially homologous to a DNA molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 50.
In another aspect of the invention a transgenic plant containing the DNA construct is provided wherein the transgenic plant exhibits enhanced stress tolerance. The transgenic plant is particularly tolerant to cold stress.
The transgenic plant is selected from the group consisting of: Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, cauliflower, celery, cherry, cilantro, citrus, clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, forest tree, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, loblolly pine, mango, melon, millet, mushroom, nut, oat, okra, onion, orange, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum, southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, turf, a vine, watermelon, wheat, yams, and zucchini.
The present invention also provides a transgenic plant with enhanced stress tolerance compared to a plant of the same plant species comprising a DNA construct wherein the DNA construct comprises a promoter that functions in plants operably linked to a DNA molecule that encodes a protein substantially homologous to a protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 and operably linked to a 3′termination region.
The present invention also provides a DNA construct wherein the DNA construct comprises a promoter that functions in plants operably linked to a DNA molecule that encodes a protein substantially homologous to a protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51, and operably linked to, a 3′termination region.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Plasmid map of plant expression vector pMON 72472
FIG. 2 Plasmid map of plant expression vector pMON 80878
FIG. 3 Plasmid map of plant expression vector pMON 53616
FIG. 4 Plasmid map of plant expression vector pMON 71709
FIG. 5 Plasmid map of plant expression vector pMON 71712
FIG. 6 Plasmid map of plant expression vector pMON 83200
FIG. 7 Plasmid map of plant expression vector pMON 71710
FIG. 8 Plasmid map of plant expression vector pMON 71713
FIG. 9 Plasmid map of plant expression vector pMON 83201
FIG. 10 Plasmid map of plant expression vector pMON 82629



The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing that form a part of this application.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, in part, on the identification of polynucleic acid molecules encoding polypeptides of the present invention from plants including maize, rice and soybean and utilizing these molecules to enhance abiotic stress tolerance in plants by ectopic expression of polypeptides of the invention leading to potential enhancement in yield.
Isolated Polynucleic Acid Molecules of the Present Invention
The term “polynucleic acid molecule” as used herein means a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Both DNA and RNA molecules are constructed from nucleotides linked end to end, wherein each of the nucleotides contains a phosphate group, a sugar moiety, and either a purine or a pyrimidine base. Polynucleic acid molecules can be single or double-stranded polymers of nucleotides read from the 5′ to the 3′ end. Polynucleic acid molecules may also optionally contain synthetic, non-natural or altered nucleotide bases that permit correct read through by a polymerase and do not alter expression of a polypeptide encoded by that polynucleic acid molecule.
The term “an isolated polynucleic acid molecule” as used herein, means a polynucleic acid molecule that is no longer accompanied by some of materials with which it is associated in its natural state, or to a polynucleic acid molecule for which the structure of which is not identical to that of any naturally occurring polynucleic acid molecule. It is also contemplated by the inventors that the isolated polynucleic acid molecules of the present invention also include known types of modifications.
The term “nucleotide sequence” as used herein means the linear arrangement of nucleotides to form a polynucleotide of the sense and complementary strands of a polynucleic acid molecule either as individual single strands or in the duplex
As used herein both terms “a coding sequence” and “a structural polynucleotide molecule” mean a polynucleotide molecule that is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory molecules. The boundaries of the coding sequence are determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to, genomic DNA, cDNA, and recombinant polynucleotide sequences.
The term “recombinant DNAs” as used herein means DNAs that contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, or other methods known in the art for manipulation of DNA molecules.
The term “synthetic DNAs” as used herein means DNAs assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art.
Both terms “polypeptide” and “protein”, as used herein, mean a polymer composed of amino acids connected by peptide bonds. An amino acid unit in a polypeptide (or protein) is called a residue. The terms “polypeptide” and “protein” also apply to any amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to any naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a polypeptide, that polypeptide is specifically reactive to antibodies elicited to the same polypeptide but consisting entirely of naturally occurring amino acids. It is well known in the art that proteins or polypeptides may undergo modification. Exemplary modifications are described in most basic texts, such as, for example, Proteins—Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York (1993. Many detailed reviews are available on this subject, for example, those provided by Wold, F., Post-translational Protein Modifications. Perspectives and Prospects, pp. 1-12 in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-M (1990) and Rattan et al., Protein Synthesis: Post-translational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992).
The term “amino acid sequence” means the sequence of amino acids in a polypeptide (or protein) that is written starting with the amino-terminal (N-terminal) residue and ending with the carboxyl-terminal (C-terminal) residue.
“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (that does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The terms “substantially identical”, “substantially homologous” and “substantial identity”, used in reference to two polypeptide sequences or two polynucleotide sequences, mean that one polypeptide sequence or one polynucleotide sequence has at least 75% sequence identity compared to the other polypeptide sequence or polynucleotide sequence as a reference sequence using the Gap program in the WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc. based on the method of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970) using the set of default parameters for pairwise comparison (for amino acid sequence comparison: Gap Creation Penalty=8, Gap Extension Penalty=2; for nucleotide sequence comparison: Gap Creation Penalty=50; Gap Extension Penalty=3) or using the TBLASTN program in the BLAST 2.2.1 software suite (Altschul et al., Nucleic Acids Res. 25:3389-3402), using BLOSUM62 matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919, 1992) and the set of default parameters for pair-wise comparison (gap creation cost=11, gap extension cost=1.)
One aspect of the present invention provides an isolated polynucleic acid molecule comprising a nucleotide sequence or complement thereof, wherein the nucleotide sequence encodes a polypeptide from a crop plant having an amino acid sequence that has at least 75% sequence identity, or 80% sequence identity, or at least 85% or 90% sequence identity, or at least 95% sequence identity, or at least 98% sequence identity to a member selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51.
Polypeptides that are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes. “Conservative amino acid changes” and “Conservative amino acid substitution” are used synonymously to describe the invention. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. “Conservative amino acid substitutions” mean substitutions of one or more amino acids in a native amino acid sequence with another amino acid(s) having similar side chains, resulting in a silent change. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
One skilled in the art will recognize that the values of the above substantial identity of nucleotide sequences can be appropriately adjusted to determine the corresponding sequence identity of two nucleotide sequences encoding the polypeptides of the present invention by taking into account codon degeneracy, conservative amino acid substitutions and reading frame positioning. Substantial identity of nucleotide sequences for these purposes normally means sequence identity of at least 75%.
The term “codon degeneracy” means divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for ectopic expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of codon usage of the host cell as observed in a codon usage table.
The polynucleic acid molecules encoding a polypeptide of the present invention may be combined with other non-native, or “heterologous” sequences in a variety of ways. By “heterologous” sequences it is meant any sequence that is not naturally found joined to the nucleotide sequence encoding polypeptide of the present invention, including, for example, combinations of nucleotide sequences from the same plant that are not naturally found joined together, or the two sequences originate from two different species.
The term “operably linked”, as used in reference to a regulatory molecule and a structural polynucleotide molecule, means that the regulatory molecule causes regulated expression of the operably linked structural polynucleotide molecule. “Expression” means the transcription and stable accumulation of sense or antisense RNA derived from the polynucleic acid molecule of the present invention. Expression may also refer to translation of mRNA into a polypeptide. “Sense RNA” means RNA transcript that includes the mRNA and so can be translated into polypeptide or protein by the cell. “Antisense RNA” means a RNA transcript that is complementary to all or part of a target primary transcript or complementary to mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-translated sequence, introns, or the coding sequence. “RNA transcript” means the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.
The DNA construct of the present invention can, in one embodiment, contain a promoter which causes the over-expression of the polypeptide of the present invention, where “over-expression” means the expression of a polypeptide either not normally present in the host cell, or present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide. Promoters that can cause the over-expression of the polypeptide of the present invention are generally known in the art.
The DNA construct of the present invention can, in another embodiment, contain a promoter which causes the ectopic expression of the polypeptide of the invention, where “ectopic expression” means the expression of a polypeptide in a cell type other than a cell type in which the polypeptide is normally expressed; at a time other than a time at which the polypeptide is normally expressed; or at a expression level other than the level at which the polypeptide normally is expressed. Promoters that can cause ectopic expression of the polypeptide of the present invention are generally known in the art. The expression level or pattern of the promoter of the DNA construct of the present invention may be modified to enhance its expression. Methods known to those of skill in the art can be used to insert enhancing elements (for example, subdomains of the CaMV 35S promoter, Benfey et. al, 1990 EMBO J. 9: 1677-1684) into the 5′ sequence of genes. In one embodiment, enhancing elements may be added to create a promoter that encompasses the temporal and spatial expression of the native promoter of the gene of the present invention but have altered levels of expression as compared to the native levels of expression. Similarly, tissue specific expression of the promoter can be accomplished through modifications of the 5′ region of the promoter with elements determined to specifically activate or repress gene expression (for example, pollen specific elements, Eyal et al., 1995 Plant Cell 7: 373-384).
The term “a gene” means the segment of DNA that is involved in producing a polypeptide. Such segment of DNA includes regulatory molecules preceding (5′ non-coding DNA molecules) and following (3′ non-coding DNA molecules) the coding region, as well as intervening sequences (introns) between individual coding segments (exons). A “native gene” means a gene as found in nature with its own regulatory DNA sequences. “Chimeric gene” means any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” means a native gene in its natural location in the genome of an organism. A “foreign gene” means a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure resulting in a transgenic organism.
“Regulatory sequences” means polynucleotide molecules located upstream (5′ non-coding sequences), within, or downstream (3′ non-translated sequences) of a structural polynucleotide sequence, and that influence the transcription, RNA processing or stability, or translation of the associated structural polynucleotide sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
The term promoter sequence or promoter means a polynucleotide molecule that is capable of causing expression of one or more genes when present in “cis” location of the structural polynucleotide capable of expressing polypeptide. Such promoter regions are typically found upstream of the trinucleotide, ATG, at the start site of a polypeptide coding region. Promoter molecules can also include DNA sequences from which transcription of transfer RNA (tRNA) or ribosomal RNA (rRNA) sequences are initiated. Transcription involves the synthesis of a RNA chain representing one strand of a DNA duplex which provides the template for its synthesis. Transcription takes place by the usual process of complementary base pairing, catalyzed and scrutinized by the enzyme RNA polymerase. The reaction can be divided into three stages described as initiation, elongation and termination. Initiation begins with the binding of RNA polymerase to the double stranded (DS or ds) DNA. The polynucleotide sequence of DNA required for the initiation reaction defines the promoter. The site at which the first nucleotide is incorporated is called the start-site or start-point of transcription. Elongation describes the phase during which the enzyme moves along the DNA and extends the growing RNA chain. Elongation involves the disruption of the DNA double stranded structure in which a transiently unwound region exists as a hybrid RNA-DNA duplex and a displaced single strand of DNA. Termination involves recognition of the point at which no further bases should be added to the chain. To terminate transcription, the formation of phosphodiester bonds must cease and the transcription complex must come apart. When the last base is added to the RNA chain, the RNA-DNA hybrid is disrupted, the DNA reforms into a duplex state, and the RNA polymerase enzyme and RNA molecule are both released from the DNA. The sequence of DNA required for the termination reaction is called the transcription termination region.
The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
Promoters that are known or are found to cause transcription of DNA in plant cells can be used in the present invention. Such promoters may be obtained from a variety of sources such as plants and plant viruses. A number of promoters, including constitutive promoters, inducible promoters and tissue-specific promoters, that are active in plant cells have been described in the literature. It is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide to cause the desired phenotype. In addition to promoters that are known to cause transcription of DNA in plant cells, other promoters may be identified for use in the current invention by screening a plant cDNA library for genes that are selectively or preferably expressed in the target tissues and then determine the promoter regions.
The term “constitutive promoter” means a regulatory sequence that causes expression of a structural nucleotide sequence in most cells or tissues at most times. Constitutive promoters are active under most environmental conditions and states of development or cell differentiation. A variety of constitutive promoters are well known in the art. Examples of constitutive promoters that are active in plant cells include but are not limited to the nopaline synthase (NOS) promoters; the cauliflower mosaic virus (P-CaMV) 19S and 35S (U.S. Pat. No. 5,858,642); the figwort mosaic virus promoter (P-FMV, U.S. Pat. No. 6,051,753); and actin promoters, such as the rice actin promoter (P-Os.Act1, U.S. Pat. No. 5,641,876).
The term “inducible promoter” means a regulatory sequence that causes conditional expression of a structural nucleotide sequence under the influence of changing environmental conditions (U.S. Pat. Nos. 5,922,564 and 5,965,791), or developmental conditions. The term “tissue-specific promoter” means a regulatory sequence that causes transcriptions or enhanced transcriptions of DNA in specific cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots, leaves or stems, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistils, flowers, or any embryonic tissue. Reproductive tissue specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. One skilled in the art will recognize that a tissue-specific promoter may drive expression of operably linked DNA molecules in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can be used to express the polynucleic acid molecules of the present invention. Examples of tuber-specific promoters include, but are not limited to the class I and II patatin promoters (Bevan et al., EMBO J. 8:1899-1906, 1986; Koster-Topfer et al., Mol Gen Genet. 219:390-396, 1989; Mignery et al., Gene. 62:27-44, 1988; Jefferson et al., Plant Mol. Biol. 14: 995-1006, 1990). Examples of leaf-specific promoters include but are not limited to the ribulose biphosphate carboxylase (RBCS or RuBISCO) promoters (see, e.g., Matsuoka et al., Plant J. 6:311-319, 1994); the light harvesting chlorophyll a/b binding protein gene promoter (see, e.g., Shiina et al., Plant Physiol. 115:477-483, 1997). Examples of root-specific promoters include, but are not limited to, the promoter for the acid chitinase gene (Samac et al., Plant Mol. Biol. 25:587-596, 1994); the root specific subdomains of the CaMV35S promoter that have been identified (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894, 1989).
Promoters derived from genes encoding embryonic storage proteins, which includes the gene encoding the 2S storage protein from Brassica napus (Dasgupta et al., Gene 133:301-302, 1993); the gene encoding oleosin 20 kD from Brassica napus (GenBank No. M63985); the genes encoding oleosin A (GenBank No. U09118) and oleosin B (GenBank No. U09119) from soybean; the gene encoding oleosin 18 kD from maize (GenBank No. J05212, Lee, Plant Mol. Biol. 26:1981-1987, 1994); and the gene encoding low molecular weight sulphur rich protein from soybean (Choi et al., Mol. Gen. Genet. 246:266-268, 1995), can also be used. Promoters derived from zein encoding genes (including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD, and gamma genes, Pedersen et al., Cell 29:1015-1026, 1982) can be also used. The zeins are a group of storage proteins found in maize endosperm.
It is recognized that additional promoters that may be utilized are described, for example, in U.S. Pat. Nos. 5,378,619, 5,391,725, 5,428,147, 5,447,858, 5,608,144, 5,608,144, 5,614,399, 5,633,441, 5,633,435, and 4,633,436, all of which are herein incorporated in their entirety. In addition, a tissue specific enhancer may be used (Fromm et al., The Plant Cell 1:977-984, 1989). It is further recognized that the exact boundaries of regulatory sequences may not be completely defined and DNA fragments of different lengths may have identical promoter activity.
The “translation leader sequence” means a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences include maize and petunia heat shock protein leaders, plant virus coat protein leaders, and plant rubisco gene leaders among others (Turner and Foster, Molecular Biotechnology 3:225, 1995).
The “3′ non-translated sequences” or “3′ termination region” means DNA sequences located downstream of a structural nucleotide sequence and include sequences encoding polyadenylation and other regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3′ end of the mRNA precursor. The polyadenylation sequence can be derived from the natural gene, from a variety of plant genes, or from T-DNA. An example of the polyadenylation sequence is the nopaline synthase 3′ sequence (nos 3′; Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983). Ingelbrecht et al. exemplify the use of different 3′ non-translated sequences (Plant Cell 1:671-680, 1989).
The laboratory procedures in recombinant DNA technology used herein are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), herein referred to as Sambrook et al., (1989).
A “substantial portion” of a polynucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a polynucleic acid molecule comprising the sequence. Polynucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. J. Mol. Biol. 215:403-410, 1993). In general, a sequence of thirty or more contiguous nucleotides is necessary in order to putatively identify a nucleotide sequence as homologous to a gene. Moreover, with respect to polynucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular polynucleic acid molecule comprising the primers. The skilled artisan having the benefit of the polynucleic acid molecules as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete polynucleotide sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
Isolation of polynucleic acid molecules encoding homologous polypeptides using polynucleotide sequence-dependent protocols is well known in the art. Examples of polynucleotide sequence-dependent protocols include, but are not limited to, methods of polynucleic acid molecule hybridization, and methods of DNA and RNA amplification as exemplified by various uses of polynucleic acid molecule amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
For example, structural polynucleic acid molecules encoding additional polypeptides of the present invention, either as cDNAs or genomic DNAs, could be isolated directly by using all or a substantial portion of the polynucleic acid molecules of the present invention as DNA hybridization probes to screen cDNA or genomic libraries from any desired plant employing methodology well known to those skilled in the art. Methods for forming such libraries are well known in the art. Specific oligonucleotide probes based upon the polynucleic acid molecules of the present invention can be designed and synthesized by methods known in the art. Moreover, the entire sequences of the polynucleic acid molecules can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length cDNA or genomic DNAs under conditions of appropriate stringency.
Alternatively, the polynucleic acid molecules of interest can be isolated from a mixture of polynucleic acid molecules using amplification techniques. For instance, the disclosed polynucleic acid molecules may be used to define a pair of primers that can be used with the polymerase chain reaction (Mullis, et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273, 1986; EP 50,424; EP 84,796, EP 258,017, EP 237,362, EP 201,184; U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788, and U.S. Pat. No. 4,683,194) to amplify and obtain any desired polynucleic acid molecule directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleotide sequences that encode for polypeptides to be expressed, to make polynucleic acid molecules to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
In addition, two short segments of the polynucleic acid molecules of the present invention may be used in polymerase chain reaction protocols to amplify longer polynucleic acid molecules encoding homologs of a polypeptide of the invention from DNA or RNA. For example, the skilled artisan can follow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998, 1988) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the polynucleic acid molecules of the present invention. Using commercially available 3′RACE or 5′RACE systems (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.), specific 3′ or 5′ cDNA fragments can be isolated. Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin, Techniques 1:165, 1989).
Polynucleic acid molecules of interest may also be synthesized, either completely or in part, especially where it is desirable to provide modifications in the polynucleotide sequences, by well-known techniques as described in the technical literature, see, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Thus, all or a portion of the polynucleic acid molecules of the present invention may be synthesized using a codon usage table of a selected plant host. Other modifications of the coding gene sequences may result in mutants having slightly altered activity.
After transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenic plants for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the polypeptide encoded by the gene being expressed. Alternatively, one could establish assays that specifically measure enzyme activity. A preferred method will be one that allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.
All or a substantial portion of the polynucleic acid molecules of the present invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the polynucleic acid molecules of the present invention may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook et al., 1989) of restriction-digested plant genomic DNA may be probed with the polynucleic acid fragments of the present invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al., Genomics 1:174-181, 1987), in order to construct a genetic map. In addition, the polynucleic acid fragments of the present invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the polynucleotide sequence of the present invention in the genetic map previously obtained using this population (Botstein et al., Am. J. Hum. Genet. 32:314-331, 1980).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (Plant Mol. Biol. Reporter 4:37-41, 1986). Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, exotic germplasms, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
Polynucleic acid probes derived from the polynucleic acid molecules of the present invention may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al., In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346).
In another embodiment, polynucleic acid probes derived from the polynucleic acid molecules of the present invention may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask, Trends Genet. 7:149-154, 1991). Although current methods of FISH mapping favor use of large clones (several to several hundred kilobases; see Laan et al., Genome Res. 5:13-20, 1995), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of polynucleic acid amplification-based methods of genetic and physical mapping may be carried out using the nucleotide molecules of the present invention. Examples include allele-specific amplification (Kazazian et al., J. Lab. Clin. Med. 11:95-96, 1989), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al., Genomics 16:325-332, 1993), allele-specific ligation (Landegren et al., Science 241:1077-1080, 1988), nucleotide extension reactions (Sokolov et al., Nucleic Acid Res. 18:3671, 1990), Radiation Hybrid Mapping (Walter et al., Nat. Genet. 7:22-28, 1997) and Happy Mapping (Dear and Cook, Nucleic Acid Res. 17:6795-6807, 1989). For these methods, the sequence of a polynucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the nucleotide sequence. However, this identification is generally not necessary for mapping methods.
Isolated polynucleic acid molecules of the present invention may find use in the identification of loss of function mutant phenotypes of a plant, due to a mutation in one or more endogenous genes encoding polypeptides of the present invention. This can be accomplished either by using targeted gene disruption protocols or by identifying specific mutants for these genes contained in a population of plants carrying mutations in all possible genes (Ballinger and Benzer, Proc. Natl. Acad Sci USA 86:9402-9406, 1989; Koes et al., Proc. Natl. Acad. Sci. USA 92:8149-8153, 1995; Bensen et al., Plant Cell 7:75-84, 1995). The latter approach may be accomplished in two ways. First, short segments of the polynucleic acid molecules of the present invention may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which mutator transposons or some other mutation-causing DNA element has been introduced. The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding polypeptides. Alternatively, the polynucleic acid molecules of the present invention may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adapter.
The polypeptides of the present invention may also include fusion polypeptides. A polypeptide that comprises one or more additional polypeptide regions not derived from that polypeptide is a “fusion” polypeptide. Such molecules may be derivatized to contain carbohydrate or other moieties (such as keyhole, limpet, hemocyanin, etc.). Fusion polypeptides of the present invention are preferably produced via recombinant means.
The polypeptide molecules of the present invention may also include polypeptides encoded by all or a substantial portion of polypeptide-encoding sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 or complements thereof or, fragments or fusions thereof in which conservative, non-essential, or not relevant, amino acid residues have been added, replaced, or deleted. An example of such a homolog is the homolog polypeptide (or protein) from different species. Such a homolog can be obtained by any of a variety of methods. For example, as indicated above, one or more of the disclosed sequences, all or a substantial portion of a polypeptide-encoding sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 50 and complements thereof will be used to define a pair of primers that may be used to isolate the homolog encoding polynucleic acid molecules from any desired species. Such molecules can be expressed to yield homologs by recombinant means.
Polynucleic acid molecules that encode all or part of the polypeptides of the present invention can be expressed, via recombinant means, to yield polypeptides that can in turn be used to elicit antibodies that are capable of binding the expressed polypeptides. It may be desirable to derivatize the obtained antibodies, for example with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope or an enzyme). Such antibodies may be used in immunoassays for that polypeptide. In a preferred embodiment, such antibodies can be used to screen cDNA expression libraries to isolate full-length cDNA clones of the present invention (Lemer, Adv. Immunol. 36:1, 1984; Sambrook et al., 1989).
Plant Recombinant DNA Constructs and Transformed Plants
The isolated polynucleic acid molecules of the present invention can find particular use in creating transgenic crop plants in which polypeptides of the present invention are overexpressed. Overexpression of these polypeptides in a plant can enhance plant stress tolerance and thereby lead to improvement in the yield of the plant. It will be particularly desirable to enhance plant drought and osmotic stress tolerance in crop plants that undergo such stresses over the course of a normal growing season. Crop plants are defined as plants which are cultivated to produce one or more commercial products. Examples of such crops or crop plants include soybean, canola, rape, cotton (cottonseeds), sunflower, and grains such as corn, wheat, rice, rye, and the like.
The term “transgenic crop plant” means a plant that contains an exogenous polynucleic acid, which can be derived from the same plant species or from a different species. By “exogenous” it is meant that a polynucleic acid molecule originates from outside the plant into which the polynucleic acid molecule is introduced. An exogenous polynucleic acid molecule can have a naturally occurring or non-naturally occurring nucleotide sequence. One skilled in the art understands that an exogenous polynucleic acid molecule can be a heterologous polynucleic acid molecule derived from a different plant species than the plant into which the polynucleic acid molecule is introduced or can be a polynucleic acid molecule derived from the same plant species as the plant into which it is introduced.
Crop plant cell, as used herein, includes without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
The term “genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components of the cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized. The term “genome” as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs of the present invention introduced into bacterial or microbial host cells can therefore be either chromosomally integrated or plasmid-localized.
Exogenous polynucleic acid molecules may be transferred into a crop plant cell by the use of a recombinant DNA construct (or vector) designed for such a purpose. The present invention also provides a plant recombinant DNA construct (or vector) for producing transgenic crop plants, wherein the plant recombinant DNA construct comprises a structural nucleotide sequence encoding an polypeptide of the present invention. Methods that are well known to those skilled in the art may be used to prepare the crop plant recombinant DNA construct (or vector) of the present invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., (1989). The GATEWAYTM cloning technology (Invitrogen Life Technologies, Carlsbad, Calif.) is also used for construction of a few vectors of the invention. GATEWAYTM technology uses phage lambda base site-specific recombination for vector construction, instead of restriction endonucleases and ligases. Using the GATEWAYTM cloning technology, a desired DNA sequence, such as a coding sequence, may be amplified by PCR with the phage lambda attB 1 sequence added to the 5′ primer and the attB2 sequence added to the 3′ primer. Alternatively, nested primers comprising a set of attB1 and attB2 specific primers and a second set of primers specific for the selected DNA sequence can be used. Sequences, such as coding sequences, flanked by attB1 and attB2 sequences can be readily inserted into plant expression vectors using GATEWAYTM methods. Assembly of DNA constructs are done by standard molecular biology techniques as described in Sambrooks et al.
A plant recombinant DNA construct of the present invention contains a structural nucleotide sequence encoding a polypeptide of the present invention and operably linked to regulatory sequences. Exemplary regulatory sequences include but are not limited to promoters, translation leader sequences, introns and 3′ non-translated sequences. The promoters can be constitutive, inducible, native, or tissue-specific promoters.
A plant recombinant DNA construct of the present invention will typically comprise a selectable marker that confers a selectable phenotype on plant cells. Selectable markers may also be used to select for plants or plant cells that contain the exogenous polynucleic acid molecules encoding polypeptides of the present invention. The marker may encode biocide resistance, antibiotic resistance (e.g, kanamycin, G418, bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate, glufosinate, etc.). Examples of selectable markers include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985) that codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene that codes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., Bio/Technology 6:915-922 (1988)) that encodes glyphosate resistance; a nitrilase gene that confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263:6310-6314 (1988) a mutant acetolactate synthase gene (ALS) that confers imidazolinone or sulphonylurea resistance, and a methotrexate resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).
A plant recombinant DNA construct of the present invention may also include a screenable marker. Screenable markers may be used to monitor expression. Exemplary screenable markers include a β-glucuronidase or uidA gene (GUS:1) that encodes an enzyme for which various chromogenic substrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405 (1987)); an R-locus gene that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., Stadler Symposium 11:263-282 (1988)); a β-lactamase gene (Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741 (1978)), a gene that encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al., Science 234:856-859 (1986)); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105 (1983)) that encodes a catechol dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikatu et al., Bio/Technol. 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone that in turn condenses to melanin; and an α-galactosidase that will turn over a chromogenic α-galactose substrate.
Included within the terms “selectable or screenable marker genes” are also genes that encode a secretable marker whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes that can be detected catalytically. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA, small active enzymes detectable in extracellular solution (e.g., α-amylase, β-lactamase, phosphinothricin transferase), or proteins that are inserted or trapped in the cell wall (such as proteins that include a leader sequence such as that found in the expression unit of extension or tobacco PR-S). Other possible selectable and/or screenable marker genes will be apparent to those of skill in the art.
In addition to a selectable marker, it may be desirable to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes that are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Wising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the beta-glucuronidase (GUS) of the uidA locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis. An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uidA locus of E. coli as described by Jefferson et al., (Biochem. Soc. Trans. 15, 17-19 (1987) to identify transformed cells, referred to herein as GUS:1.
In preparing the recombinant DNA constructs (vectors) of the present invention, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector, e.g., a plasmid that is capable of replication in a bacterial host, e.g., E. coli. Numerous cloning vectors exist that have been described in the literature, many of which are commercially available. After each cloning, the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components of the desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell.
The present invention also provides a transgenic plant comprising in its genome an isolated polynucleic acid that comprises: (a) a 5′ non-coding sequence that functions in the cell to cause the production of a mRNA molecule; that is operably linked to (b) a structural polynucleotide sequence encoding a polypeptide of this invention that is operably linked to (c) a 3′ non-translated sequence that functions in said cell to cause termination of transcription. Preferably, the amino acid sequence of the polypeptide has at least 75% sequence identity, about 85% sequence identity, or about 95% or about 98% sequence identity to a member selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51. The polypeptide can also have one of the sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 with conservative amino acid substitutions.
Transgenic crop plants of the present invention have incorporated into their genome, or transformed into their chloroplast or plastid genomes, an exogenous polynucleic acid molecule that comprises at least a structural nucleotide sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51. Transgenic crop plants are also meant to comprise progeny (descendant, offspring, etc.) of any generation of such a transgenic plant. A seed of any generation of all such transgenic crop plants wherein said seed comprises a DNA sequence encoding the polypeptide of the present invention is also an important aspect of the invention.
In one embodiment, the transgenic crop plants of the present invention will have enhanced tolerance to environmental stress due to the expression of an exogenous polynucleic acid molecule encoding a polypeptide of the present invention. The transgenic crop plants of the present invention will have tolerance to abiotic stresses, for example, variations from optimal condition to sub-optimal conditions for water, humidity, temperature, light or other radiations, organic and inorganic nutrients, and salinity. “Cold” is defined as sub-optimal thermal conditions needed for normal growth of natural plants. As used herein, “cold germination” is germination occurring at temperatures below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant. As used herein, “cold tolerance” is defined as the ability of a plant to continue growth for a significant period of time after being placed at a temperature below that normally encountered by a plant of that species at that growth stage. As used herein “enhanced” is defined as to increase or improve in value, quality, desirability, or attractiveness of one or more desired traits in a transgenic plant as compared to a nontransgenic plant of comparable variety. The transgenic plants of the present invention will have higher tolerance to cold, higher germination in cold temperature and a higher yield of agricultural products under stressed conditions. Similarly “water stress” is defined as a sub-optimal amount of water needed for normal growth of natural plants. As used herein “water-stress” is a plant condition characterized by water potential in a plant tissue of less than about −0.5 megapascals (MPa). Water potential in maize is conveniently measured by clamping a leaf segment in a pressurizable container so that a cut cross section of leaf is open to atmospheric pressure. Gauge pressure (above atmospheric pressure) on the contained leaf section is increased until water begins to exude from the atmospheric-pressure-exposed cross section. The gauge pressure at incipient water exudation is reported as negative water potential in the plant tissue. The transgenic plants of the present invention will have a higher tolerance to water stress as compared to natural plants of same species and will have a higher yield of agricultural products under water stressed conditions.
The DNA construct of the present invention may be introduced into the genome of a desired plant host by a variety of conventional transformation techniques that are well known to those skilled in the art. Methods of transformation of plant cells or tissues include, but are not limited to the Agrobacterium mediated transformation method and the Biolistics or particle-gun mediated transformation method. Suitable plant transformation vectors for the purpose of Agrobacterium mediated transformation include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella et al., Nature 303:209 (1983); Bevan, Nucleic Acids Res. 12: 8711-8721 (1984); Klee et al., Bio-Technology 3(7): 637-642 (1985); and EP 120,516. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, but are not limited to, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen.
A plasmid expression vector suitable for the introduction of a polynucleic acid encoding a polypeptide of present invention in monocots using electroporation or particle-gun mediated transformation is composed of the following: a promoter that is constitutive or tissue-specific; an intron that provides a splice site to facilitate expression of the gene, such as the maize Hsp70 intron (U.S. Pat. No. 5,593,874, herein incorporated by reference in its entirety); and a 3′ polyadenylation sequence such as the nopaline synthase 3′ sequence (nos 3; Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983). This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA.
An example of a useful Ti plasmid cassette vector for plant transformation is pMON17227. This vector is described in U.S. Pat. No. 5,633,435, herein incorporated by reference in its entirety, and contains a gene encoding an EPSPS enzyme with glyphosate resistance (herein referred to as aroA:CP4), that is an excellent selection marker gene for many plants. The gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (At. EPSPS:CTP2) and expressed from the Figwort mosaic virus (P-FMV) promoter as described therein.
When adequate numbers of cells containing the exogenous polynucleic acid molecule encoding polypeptides from the present invention are obtained, the cells can be cultured, then regenerated into whole plants. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Regeneration techniques are described generally in Klee et al., Ann. Rev. Plant Phys. 38:467-486 (1987).
The development or regeneration of transgenic crop plants containing the exogenous polynucleic acid molecule that encodes a polypeptide of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic crop plants, as discussed above. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
Plants that can be made to have enhanced stress tolerance by practice of the present invention include, but are not limited to, Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, cauliflower, celery, cherry, cilantro, citrus, clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, forest trees, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nut, oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, turf, a vine, watermelon, wheat, yams, and zucchini.
The following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications, additions, substitutions, truncations, etc., can be made to the methods and genes described herein while not departing from the spirit and scope of the present invention.
EXAMPLES
Example 1
Stock Rice Plants and Growth Conditions
Rice seeds (Oryza sativa, “Kasalath” cultivar) were obtained from the National Institute of Agrobiological Resources MAFF1-2Kannondai2-Chome, Tsukuba Ibrai-3058602 Japan. For increasing the stock size of initial seeds, seeds were planted to raise seedling and seedlings were subsequently transplanted in 6 inch (6′′) pots for obtaining mature plants bearing panicles and mature seeds.
Seed Propagation
For obtaining mature seeds, rice plants, plant organs or immature embryos at the desired developmental stage, approximately 100 seeds of each variety were soaked in distilled water for 30 to 60 minutes at room temperature. During the soaking period floating chaff and impurities from the seeds were removed, water was decanted and the seeds were placed in properly labeled pre-irrigated 6′′ pots filled with red soil. After placing 1-2 seed(s)/pot on top of the soil, the seeds were covered with fine sand and then gently patted. Each seeded pot was covered with newspaper and was irrigated regularly with rose-can tin in order to maintain humidity in the soil. After 4-6 days, paper covers from the pots were removed, exposing germinated seeds to the light. The germinated seeds were allowed to grow 1′′-2′′ in height which usually occurred 7-8 days after planting seeds. Pots were then transferred to a water tray for proper water and nutrient treatments. Initial fertilizer was prepared by mixing 10 grams (gm) urea, 30 gm of 17:17:17 N:P:K fertilizer, 2.5 gm of Multiplex -a micro nutrient (Karnataka Agro chemicals, Bangalore, India), 0.25 gm of FeSO4 in one liter of water and adjusting the pH to 6.2. Approximately 1 liter of this solution was used to fertilize pots placed on 1 square meter of water trays. Water level was maintained in trays with potted seedlings. Seedlings were allowed to grow for 20 days under natural sunlight (400-800 g mole/m2/sec)/10-12 hr day. Day temperature was observed at 28° C.-30° C., night temperature at 19° C.-20° C. with a relative humidity of 60-70% in the greenhouses.
Transplanting of Rice Seedlings
For transplanting rice seedlings to generate mature plants, a red and black soil mixture was used as potting mix in 6′′ pots. Red and black soils were mixed in 3:1 ratio to bring soil pH between pH 6 and pH 7. Ten grams of farm yard manure, (Varsha Agro. Industries, Bangalore, India, from now on referred to as FYM) was added per 0.003 cubic meter of soil (which is roughly equivalent to a full 6′′ pot soil). This mixture of soil was used to fill 6′′ pots for transplanting. Potted soil was saturated with water and then allowed to drain before packing the soil to the desired density. Then soil in the pot was drenched with the fungicide “Carbendzim” at the concentration of 1 gm/L (Carbendzim, 50% WP, BASF India Ltd. Mumbai) and the insecticide Monocrotophos (Monocrotophos 36% SL, Bayer India Ltd, Mumbai India) 1 ml/L for disinfection. During or prior to the disinfection procedure, all clumps of soil in pot were eliminated to maximize the treatment.
For transplanting, entire growing rice seedlings along with the old soil were carefully removed from the pots. Excess soil from the seedlings was removed by gentle tapping. Two seedlings were planted (3-6 cm deep) in pots with new soil mix. For the first 10 days approximately 1′′ water level followed by 2′′ water level was maintained until 10 days before harvesting. Before harvesting ripe panicles with seed, water was siphoned out of the trays. Siphoning was done by draining all the water from the tray on the 30th day of heading and 10 days before harvesting. Fertilizer application for growing rice was done as per the following table:







TABLE 1







Composition of Different plant growth medium used for growing


rice plants and seedlings.

















Micro











Nutrient





S.
M.
Application


Fertilizers
(Multiplex)
N
P
K
17:17:17
Urea
Phosphate
Potash
Time



















Total Doses

15
5
7.5







gm/m2











Basal Doses
2.5
2.5
5
2.5
14.71

11.90
0
DOP + 1 = A


gm/ m2











gm/ m2
2.5
2.5


14.71



A + 15 = B


Doses at Active
2.5
2.5

2.5

5.4

4.17
B + 15 = C


Tillering gm/











m2











gm/ m2
2.5
2.5

2.5

5.4

4.17
C + 15 = D


Doses at
2.5
2.5



5.4

4.17
D + 15 = E


Panicle











Initiation Stage











gm/ m2











Doses at
2.5
2.5



5.4


E + 15


Heading gm/m2





Table legend:


Micro Nutrient (Multiplex - (Karnataka Agro chemicals, Bangalore, India),. N, P, K (Nitrogen, P2O5 , K2O in the form of complex fertilizer 17:17:17 Madras Fertilizers Ltd, Chennai). Superphosphate. Phosphate(P2O5 16%, EID Parry India Ltd, Chennai India), Muriate of Potash (K2O 60% Zuari Agro industries, Goa, India) Date of sowing (DOS). Date of transplanting (DOP).






Seeds from transgenic or non-transgenic rice plants were kept segregated from the time of harvest until next use as per standard practices well know in the art.
Example 2
This example demonstrates how rice OsPK7 was cloned to express in rice plants. OsPK7 cDNA specific primers were designed based on the gene sequences as shown in SEQ ID NO: 1. DNASTAR software (DNASTAR, Inc. Madison, Wis., USA) was used for primer design. The sequences of the 5′ and 3′ primer were SEQ ID NO: 48 and SEQ ID NO: 49 respectively. Total RNA was purified from pooled rice (var. Nipponbare) coleoptile tissue by using Trizol reagent from Life Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A. from now on referred to as Gibco), essentially as recommended by the manufacturer. Total RNA was used as the template to synthesize rice cDNA molecules by using a RT-PCR kit manufactured by Life Technologies as per the instructions of manufacturer of the kit. This cDNA was used as template DNA in a PCR reaction to amplify cDNA molecules which were purified on a low melting agarose gel by electrophoresis as described by Sambrook et al. Purified cDNA molecules of Seq ID NO: 1 were cloned in pCRTOPO 2.1 vector as per the manufacturer's instructions (Invitrogen, Carlsbad, Calif. 92008). After confirming the sequence, cloned molecules were excised and re-cloned in the publicly available rice binary expression vector pCAMBIA 1300 (CAMBIA, Canberra, Australia) to generate rice transforming vector molecules. Restriction analysis was performed to identify the transforming vector with SEQ ID NO: 1 in proper orientation which would encode polypeptide molecules as shown in SEQ ID NO 2.
Example 3
Identification of Homologs, Paralogs or Orthologs
This example explains how to isolate homologs, orthologs, or paralogs of SEQ ID NO: 1 by generating cDNA libraries, sequencing cDNA clones to generate a database for identification of desired clones from desired plant species.
For construction of cDNA libraries from plants, plant tissues are harvested and immediately frozen in liquid nitrogen and stored at −80° C. until total RNA extraction. Total RNA is purified from tissues using Trizol reagent from Life Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.), essentially as recommended by the manufacturer. Poly A+ RNA (mRNA) is purified using magnetic oligo dT beads essentially as recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake Success, N.Y. U.S.A.).
Construction of plant cDNA libraries is well known in the art and a number of cloning strategies exist. A number of cDNA library construction kits are commercially available. The SuperscriptTM Plasmid System for cDNA synthesis and Plasmid Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is used, following the conditions suggested by the manufacturer.
The cDNA libraries are plated on LB agar containing the appropriate antibiotics for selection and incubated at 37° for sufficient time to allow the growth of individual colonies. Single selective-media colonies are individually placed in each well of 96-well microtiter plates containing LB liquid including the selective antibiotics. The plates are incubated overnight at approximately 37° C. with gentle shaking to promote growth of the cultures.
The plasmid DNA is isolated from each clone using Qiaprep plasmid isolation kits, using the conditions recommended by the manufacturer (Qiagen Inc., Santa Clara, Calif. U.S.A.).
The template plasmid DNA clones are used for subsequent sequencing. For sequencing the cDNA libraries, a commercially available sequencing kit, such as the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS, is used under the conditions recommended by the manufacturer (PE Applied Biosystems, Foster City, Calif.). The cDNAs of the present invention are generated by sequencing initiated from the 5′ end or 3′ end of each cDNA clone. Entire inserts or only part of the inserts (ESTs or expressed sequenced tags) are sequenced.
A number of DNA sequencing techniques are known in the art, including fluorescence-based sequencing methodologies. These methods have the detection, automation and instrumentation capability necessary for the analysis of large volumes of sequence data. Currently, the 377 and 3700 DNA Sequencer (Perkin-Elmer Corp., Applied Biosystems Div., Foster City, Calif.) allow the most rapid electrophoresis and data collection. With these types of automated systems, fluorescent dye-labeled sequence reaction products are detected and data are entered directly into the computer, producing a chromatogram that is subsequently viewed, stored, and analyzed using the corresponding software programs. These methods are known to those of skill in the art and have been described and reviewed (Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.).
The generated ESTs (including any full-length cDNA inserts or complete coding sequences) are combined with ESTs and full-length cDNA sequences in public databases such as GenBank. Duplicate sequences are removed, and duplicate sequence identification numbers are replaced. The combined dataset is then clustered and assembled using Pangea Systems (DoubleTwist, 2001 Broadway, Oakland, Calif. 94612) tool identified as CAT v.3.2.
First, the EST sequences are screened and filtered, e.g. high frequency words are masked to prevent spurious clustering; sequence common to known contaminants such as cloning bacteria are masked; high frequency repeated sequences and simple sequences are masked; unmasked sequences of less than 100 base pairs are eliminated. The thus-screened and filtered ESTs are combined and subjected to a word-based clustering algorithm that calculates sequence pair distances based on word frequencies and uses a single linkage method to group like sequences into clusters of more than one sequence, as appropriate. Clustered sequences are assembled individually using an iterative method based on PHRAP/CRAW/MAP providing one or more self-consistent consensus sequences and inconsistent singleton sequences. The assembled clustered sequence files are checked for completeness and parsed to create data representing each consensus contiguous sequence (contig), the initial EST sequences, and the relative position of each EST in a respective contig. The sequence of the 5′ most clone is identified from each contig. The initial sequences that are not included in a contig are separated out.
Above described databases with nucleotide and peptide sequences are queried with sequences of present invention to get the following homologs, orthologs or paralogs as shown in Table 2. The BLAST 2.2.1 software (Altschul, et. al., Nucleic Acids Res. 25: 3389-3402 (1997), with BLOSUM62 matrix and “no Filter” options, is used in the queries. When necessary, frame-shifts in the DNA sequences of the homologs are detected by aligning the DNA sequence of the homolog in question to the protein sequence of present invention, using the “frame+_n2p” program with default parameters in the GenCore software package (Compugen Inc., 25 Leek Crescent, Richmond Hill, Ontario, L4B 4B3, Canada, 1998). Such frame-shifts are conceptually corrected to yield open reading frames. The “translate” program with default parameters in the same package is used to translate open reading frames to corresponding peptide sequences based on standard genetic codes.







TABLE 2







Description of homologs, orthologs or paralogs of SEQ ID NO: 1










SEQ ID NO
Genus species







1 to 6
Oryza sativa



 7 to 14
Zea mays



15 to 20
Glycine max



21 and 22
Gossypium hirsutum



23 to 27
Triticum aestivum



28 and 29
Hordeum vulgare



30 to 33
Allium porrum



34 and 35
Brassica napus



36 and 37
Pisum sativum



38 and 39
Medicago truncatula



40 to 47
Arabidopsis thaliana










Example 4
Isolation of Polynucleotide Molecules of the Present Invention and their Modification
For isolating polynucleotide molecules of the present invention, total RNA is isolated from the appropriate crop and other desired plant species by pooling tissues of different developmental stages of all vegetative and reproductive organs. RNA is prepared from pooled plant tissue by the Trizol method (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) essentially as recommended by the manufacturer. Sequences are amplified out from total RNA by using the Superscript II kit (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) according to the manufacturer's directions. Design of appropriate PCR primers for isolating sequences of present invention is based on the sequence information provided in the sequence listing of this disclosure. Design of primers and reaction conditions are determined as described in the art. (PCR Strategies, Edited by Michael A. Innis; David H. Gelfand; & Johm J. Sninsky; Academic Press 1995 and PCR Protocols, A Guide to Method and Applications, Edited by Michael A. Innis; David H. Gelfand; Johm J. Sninsky; & Thomas J. White Academic Press 1990). All reagents for isolating sequences of the invention can be procured from Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.
Example 5
This example explains transformation of rice plants to generate plants of the present invention.
Transgenic rice plants were produced by an Agrobacterium mediated transformation method. A disarmed Agrobacterium strain C58 (EHA105) harboring the plant transformation construct was produced by the standard electroporation method (Bio-Rad) of transforming bacteria. Transformed bacterial cells were grown overnight in LB medium (Gibco) containing 5 gm/L hygromycin at 25° C., centrifuged and suspended in Co-cultivation medium (Table 3 shown as CC1 medium) supplemented with acetosyringone (100 uM) at an OD600 of 1. This suspension was used for transforming rice tissue.
Tissue Preparation for Rice Transformation:
Panicles of Kasalath rice were collected 10-15 days after anthesis. First, panicles were thoroughly washed with deionized water containing a few drops of Tween 20, surface-sterilized with 70% ethanol for 3 minutes, and washed again at room temperature with deionized water before treating with 2% Sodium hypochlorite for 10 minutes. Sterilized panicles were washed with water repeatedly to remove all sodium hypochorite. The husk was manually removed to isolate immature seed, washed again with deionized sterile water before a second sterilization with 70% ethanol followed by three washes with sterile deionized water. Finally, immature seeds were surface-sterilized with 2% Sodium hypochlorite for 30-40 minutes, washed with deionized water remove traces of sterilant. Immature seeds remained in sterilized water during entire subsequent operation. Immature embryos or immature seeds were placed on MSAg medium (Table 3) until the co-cultivation.







TABLE 3







Describes composition of different media used for examples of the invention.




















Regener-
Plant


Component/L
MSAg
CC-1
CC-2
Delay
Selection
ation
development
























MS Salts
4.2
g
4.2
g
4.2
g
4.2
g
4.2
g
4.2
g
2.1
g


(Hi media, India)
















CaC12•2H2O
440
mg
440
mg
440
mg
440
mg
440
mg
440
mg
0



Thiamine HC1
1.0
mg
0.5
mg
0.5
mg
0

1.0
mg
0

0



Glutamine
500
mg
0

0

0

500
mg
0

0



Myo-Inositol
0

0

0

0

0

100
mg
0



Magnesium chloride
750

0

0

0

750

0

0



Casein Hydrolysate
100
mg
0

0

0

100
mg
0

0



Sucrose
20
g
20
g
20
g
20
g
20
g
30

15
g


Glucose
0

10
g
10
g
0

0

0

0



2,4-D
2
mg
2
mg
2
mg
1.5
mg
2
mg
0

0



Kinetin
0

0

0

0.2
mg
0

2.0
mg
0



NAA
0

0

0

0

0

2.0
mg
0



BAP
0

0

0

0

0

4.0
mg
0



Phytagel
2.0
g
0

2.0
g
2
g
2.0
g
0

0



L-Proline
0

115
mg
115
mg
500

0

0

0



Acetosyringone
0

0

0

0

0

0

0



Cefataxime
0

0

0

250
mg
250

250

250
mg


Hygromycin
0

0

0

0

50
mg
25
mg
25
mg









Infection of Rice Plants
Freshly isolated embryos were incubated with bacterial culture (100 μl per 10 embryos) for 10 minutes. Individual embryos were handpicked and cultured on CC2 medium after removing bacterial suspension. Embryos were incubated for three days in the dark, washed with sterilized water supplemented with Cefotaxime (Sigma Chemical Co Catalog No. 22128) and then blotted dry before culturing on delay medium (Table 3). After one week, roots were excised and scutellar calli were subcultured on selection medium (Table 3)
Selection and Regeneration of Rice Plants
Putative calli were selected by culturing treated calli on selection medium (7-10 day interval) for two to three months or until calli attained 10 mm size. These were then transferred to regeneration medium for a week under darkness. For shoot regeneration, calli were transferred to light. Once plants attained a size of 5-10 mm, they were transferred to bottles containing 1⁄2 X, Murashige and Skoog basal salts medium (Now on referred as MS medium or MS. MS can be procured from Sigma Chemical Co. Saint Louis, Mo., Catalog No. M8900). Selection pressure with hygromycin was maintained in vitro throughout. Once plants attained a height of 4-6 inches, they were transferred to the greenhouse for hardening. These plants are referred to as R0 plants.
Acclimatization:
Primary Acclimatization
R0 plants were acclimatized by placing plant in greenhouse under covered tunnel for 3-4 days. At the end of this period plants were removed from agar medium, and all adhering agar was carefully removed from roots by washing with water to avoid future fungus and other plant infection. Root were dipped in Bavistin (Carbendzim, 50% WP, BASF India Ltd. Mumbai) solution (1.0 gm/L) for 1⁄2-1 minute before transplanting in net pots containing “Soilrite Mix” (Chougule Industries, Bangalore, India), or “Cocopeat” (Varsha Agro Industries, Bangalore India). A suitable number (50 or 98) of plants in net pots were placed on portray (a plastic tray, of dimension 52.5 cm length×25.25 cm width, containing 50 plug holes and each plug hole, with a dimension of 5 cm diameter & 5 cm depth, was fitted with a net pot (5 cm diameter×4.7 cm depth) and drenched with fungicide solution Bavistin/Dithane M 45 (1.0 gm/L) (Carbendzim, 50% WP, BASF India Ltd. Mumbai, India/Mancozeb 75% WP Indofil Chemicals Ltd. Mumbai, India).
Newly transplanted R0 plants on tray were kept for 7-10 days in a humid chamber with 80%-90% relative humidity, 24° C.-25° C. temperature and 800-100 Lux light intensity. During this period, every 3-4 days the plants were treated with Hoagland nutrient solution (Sigma Chemical Co. Catalog No. H2395). After initial period of 7-10 days the relative humidity was dropped to 70%-80% and the light intensity was increased to 1100-1500 Lux. Then the plants were treated with 10:52:10 (N:P:K) fertilizer solution at 100 ppm N level and a mild spray of Bavistin (0.5 gm/L).
Secondary Acclimatization
After primary acclimatization plants were acclimatized for 7 days at a light intensity of 1200-1800 Lux, 65%-75% relative humidity and a temperature between 25° C.-26° C. After secondary acclimatization plants were transferred to 6′′ pots and were grown as described earlier.
Details on number of lines, total plants received and survival status during acclimatization are shown in Table 4.







TABLE 4







Survival Status of transgenic rice plant lines after the acclimatization.


















Survival status
Date of















Date of

No. of
No. of
Primary
Secondary
transplanting


Batch no.
receipt
GOI
lines
plants
Acclimatization
Acclimatization
to pot

















B.N. 2001-9
May 28, 2001
Ospk7
11
33
31
31
Jun. 7, 2001


B.N. 2001-9
Jun. 1, 2001
Ospk7
8
22
22
22
Jun. 7, 2001


B.N. 2001-10
Jun. 6, 2002
Ospk7
1
3
3
3
Jun. 18, 2001









Example 6
This example describes a method of determining in-planta sequence of OsPK7 gene in a rice plant transformed with the OsPK7 gene or its homolog. The basic methodology presented in this example can be used for determining in planta sequence in any plant of the invention.
DNA Isolation
Rice plant DNA was prepared using the Phenol extraction method, modified from Sambrook et al., (1989). 0.5 to 1.0 g leaf tissue was grinded with liquid nitrogen into a fine powder, and then was mixed with extraction buffer immediately (at 1:5 w/v ratio, and buffer composition: 500 mM NaCl, 100 mM Tris-Hcl (PH 8.0), 0.5% SDS, 50 mM EDTA, 80 mM Beta-Mercaptoethanol) and incubated at 65° C. for 10 minutes. Equal volume of phenol:chloroform (1:1) was added and gently mixed for 3 to 5 minutes, centrifuged at 10,000 rpm for 10 minutes and the aqueous phase was transferred into a fresh tube. The aqueous phase was extracted one more time using only chloroform, and then added with two volumes of chilled ethanol and gently mixed. DNA precipitates were spooled into a fresh 1.5 ml tube and dissolved in 800 ul Tris-EDTA (TE) buffer at room temperature. 5 ul of RNAase (10 mg/ml) was added and incubated at 37° C. for 30 minutes. The DNA sample was then extracted with Phenol:chloroform (1:1) twice and chloroform once, and precipitated using one tenth volume of 3M sodium acetate (pH 5.4) and two volumes of ethanol. DNA was spooled into a fresh 1.5 ml microfuge tube and washed with 70% ethanol. DNA pellet was then dissolved in 60 to 100 ul TE buffer pH 8.0.
Amplification of Gene from Isolated DNA:
Nested sets of PCR primers were designed based on the expression cassette of the plant transformation construct. Designing of primer pairs is well known in the art and is also briefly described in example two of the present disclosure. Approximately 10 ng of isolated genomic DNA from each transgenic rice plant was used in a standard PCR reaction for amplification of in planta gene. Reaction mixture with genomic DNA, appropriated primer pairs, and enzyme in reaction buffer was subjected to initial denaturation of DNA by heating the mixture at 94° C., 2 minutes in a PCR machine, followed by 40 cycles of reaction. Each cycle consisted of denaturation at 94° C. for 30 seconds, annealing at 61° C. for 30 seconds followed by primer extension at 72° C. for 90 seconds. Amplified DNA was isolated at the end of PCR reaction by using QIAquick Gel extraction kit (Qiagen, Cat No. 28704, Qiagen Inc., Santa Clara, Calif. U.S.A.). The DNA was eluted in TE buffer pH 8.0 and stored at −20° C. till further use.
Sequencing of Isolated In-Planta Gene:
Amplified DNA was used as a template in standard sequencing reaction. Standard method of sequencing is described in Example 3 of the present disclosure. The DNA was sequenced by using sequencing primers designed on the basis of expression cassette of the gene in rice plants. In planta gene sequences from two of the events in rice plants were confirmed to be same and are presented as SEQ ID No: 50 and its translation is presented as SEQ ID NO 51.
In some cases sequencing the in planta gene from different events of transgenic plants demonstrates minor variation in gene sequences. Minor sequence variation is capable of providing variation in the level of the desired phenotype in plants. Some sequence variations were observed when comparing the gene sequence from the transformation construct isolated from agrobacterium and the gene sequence isolated from transgenic rice events transformed with the construct.
Example 7
This example describes the morphological assay and observations performed on rice plants of the present invention.
Transgenic and non-transgenic isolines were segregated based on the southern analysis of genomic DNA isolated from plants. Southern analysis of plant genomic DNA was performed by standard procedures as described in Molecular Cloning, A Laboratory Manual, Sambrook et al., (1989) and using a hpt DNA fragment as a non-radioactive probe (using material and protocol supplied in AlkPhos Direct labeling and detection kit, Amersham pharmacia).
Morphological Assay on R1 Seeds
R1 seeds were germinated on MS medium with 50 mg/L hygormycin to separate transgenic seeds from non-transgenic, and for further physiological/phenotypical analysis. A subset of these seeds with the transgene was allowed to mature for production of R2 seeds. 15-20 seeds from 10 independent lines with different copy numbers of genes were de-husked, surface sterilized and inoculated on MS medium in culture bottles. Bottles were incubated in the dark for 2 days and later on transferred to light. At the end of the incubation period (13 Days) the plants were removed from the bottles and washed under a gentle flow of water and used for transplanting. The first ten tallest seedlings were transplanted to pots for further morphological analysis of R1 plants.
Morphological data on R1 plants were recorded. Results are shown in Table 5.







TABLE 5







Morphological observation of R1 plants















NUMBER OF TILLERS
PLANT
PANICLE
SEED WT.
YIELD PER PLANT















Plant ID
DOH
TOTAL NO.
PROD.
HEIGHT
LENGTH
PER 1000
TOTAL Yield
Seed Yield





WT (Kasalath)
  74 ± 0.00
13.00 ± 1.49
11.70 ± 0.95
149.29 ± 6.05
26.82 ± 0.54
16.13 ± 0.65
18.91 ± 4.36
18.08 ± 4.48


653-4-1
76.80 ± 4.08
23.40 ± 3.95
21.80 ± 3.85
141.65 ± 6.87
23.40 ± 1.28
16.76 ± 0.54
12.67 ± 8.77
10.66 ± 9.22


652-1-1
77.62 ± 5.41
13.62 ± 5.41
 12.54 ± 4s.61
 145.55 ± 11.55
23.16 ± 2.44
16.35 ± 0.52
14.94 ± 4.09
13.86 ± 3.89


652-5-1
78.50 ± 5.61
11.17 ± 1.17
10.33 ± 1.51
153.61 ± 7.25
22.02 ± 0.88
17.12 ± 0.94
 9.71 ± 4.49
 8.52 ± 4.75


652-6-1
82.30 ± 5.10
12.50 ± 2.17
11.40 ± 1.84
141.35 ± 5.97
25.08 ± 1.30
17.78 ± 2.25
 8.21 ± 6.08
 6.95 ± 5.99


610-1-1
87.00 ± 0.00
12.38 ± 3.85
11.13 ± 3.56
146.83 ± 8.04
24.98 ± 0.69
16.58 ± 1.52
 5.68 ± 3.67
 4.35 ± 3.99


610-2-3
76.44 ± 6.88
15.11 ± 2.89
13.44 ± 3.68
143.41 ± 6.06
25.14 ± 0.64
17.31 ± 0.43
 8.07 ± 4.76
 6.25 ± 5.22


612-1-1
76.63 ± 1.06
 9.88 ± 1.46
 9.00 ± 1.77
142.91 ± 6.24
25.40 ± 0.90
16.26 ± 0.42
11.15 ± 3.19
10.47 ± 3.31


647-1-1
80.88 ± 3.23
15.88 ± 2.30
14.75 ± 2.31
136.51 ± 5.60
24.39 ± 1.14
16.76 ± 0.41
 6.42 ± 3.87
 5.39 ± 3.85





Table legend:


DOH (Day of heading)- this explains how many days the plant has taken for flowering after transplanting. Data given here is an average of 8-12 plants from each event with standard deviation.


WT- Wild type is control set.






Example 8
This example explains the selection of homozygous rice line for performing physiological experiments on transgenic plants of the present invention.
Homozygosity Test for R2 Seeds
Rice is a self-pollinated crop. Hence the R1 seed pool from a R0 transgenic plant with a single copy of the transgene will harbor the transgene in 1:2:1 ratio i.e one homozygous, 2 heterozygous and one null segregant. R1 homozygous plants will produce R2 seeds where all the seeds are transgenic and homozygous. Therefore homozygous lines were identified in the R2 generation by germinating 30 R2 seeds from individual clones from different events on 1⁄2 strength MS medium supplemented with hygormycin as described earlier. A line with more than 80% germination is considered homozygous as germination is also affected by seed quality. Seeds from these homozygous lines were used in different physiological assays.







TABLE 6







Homozygosity test












SI



No. of seeds
No. of seeds


No.
Plant ID
GOI
Variety
Inoculated
Germinated















1
T1610-1-1-1
OSPK-7
41
30
30


2
T1610-1-1-2
OSPK-7
41
30
22


3
T1610-1-1-3
OSPK-7
41
30
28


4
T1610-2-3-1
OSPK-7
41
30
28


5
T1610-2-3-3
OSPK-7
41
30
19


6
T1610-2-3-4
OSPK-7
41
30
22


7
T1612-1-1-1
OSPK-7
41
30
20


8
T1612-1-1-2
OSPK-7
41
30
27


9
T1612-1-1-3
OSPK-7
41
30
19


10
T1647-1-1-1
OSPK-7
41
30
26


11
T1647-1-1-2
OSPK-7
41
30
19


12
T1647-1-1-3
OSPK-7
41
30
23


13
T1652-1-1-1
OSPK-7
41
30
28


14
T1652-1-1-5
OSPK-7
41
30
0


15
T1652-1-1-6
OSPK-7
41
30
0


16
T1652-3-1-1
OSPK-7
41
30
26


17
T1652-3-1-2
OSPK-7
41
30
0


18
T1652-3-1-3
OSPK-7
41
30
0


19
T1652-3-1-5
OSPK-7
41
30
5


20
T1652-5-1-1
OSPK-7
41
30
30


21
T1652-5-1-2
OSPK-7
41
30
29


22
T1652-5-1-3
OSPK-7
41
30
17


23
T1652-5-1-6
OSPK-7
41
30
30


24
T1652-6-1-1
OSPK-7
41
30
24


25
T1652-6-1-2
OSPK-7
41
30
23


26
T1652-6-1-3
OSPK-7
41
30
19


27
T1653-4-1-1
OSPK-7
41
30
23


28
T1653-4-1-2
OSPK-7
41
30
23


29
T1653-4-1-3
OSPK-7
41
30
25


30
T1653-4-1-5
OSPK-7
41
30
30


31
T1653-4-1-6
OSPK-7
41
35
34


32
T1653-4-1-7
OSPK-7
41
30
30


33
41 control
OSPK-7
41
27
25


34
41 control
OSPK-7

35
0









Example 9
This example explains the water stress test for analyzing transgenic rice plants of the invention.
R2Generation Water Stress Test—Rapid Stress:
Germinated seedlings were planted in portrays. For plating seedlings each net pot was filled with 75 g of red sandy loam soil (dry) and the entire tray was drenched to saturation level with water containing fungicide Bavistin (1 gm/l). Excess water was drained before weighing the entire tray as well as individual net pots. Individual net pots with water-saturated soil weighing about 95 to 100 grams were considered at 100% field water capacity. Germinated seedlings were further grown in the greenhouse with conditions as described in example 1. Every day during the growth period lost water was measured (by weighing pots) and replenished to maintain 100% of field water capacity in the desired pots. Loss of water in pots with plants was due to evaporation and transpiration. Ten net pots were maintained without plants to calculate the amount of water lost due to evaporation. Plants were fertilized once every three days with a solution containing 3 gm urea, 6 gm N:P:K (17:17:17), 0.5 gm FeSO4 and 2.5 gm micronutrient mix/32 L. Fifteen-day-old seedlings were subjected to water stress by withholding irrigation for 4 days. Subsequently net pots were saturated with water and excess water was drained to attain 100% field water capacity for alleviating stress. The plats were maintained at 100% field capacity throughout the recovery period by weighing the pot every day and replenishing the amount of water lost through evaporation/transpiration. The plants were allowed to recover for twelve days. At the end of recovery i.e., the 12th day, growth was measured by weighing only the shoot (above soil, i.e without root). Growth was recorded as fresh weight in milligrams as shown in Table 7. The transgenic lines of the present invention were observed to have significantly higher biomass at the end of recovery as compared to the wild type rice line.







TABLE 7







Result of the R2 generation water stress test.








lines
Fresh. Wt. (mg)





R2-610-1-1-3
311.0 ± 68.4


R2-610-2-3-1
445.5 ± 95.5


R2-612-1-1-2
390.3 ± 71.3


R2-652-5-1-1
343.8 ± 53.8


R2-652-3-1-1
332.5 ± 51.8


R2-653-4-1-5
297.2 ± 41.8


WT- kasalath (wild type non-tansgenic control
170.4 ± 70.1









Example 10
This example demonstrates the rate of survival of transgenic rice plants as compared to non-transgenic rice plants after the water stress.
Three-leaf or 12 days old rice seedlings grown as per the earlier description and were subjected to water stress by withholding irrigation for two days and allowing the plant to recover for 8 days. At the end of recovery, surviving seedling were counted and expressed as percent seedling survival. For determining percent survival of transgenic rice plants, five different sets of experiments designated as 2a, 2b, 2c, 2d, and 2e, were conducted as described above. Ten plants/set were used for this experiment. The results of this experiment are shown in Table 8 indicating all transgenic lines except R2-610-2-3-1 exhibited a significantly high rate of survival at the end of water stress compared to that of wild type.







TABLE 8







Showing the survival of transgenic rice seedlings as compared to non-


transgenic rice seedlings after water stress treatment.











Survival at the end of recovery (%)













Line

Exp.
Exp.
Exp.
Exp.
Exp.


code
Lines
2a
2b
2c
2d
2e
















1
R2-610-1-1-3
30
27
40
ND
ND


2
R2-610-2-3-1
0
20
0
ND
ND


3
R2-612-1-1-2 *
100
100
100
50
30


4
R2-647-1-1-1
50
54
20
80
60


5
R2-652-1-1-1
ND
ND
60
ND
ND


6
R2-652-5-1-1
ND
ND
60
80
80


7
R2-652-3-1-1
66
54
60
60
20


8
R2-653-4-1-5
83
63
80
70
10


9
WT- kasalath (wild type
16
41
0
0
0



non-tansgenic control)









Example 11
This example demonstrates the effect of water stress on plant biomass in transgenic rice plants of the invention in comparison with wild type rice plants.
Three-leaf or 12 day-old rice seedlings, grown as per the description of Example 7, were subjected to water stress by withholding irrigation for two days and allowing plants to recover for 10 days. At the end of recovery, growth was measured in terms of fresh weight. Results of this experiment are shown in Table 9. The transgenic lines of the present invention maintained higher average biomass at the end of recovery compared to that of the wild type.







TABLE 9







Biomass of rice seedlings as compared to non-transgenic rice 


seedlings after water stress treatment. Biomass 


of plant is indicated as fresh weight in milligrams.


WT Kasalath is natural, wild type rice plant.









Line code
Lines
Fresh weight (mg)





3
R2-612-1-1-3
243.89 ± 227.45


4
R2-647-1-1-2
438.44 ± 273.98


6
R2-652-5-1-1
582.00 ± 374.53


7
R2-652-3-1-1
417.44 ± 327.82


8
R2-653-4-1-5
318.22 ± 271.63


WT
WT- Kasalath
152.89 ± 112.52









Example 12
This example demonstrates the effect of water stress on plant biomass in older transgenic rice plants of the invention in comparison with wild type rice plants.
Five-leaf or 20 day-old rice seedlings, grown as per the description of Example 7 were subjected to water stress by withholding irrigation for two days and allowing plants to recover for 6 days. At the end of recovery, growth was measured in terms of fresh weight. Results of this experiment are shown in Table 10. The transgenic lines of the present invention maintained higher average biomass at the end of recovery compared to that of the wild type.







TABLE 10







Biomass of older rice seedling as compared to non-transgenic


rice seedlings after water stress treatment. Biomass of the


plant is indicated as fresh weight in milligrams.


WT-kasalath is non-transgenic.









Line code
Lines
Fresh weight (mg)





3
R2-612-1-1-3
153.7 ± 40.8 


4
R2-647-1-1-2
363.4 ± 109.79


6
R2-652-5-1-1
484.5 ± 180.59


7
R2-652-3-1-1
266.5 ± 96.03 


8
R2-653-4-1-5
215.4 ± 78.33 


WT
WT- Kasalath
252.9 ± 93.28 









Example 13
This example describes the effect of long term stress on R2 plants of the present invention.
Germinated seedlings were transferred to plastic pots (10 cm diameter×4 cm depth) containing 100 g of red sandy loam soil with two different levels of water content. The two levels are 25 percent field capacity (FC25), 9.3 ml/100 g soil and 100 percent field capacity (FC100), 37.5 ml/100 g soil. The seedlings were allowed to adapted in two different water regimes for 15 days. The seedlings were adapted in the greenhouse. During the growth period the water level was maintained at designated field capacity by weighing the pots every day and replenishing the amount of water lost through evaporation/transpiration. Ten pots were maintained without plants to calculate the amount of water lost due to evaporation. During this period plants were fertilized once every three days with solution as described in Example 7. On the 15th day the difference in growth rate between transgenic and wild type was assessed in terms of leaf extension growth by measuring the length of the 4th leaf. All transgenic lines were observed to have significant leaf growth differences as compared to non-transgenic lines under experimental stress conditions as described in this example. Results are show below in table 11.







TABLE 11







Effect of Long term Stress on R2 rice plants of present invention


as compared to non transgenic WT-kasalath rice plants.










Line code
Lines
Stressed (FC 25)
Non-stressed (FC-100)





1
R2-610-1-1-3
11.45 ± 3.5 
36.48 ± 3.82


3
R2-612-1-1-2
9.34 ± 2.51
33.55 ± 3.15


4
R2-652-5-1-1
10.25 ± 1.96 
33.76 ± 2.03


7
R2-652-3-1-1
8.07 ± 2.89
32.18 ± 3.84


9
WT- kasalath
5.74 ± 1.86
33.52 ± 3.59









Example 14
This example demonstrates the effect of cold stress on rice plants of the present invention.
Twelve-day-old or three leaf stage seedlings were grown according to Example 7 and were exposed to cold temperature at 12° C. for 24 hours in the presence of 1000 micro mol/mt2/Sec.light. Subsequently, the plants were allowed to recover in the greenhouse for 20 days. The growth observations such as the length of the 4th leaf on the 7th day and plant height (pl. ht), fresh weight and dry weight were recorded on the 20th day of recovery. The cold stressed OSPK-7 transgenic lines exhibited significantly higher initial recovery growth measured in terms of the length of the 4th leaf at the end of recovery. Further, the transgenic lines exhibited significantly higher plant height and marginally higher total biomass at the end of recovery compared to that of the wild type. Results are shown in Tables 12 and 13.







TABLE 12







Results of recovery growth in terms of the length of the


4th leaf of the plant after exposure to cold temperature.










Lines code
Lines
Stress
Non-stress





1
R2-610-1-1-3
18.2 ± 3.5
16.9 ± 4.8


2
R2-610-2-3-1
17.9 ± 2.1
21.6 ± 3.2


3
R2-612-1-1-2
22.1 ± 3.2
21.2 ± 3.2


4
R2-652-5-1-1
19.1 ± 2.0
19.4 ± 6.2


7
R2-652-3-1-1
30.9 ± 3.1
18.8 ± 6.2


8
R2-653-4-1-5
15.3 ± 2.5
21.0 ± 3.5


9
WT- kasalath
 3.7 ± 2.2
23.3 ± 3.3
















TABLE 13







Results of recovery in terms of plant height, fresh


weight, and dry weight after exposure to cold temperature.










Lines
Plant ht.(cm)
Fresh weight (mg)
Dry weight (mg)





R2-610-1-1-3
32.5 ± 5.5
354.9 ± 77.0
86.9 ± 19.5


R2-610-2-3-1
35.3 ± 3.1
389.8 ± 26.8
87.8 ± 8.2 


R2-612-1-1-2
37.2 ± 4.2
387.1 ± 46.0
104.8 ± 42.7 


R2-652-5-1-1
35.3 ± 2.6
325.8 ± 56.6
86.8 ± 10.5


R2-652-3-1-1
39.0 ± 5.0
488.5 ± 51.1
113.2 ± 13.6 


R2-653-4-1-5
37.7 ± 5.1
432.9 ± 94.5
91.5 ± 18.0


WT- kasalath
29.9 ± 2.9
364.6 ± 61.6
86.0 ± 13.6









Example 15
Genetic Elements of Plant Expression Vectors pMON 80878 (FIG. 2), pMON 71709 (FIG. 4), pMON 71712 (FIG. 5), pMON 83200 (FIG. 6), pMON 71710 (FIG. 7), pMON 71713 (FIG. 8), pMON 83201 (FIG. 9), and pMON 82629 (FIG. 10)
The DNA constructs are double border plant transformation constructs that also contain DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an E. coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spc/Str that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host bacterial strain is Agrobacterium tumefaciens ABI or LBA4404.
The polylinker regions in these DNA constructs provide for multiple restriction endonuclease cut sites that digest the DNA to provide a cloning site. Examples of such cloning sites may include BglII, NcoI, EcoRI, SalI, NotI, XhoI and other sites known to those skilled in the art of molecular biology. pMON 72472 plant expression vector (FIG. 1) is modified for cloning and expression of SEQ ID NO: 1 from rice plants by changing multiple cloning sites to accept a DNA fragment with Not 1 and SalI restriction enonuclease fragment. SEQ ID NO:1 is in cloned pMON 72472 (FIG. 1) or pMON53616 (FIG. 3) at a restriction site resulting in a plant expression vector pMON 80878 (FIG. 2) pMON 71709 (FIG. 4). The construct is used for transforming wild type corn plants to generated transgenic corn plants. Orthologs of SEQ ID NO:1 are cloned in vector pMON 53616 or pMON 72472 by replacing an existing expression cassette of the construct with a desired expression cassette containing a desired promoter, the polynucleotide of the present invention and desired 3′ terminator resulting in constructs pMON 71712, pMON 83200, pMON 71710, pMON 71713, pMON 83201 or pMON 71709 as shown in FIGS. 5 to 10 and Table 14.







TABLE 14







Construction of plant transforming vectors.














Gene/Homolog
Plant of
Construct
Vector for

Transformed
Construct's



name
origin
name
the construct
Cloning sites
plant
FIGURE
Promoter





OsPK7
Oryza sativa
pMON80878
pMON 72472
attB1 and attB2
LH59 corn
FIG. 1
rACT (promoter









leader, intron)


OsPK7
Oryza sativa
pMON71709
pMON 53616
5′ BsiWI; 3′ XhoI
LH244 corn,
FIG. 2
rACT (promoter






(destroyed by
haploid LH244

leader, intron)






ligation to Not1)
corn




OsPK7
Oryza sativa
pMON71712
pMON 53616
5′ BsiWI; 3′ XhoI
LH244 corn
FIG. 3
CVY-CIK1






(destroyed by


(promoter, intron






ligation to SalI)


leader)


OsPK7
Oryza sativa
pMON 83200
pMON 53616
5′ BsiWI; 3′ XhoI
LH244 corn
FIG. 4
Rab17






(destroyed by









ligation to SalI)





ZmPK4
Zea mays
pMON71710
pMON 53616
5′ BsiWI; 3′ XhoI
LH244 corn
FIG. 5
rACT (promoter






(destroyed by


leader, intron)






ligation to SalI)





ZmPK4
Zea mays
pMON71713
pMON 53616
5′ BsiWI; 3′ XhoI
LH244 corn
FIG. 6
CVY-CIK1






(destroyed by


(promoter, intron






ligation to SalI)


leader)


ZmPK4
Zea mays
pMON83201
pMON 53616
5′ BsiWI; 3′ XhoI
LH244 corn
FIG. 7
Rab17






(destroyed by









ligation to SalI)





ZmPK4
Zea mays
pMON82629
pMON 72472
attB1 and attB2
LH59 corn
FIG. 8
rACT (promoter









leader, intron)









The DNA constructs used in the method of the current invention comprise any promoter known to function to cause transcription in plant cells and any antibiotic or herbicide tolerance encoding polynucleotide sequence known to confer antibiotic or herbicide tolerance to plant cells. The antibiotic tolerance polynucleotide sequences include, but are not limited to polynucleotide sequences encoding for proteins involved in tolerance to kanamycin, neomycin, hygromycin, and other antibiotics known in the art. An antibiotic tolerance gene in such a vector can be replaced by a herbicide tolerance gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in U.S. Pat. Nos. 5,627,061, and 5,633,435, herein incorporated by reference in its entirety; Padgette et al. (1996) Herbicide Resistant Crops, Lewis Publishers, 53-85, and in Penaloza-Vazquez, et al. (1995) Plant Cell Reports 14:482-487), aroA (U.S. Pat. No. 5,094,945) for glyphosate tolerance, bromoxynil nitrilase (Bxn) for Bromoxynil tolerance (U.S. Pat. No. 4,810,648), phytoene desaturase (crtI) (Misawa et al, (1993) Plant Journal 4:833-840, and (1994) Plant Jour 6:481-489) for tolerance to norflurazon, acetohydroxyacid synthase (AHAS, Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193) and the bar gene for tolerance to glufosinate (DeBlock, et al. (1987) EMBO J. 6:2513-2519). Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides.
Genetic elements of transgene DNA constructs used for plant transformation and expression of transgenes in plants include, but are not limited to: plant virus promoters, e.g., P-CaMV.35S promoter (U.S. Pat. No. 5,858,742, herein incorporated by reference in its entirety), the CaMV 35S promoter with a duplicated enhancer (U.S. Pat. No. 5,539,142, herein incorporated by reference in its entirety), the Figwort mosaic virus promoter, P-FMV, as described in U.S. Pat. No. 5,378,619, herein incorporated by reference in its entirety; or the P-AtEF1a (P-AtEF1 or EF1a), the sugarcane bacilliform virus promoter, commelina yellow mottle virus or other Badnavirus promoters; heterologous plant promoters, e.g., plant actin promoters including the rice actin 1 promoter and intron (U.S. Pat. No. 5,641,876) and rice actin 2 promoter and intron (U.S. Pat. No. 6,429,357), Arabidopsis actin promoters, a promoter region from the tomato elongation factor gene and Arabidopsis thaliana elongation factor gene 1a; or the DC3 promoter region from carrot (Seffens et al., Develop. Genet. 11:65-76); or the TP12 promoter (GenBank accession no. U68483).
The genetic elements of the DNA construct further comprise 5′ leader polynucleotides for example, the Hsp70 non-translated leader sequence from Petunia hybrida as described in U.S. Pat. No. 5,362,865, herein incorporated by reference in its entirety.
The genetic elements further comprise herbicide tolerance genes that include, but are not limited to, for example, the aroA:CP4 coding region for EPSPS, a glyphosate resistant enzyme isolated from Agrobacterium tumefaciens (AGRTU) strain CP4 as described in U.S. Pat. No. 5,633,435, herein incorporated by reference in its entirety.
The genetic elements of the DNA construct further comprise 3′ termination regions that include, but are not limited to, the E9 3′ termination region of the pea RbcS gene that functions as a polyadenylation signal; the nos3′ is the 3′ end of the Ti plasmid nopaline synthase gene that functions as a polyadenylation signal; or the TML is 3′ of the end of the Ti plasmid octopine pTi15955 synthase gene (GenBank Accession AF 242881) that functions as a polyadenylation signal. The genetic elements of the DNA construct further comprise the right border (RB) and left borders (LB) of the Ti plasmid of Agrobacterium tumefaciens octopine and nopaline strains.
Example 16
The following example describes transformation of soy and corn plants with constructs expressing genes of present invention. Different plants were transformed with constructs in accordance with Table 14.
Corn
Transgenic corn can be produced by particle bombardment transformation methods as described in U.S. Pat. No. 5,424,412. The vector DNA of plasmid pMON 71709, pMON 71710, pMON 71712, pMON 71713 or pMON 80878 is digested with suitable restriction endonucleases to isolate a plant expression cassette that expresses the polypeptides of the present invention in the plant. The desired expression cassette is purified by agarose gel electrophoresis, then bombarded into embryogenic corn tissue culture cells using a Biolistic® (Dupont, Wilmington, Del.) particle gun with purified isolated DNA fragments. Transformed cells are selected on selection media such glyphosate (N-phosphonomethyl glycine and its salts) containing media and whole plants are regenerated then grown under greenhouse conditions. Fertile seed is collected, planted and the glyphosate tolerant phenotype is back crossed into commercially acceptable corn germplasm by methods known in the art of corn breeding (Sprague et al., Corn and Corn Improvement 3rd Edition, Am. Soc. Agron. Publ (1988).
Transgenic corn plants can be produced by an Agrobacterium mediated transformation method. A disarmed Agrobacterium strain C58 (ABI) harboring DNA as described earlier in the example is used for transforming plants. The construct is first transferred into Agrobacterium by a triparental mating method (Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351). Liquid cultures of Agrobacterium are initiated from glycerol stocks or from a freshly streaked plate and grown overnight at 26° C.-28° C. with shaking (approximately 150 rpm) to mid-log growth phase in liquid LB medium, pH 7.0 containing 50 mg/l kanamycin, 50 mg/l streptomycin and spectinomycin and 25 mg/l chloramphenicol with 200 μM acetosyringone (AS). The Agrobacterium cells are resuspended in the inoculation medium (liquid CM4C) and the density is adjusted to OD660 of 1. Freshly isolated Type II immature LH244 and LH59corn embryos are inoculated with Agrobacterium containing a DNA construct of the present invention and co-cultured 2-3 days in the dark at 23° C. The embryos are then transferred to delay media (N6 1-100-12/micro/Carb 500/20 μM AgNO3) and incubated at 28° C. for 4 to 5 days. All subsequent cultures are kept at this temperature. Coleoptiles are removed one week after inoculation. The embryos are transferred to the first selection medium (N61-0-12/Carb 500/0.5 mM glyphosate). Two weeks later, surviving tissues are transferred to the second selection medium (N61-0-12/Carb 500/1.0 mM glyphosate). Subculture surviving callus every 2 weeks until events can be identified. This will take 3 subcultures on 1.0 mM glyphosate. Once events are identified, bulk up the tissue to regenerate. For regeneration, callus tissues are transferred to the regeneration medium (MSOD, 0.1 μM ABA) and incubated for two weeks. The regenerating calli are transferred to a high sucrose medium and incubated for two weeks. The plantlets are transferred to MSOD media in culture vessel and kept for two weeks. Then the plants with roots are transferred into soil.
Soy Transformation:
Soybean plants are transformed using an Agrobacterium-mediated transformation method, as described by Martinell (U.S. Pat. No. 6,384,301, herein incorporated by reference). For this method, overnight cultures of Agrobacterium tumefaciens containing the plasmid that includes a gene of interest, are grown to log phase and then diluted to a final optical density at 660 nm (OD660) of 0.3 to 0.6 using standard methods known to one skilled in the art. These cultures are used to inoculate the soybean embryo explants prepared as described below.
Commercially available soybean seeds (e.g., Asgrow A3244) are germinated overnight and the meristematic tissue is excised. The excised tissue is placed in a wounding vessel and mixed with the Agrobacterium culture described above. The entire tissue is wounded using sonication. Following the wounding, explants are placed in co-culture for 2-5 days, at which point they are transferred to selection media, i.e., WPM (as described on page 19 of U.S. Pat. No. 6,211,430, incorporated herein by reference) with 75 mM glyphosate (plus antibiotics to control Agrobacterium overgrowth), for 6-8 weeks to allow selection and growth of transgenic shoots. Phenotype positive shoots are harvested approximately 6-8 weeks post transformation and placed into selective rooting media (BRM, as described in Table 3 of U.S. Pat. No. 6,384,301) with 25 mM glyphosate) for 3-5 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media (BRM without glyphosate) for up to two weeks. Roots from the shoots that produced roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil. Plants are maintained under standard greenhouse conditions until seed harvest (R1). The collected seeds are analyzed for protein and oil as described in Example.
Plant Selection:
After transformation of crop plants, positive transformants can by selected by any one, or a combination of many know techniques in the art. Plant can be selected based on the resistance provided by the transforming constructs, which may include antibiotic resistance, or herbicide resistance. Plants can also be selected by screening DNA isolated from transformed plant part with polymerase chain reaction for presence or absence of gene itself, or part of the transforming constructs. Gene or protein specific antibodies can also be utilized for selecting transformed plant expressing desired protein.
Example 17
This example describes a cold germination assay for transgenic corn seeds of the present invention.
Three sets of seeds are used for the experiment. The first set consists of twelve different positive transgenic events where the genes of the present invention are expressed in the seed. The second set consists of negative segregants from the same transgenic events as the positive seeds. The third seed set consists of two cold tolerant and two cold sensitive wild-type lines of corn. A number from one to fourteen is randomly assigned to each of the twelve transgenic events, the cold tolerant wild-type lines, and the cold-sensitive wild-type lines. Positive and negative segregants of the same event are designated as “A” and “B” randomly. Each member of the cold-tolerant or cold-sensitive pair is also designated as “A” and “B” randomly. All seeds are treated with a fungicide “Captan” (Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment. Incubations at or below 23 degrees Celsius are conducted in growth chambers (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada).
Ten Petri plates for the cold assay and 5 plates for the warm assay are used. Petri plates (Cat. #353003) can be procured from Becton, Dickinson and Company (Franklin Lakes, N.J. USA, from now on referred to as BD Biosciences). Each plate is prepared for the experiment by placing a Whatman No. 1 paper on the inner side of the lid (90 mm Catalog #1001090) and on the bottom of the plate (85 mm Catalog #1001085) manufactured by Whatman International Ltd. (Maidstone, England) and wetting them with 2 and 3 ml of sterile water respectively. Ten desired seeds per plate are placed on the bottom filter paper with the embryo side touching the paper, each plate is labeled, the lid with the moist paper is placed on the plate and plates are placed in a growth chamber set at 9.7° C. (for cold assay) or 25° C. (for warm assay) in the dark. Ten plates are laid across the bottom of a plastic box and stacked up to six layers high before placing them in growth chambers. Seeds are watered with 2 ml of deionized sterile water on the 3rd and 10th days. Warm control seeds are watered only on the 3rd day. Seeds are considered germinated if the emerged radicle size is 1 cm. Warm control seeds are scored for germination four days after planting and cold seeds are scored from days 10 to 14, days 17, 19 and 24 after planting. Scoring is conducted until all seeds have germinated or until the end of 24 days after planting. The order of plates is reversed (top to bottom, and bottom to top) on every watering and scoring day. Six radicles per set of plates are harvested at random on the last day of the experiment for analysis of RNA expression by Taqman assay.
After 24 days of data collection, a germination index is calculated for each set of seeds. The germination index is calculated as per:

Germination index=(Σ([T+1−ni]*[Pi−Pi-1]))/T 

Where: T is the total number of days for which the germination experiment is performed. The number of days after planting is defined by n. The number of times the germination has been counted, including the current day, is indicated by i. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between positive and negative selections within an event. Additionally, the germination rate is fitted to a model to determine the number of days to 50% germination and confidence intervals are used to determine the statistical significance between positive and negative selections within an event. The Taqman assay confirms the expression of the RNA of the present invention. Any event which achieved 85% or better germination in the warm is used for the cold assay; otherwise it is dropped from the cold assay.
Example 18
This example describes a cold shock assay for transgenic corn seeds of the present invention.
Experimental set-up for the cold shock assay is the same as described in above example's second paragraph, except seeds are grown in potted media for the cold shock assay.
The desired number of 2.5′′ square plastic pots are placed on flats (n=32, 4×8). Pots are filled with Metro Mix 200 soilless media containing 19:6:12 fertilizer (6 lbs/cubic yard) (Metro Mix, Pots and Flat are obtained from Hummert International, Earth City, Mo.). After planting seeds, pots are placed in a growth chamber set at 23° C., relative humidity of 65% with 12 hour day and night photoperiod (300 uE/m2-min). Planted seeds are watered for 20 minute every other day by sub-irrigation and flats are rotated every third day in a growth chamber for growing corn seedlings.
Chlorophyll fluorescence of plants is measured on the 10th day during the dark period of growth by using a PAM-2000 portable fluorometer as per the manufacturer's instructions (Walz, Germany). After chlorophyll measurements, leaf samples from each event are collected for confirming the expression of genes of the present invention. For expression analysis six V1 leaf tips from each selection are randomly harvested. Expression analysis can be done using a Taqman assay to estimate the RNA expression the 3′ termination sequence or any other part of expression cassette which will be part of the transgenic plant genome. Plants are then repositioned in one flat by alternating between the “A” and “B” selection for a total of sixteen “A” plants and sixteen “B” plants per flat (A & B are described earlier examples). The flats are moved to a growth chamber set at 5° C. The actual temperature at canopy level is 5° C. during the dark cycle and 8° C. during the light cycle. All other conditions such as humidity, day/night cycle and light intensity are kept the same in the growth chamber. The flats are sub-irrigated every day after transfer to the cold temperature. On the 4th day chlorophyll fluorescence is measured again. Plants are transferred to normal growth conditions after six days of cold shock treatment and allowed to recover for the next two days. During this recovery period the length of the V3 leaf is measured on the 1st and 3rd days. After two days of recovery V2 leaf damage is visually estimated by estimating percent of green V2 leaf.
Statistical differences in V3 leaf growth, V2 necrosis and fluorescence during pre-shock and cold shock can be used for estimation of cold shock damage on corn plants.
Example 19
This example describes the early seedling growth assay for transgenic corn seeds of the present invention.
Experimental set-up for the cold shock assay is the same as described in example 15 second paragraph, except seeds are grown in germination paper for the early seedling growth assay.
Three pieces of 12′′×18′′ germination paper (Anchor Paper #SD7606) are used for each entry in the test, “A” and “B”. For each entry the papers are numbered #1 to #3. A line is drawn using a wax pencil across the long dimension of the paper at about four inches from the top edge. Wet the papers in a solution of 0.5% KNO3 and 0.1% Thyram. For each paper, eighteen seeds are placed on the line evenly spaced down the length of the paper. The eighteen seeds are positioned on the paper such that the radical will grow downwards, e.g. longer distance to the paper's edge. The wet paper is rolled up starting from one of the short ends. The paper is rolled evenly and tight enough to hold the seeds in place. The roll is secured into place with two large paper clips, one at the top and one at the bottom. The rolls are placed on end in a tall bucket containing about one inch of the KNO3/thyram solution. The top of the bucket is covered with a plastic bag. The bag is secured such that the rolls are protected from a direct breeze or strong flow of air, but not too tight to inhibit free exchange of oxygen to the rolls.
The buckets are incubated in the growth chamber at 23° C. for three days. The chamber is set up for 65% humidity with no light cycle. For the cold stress treatment the buckets are then incubated in a growth chamber at 12° C. for fourteen days. The chamber is set up for 65% humidity with no light cycle. For the warm treatment the buckets are incubated at 23° C. for an additional three days.
After the appropriate treatment the germination papers are unrolled and the seeds are repositioned on the wax pencil line, if necessary. Seeds that did not germinate are discarded. The tip of the radicle and coleoptile are marked on the germination paper. The germination papers are allowed to dry and then the lengths of the radicle and coleoptile for each seed are measured and the data is recorded. This process can be facilitated using an automated caliper for electronic data transfer to a PC. A coleoptile sample is collected from six individual kernels of each entry for confirming the expression of genes of the present invention.
Statistical differences in the length of radical and shoot during pre-shock and cold shock are used for an estimation of the effect of the cold treatment on corn plants. The analysis is conducted independently for the warm and cold treatments.
Example 20
This example describes a wilt assay for transgenic plants of the present invention. 150 seeds from each event and a control set are imbibed by soaking in sterile water overnight. Imbibed seeds are rolled in germination paper. The seeds are placed in 3 rows on one piece of wet 38 lb 11.5′′×30′′ seed paper (Anchor Paper, St. Paul, Minn.) and overlayed with a second wet piece of seed paper. The wet papers are then placed on a 12′′×36′′ piece of wax paper from Anchor Paper, rolled up and fastened with a rubber band. The roll is placed in a 5 Liter Nalgene Pitcher with approximately 1 liter of water and allowed to germinate for 46-50 hours in a growth chamber or a greenhouse. The growth chamber is set with a day/night cycle of 16 hrs/8 hrs and 26° C. daytime/20° nighttime temperatures. The light intensity of the growth chamber is kept at 500 uE/m2-min.
One day before planting, pots are prepared for planting germinated seed. 5.25′′ square pots (Hummert Cat. No 129300) are filled with dry standard greenhouse media mix (peat moss mix) and adjusted to 330±5 grams by hand compacting the soil and hand watered thoroughly. After watering 1 germinated seed/pot is planted. Seedlings are allowed to grow for 1 week. During this period pots are watered by a capillary matting watering system. A capillary mat (Hummerts Cat. No. 18-4046) is placed on top of a piece of plywood that overlays the greenhouse bench (6 ft.×12 ft.). Watering is done every three hours, beginning at 7.00 AM, five times a day for 12-minute interval using seven 2 GPH (gallons per hour) pressure compensating drippers (Hummerts Cat. #18-4046) per bench. After one week of growth, the V1 leaf is sampled by taking a leaf tear of approximately 2 square centimeters. This leaf sample from the plant is used to determine the presence of the selectable marker, CP4. Water is turned off for several days (usually over the weekend). After 10 days plants of 8-9 cm height are selected based on the presence and absence of the CP4 gene using standard methods. For each transformation an equal number (24) of transgenic and wild type plants are selected based on matched height. These plants are placed alternating a gene positive plant with a gene negative plant on the capillary mat in a serpentine fashion and subjected to dry treatment as described. After arranging plants as per above description, 8 wettest looking pots are weighed to determine maximum current pot weight. This “maximum current pot weight” is used to calibrate all other pots by adding a desired amount of water to bring them all up to the same weight. 8 random pots are weighed every day to monitor pot weight. When the average pot weight is between 600 to 700 grams this is defined as the first day of the experiment. The height of all plants is taken as the length in cm from the top of the soil to the tip of the longest leaf on the start day of the experiment.
After start of the experiment, 8 pots from different flats are weighed. Plants are allowed to grow without any watering if the average weight of pots is greater than 500 grams. If the average weight is less than 500 grams but greater than 365 grams then 35 ml of water/pot is added. If the average weight is less than 365 grams then enough water is added to bring pot weight to 400 grams assuming that each ml of water weighs 1 gram
The treatment ends when the pots have had an average weight below 500 g for 7 days. On the 8th day when the plants weigh less than 500 grams, all plants are measured for height in cm. The difference between the height at the end of the dry treatment and the height at the beginning of the dry treatment is the key quantitative phenotype of interest for this experiment. After the first dry treatment all plants are fully watered for three days and measured again to document drought recovery.
For the second round of drought and recovery estimation plants are allowed to dry by turning off the water system for seven days. After seven days plants will develop severe drought stress exhibited by 10-25% of the plants where leaves will lean to touch the top of pots. At this stage all plants are measured and allowed to recover from stress by fully watering and resuming normal growth conditions. During the recovery phase all plants are daily monitored for recovery signs indicated by a flattening of inner whorl leaves.
After 7 days of recovery all plants are measured and sampled for protein expression analysis prior to harvesting. Harvested plants are placed in vented cellophane bags and weighed to determine the fresh weight of the plants. After determining fresh weight, plants are dried for approximately four weeks in a seed drier at  ̃90° F., 20-40% humidity and weighed to determine the dry weight of plants.

## Claims

1. A method of generating a transgenic plant with enhanced tolerance to environmental stress comprising expressing in said transgenic plant
a DNA construct comprising a promoter that functions in plants, operably linked to a DNA polynucleotide molecule selected from the group consisting of:
a) a DNA molecule encoding a polypeptide sequence at least 90% identical to SEQ ID NO:2; and
b) a DNA molecule comprising the polynucleotide sequence of SEQ ID NO:1
wherein said transgenic plant exhibits enhanced stress tolerance compared to a plant of a same plant species not containing said DNA construct.

2. The method of claim 1, wherein said promoter is a plant virus promoter.

3. The method of claim 1, wherein said promoter comprises a heterologous plant promoter.

4. The method of claim 1, wherein said DNA molecule encodes a polypeptide sequence at least 90% identical to SEQ ID NO:2.

5. The method of claim 1, wherein said enhanced stress tolerance is cold stress tolerance.

6. The method of claim 1, wherein said enhanced stress tolerance is water stress tolerance.

7. The method of claim 1, wherein said plant is a crop plant.

8. The method of claim 7, wherein said crop plant is selected from the group consisting of corn, soybean, wheat, cotton, rice and rapeseed/canola.

9. The method of claim 1, wherein said DNA molecule encodes the polypeptide of SEQ ID NO:2.