# Patent

## Title
Soybean promoters and flower-preferred expression thereof in transgenic plants

## Abstract

The promoters of a soybean lipid transfer protein LTP1 and fragments thereof and their use in promoting the expression of one or more heterologous nucleic acid fragments in plants are described.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/921,703 filed Apr. 4, 2007, which is incorporated by reference herein in its entirety.


FIELD OF THE INVENTION
The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants.
BACKGROUND OF THE INVENTION
Recent advances in plant genetic engineering have opened new doors to engineer plants to have improved characteristics or traits, such as plant disease resistance, insect resistance, herbicidal resistance, yield improvement, improvement of the nutritional quality of the edible portions of the plant, and enhanced stability or shelf-life of the ultimate consumer product obtained from the plants. Thus, a desired gene (or genes) with the molecular function to impart different or improved characteristics or qualities can be incorporated properly into the plant's genome. The newly integrated gene (or genes) coding sequence can then be expressed in the plant cell to exhibit the desired new trait or characteristic. It is important that appropriate regulatory signals be present in proper configurations in order to obtain the expression of the newly inserted gene coding sequence in the plant cell. These regulatory signals typically include a promoter region, a 5′ non-translated leader sequence and a 3′ transcription termination/polyadenylation sequence.
A promoter is a non-coding genomic DNA sequence, usually upstream (5′) to the relevant coding sequence, to which RNA polymerase binds before initiating transcription. This binding aligns the RNA polymerase so that transcription will initiate at a specific transcription initiation site. The nucleotide sequence of the promoter determines the nature of the RNA polymerase binding and other related protein factors that attach to the RNA polymerase and/or promoter, and the rate of RNA synthesis.
It has been shown that certain promoters are able to direct RNA synthesis at a higher rate than others. These are called “strong promoters”. Certain other promoters have been shown to direct RNA synthesis at higher levels only in particular types of cells or tissues and are often referred to as “tissue specific promoters”, or “tissue-preferred promoters”, if the promoters direct RNA synthesis preferentially in certain tissues (RNA synthesis may occur in other tissues at reduced levels). Since patterns of expression of a chimeric gene (or genes) introduced into a plant are controlled using promoters, there is an ongoing interest in the isolation of novel promoters that are capable of controlling the expression of a chimeric gene (or genes) at certain levels in specific tissue types or at specific plant developmental stages.
Among the most commonly used promoters are the nopaline synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci. U.S.A. 84:5745-5749 (1987)); the octapine synthase (OCS) promoter, caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987)); the CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)), and the figwort mosaic virus 35S promoter; the light inducible promoter from the small subunit of rubisco (Pellegrineschi et al., Biochem. Soc. Trans. 23(2):247-250 (1995)); the Adh promoter (Walker et al., Proc. Natl. Acad. Sci. U.S.A. 84:6624-66280 (1987)); the sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. U.S.A. 87:4144-4148 (1990)); the R gene complex promoter (Chandler et al., Plant Cell 1:1175-1183 (1989)); the chlorophyll a/b binding protein gene promoter; and the like.
A flower is a complex structure consisting of pedicel, sepal, petal, stamen, and carpel. A stamen comprises an anther, pollen and filament. A carpel comprises a stigma, style and ovary. An ovary comprises an ovule, embryo sac, and egg cell. Flower promoters in general include promoters that direct gene expression in any of the above tissues or cell types.
Lipid transfer protein (LTP) genes have been isolated from barley (Federico et al., Plant Mol. Biol. 57:35-51 (2005)), strawberry (Yubero-Serrano et al, J. Exp. Bot. 54:1865-1877 (2003)), Arabidopsis (Thoma et al., Plant Physiol. 105:35-45 (1994)), Norway spruce (Sabala et al., Plant Mol. Biol. 42:461-478 (2000)), rice (Vignols et al., Gene 142:265-270 (1994)), carrot (Toonen et al., Plant J. 12:1213-1221 (1997)), Brassica napus (Sohal et al., Plant Mol. Biol. 41:75-87 (1999)), Sorghum vulgare (Pelese-Siebenbourg et al., Gene 148:305-308 (1994)), and other plant species. The reported LTP genes are known to have various expression patterns in respective plants. However, there remains a lack of soybean LTP genes or flower-preferred expression of LTP genes.
Although advances in technology provide greater success in transforming plants with chimeric genes, there is still a need for preferred expression of such genes in desired plants. Often times it is desired to selectively express target genes in a specific tissue because of toxicity or efficacy concerns. For example, flower tissue is a type of tissue where preferred expression is desirable and there remains a need for promoters that preferably initiate transcription in flower tissue. Promoters that initiate transcription preferably in flower tissue control genes involved in flower development and flower abortion.
SUMMARY OF THE INVENTION
Compositions and methods for regulating gene expression in a plant are provided. One aspect is for an isolated polynucleotide comprising: a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1 or a full-length complement thereof; b) a nucleotide sequence comprising a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; or c) a nucleotide sequence comprising a sequence having at least 90% sequence identity, based on the BLASTN method of alignment, when compared to the sequence set forth in SEQ ID NO:1; wherein said nucleotide sequence is a promoter.
Other embodiments include recombinant DNA constructs comprising a polynucleotide sequence of the present invention operably linked to a heterologous sequence. Additionally, some embodiments provide for transgenic plant cells, transient and stable, transgenic plant seeds, as well as transgenic plants comprising the provided recombinant DNA constructs.
There are provided some embodiments that include methods of expressing a coding sequence or a functional RNA in a flowering plant comprising: introducing a recombinant DNA construct described above into the plant, wherein the heterologous sequence comprises a coding sequence; growing the plant; and selecting a plant displaying expression of the coding sequence or the functional RNA of the recombinant DNA construct.
Furthermore, some embodiments of the present invention include methods of transgenically altering a marketable flower trait of a flowering plant, comprising: introducing a recombinant DNA construct described above into the flowering plant; growing a fertile, mature flowering plant resulting from the introducing step; and selecting a flowering plant expressing the heterologous nucleotide sequence in flower tissue based on the altered marketable flower trait.
Another aspect is for an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having lipid transfer protein activity, wherein the polypeptide has at least 90% sequence identity, based on the Clustal method of alignment, when compared to the sequence set forth in SEQ ID NO:36, or (b) a full-length complement of the nucleotide sequence of (a).
A further aspect is for an isolated polypeptide having lipid transfer protein activity, wherein the isolated polypeptide has at least 90% sequence identity, based on the Clustal method of alignment, when compared to the sequence set forth in SEQ ID NO:36.



BRIEF DESCRIPTION OF SEQUENCES AND DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or application publication with color drawing(s) will be provided by the Office upon request and payment of necessary fee.
The invention can be more fully understood from the following detailed description, the accompanying drawings and Sequence Listing which form a part of this application. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2): 345-373 (1984), which are herein incorporated by reference in their entirety. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NO:1 is a DNA sequence comprising a 1136 nucleotide soybean LTP1 promoter (or full-length LTP1 promoter).
SEQ ID NO:2 is a 927 basepair truncated form of the LTP1 promoter shown in SEQ ID NO:1 (bp 209-1136 of SEQ ID NO:1).
SEQ ID NO:3 is a 738 basepair truncated form of the LTP1 promoter shown in SEQ ID NO:1 (bp 398-1136 of SEQ ID NO:1).
SEQ ID NO:4 is a 527 basepair truncated form of the LTP1 promoter shown in SEQ ID NO:1 (bp 609-1136 of SEQ ID NO:1).
SEQ ID NO:5 is a 257 basepair truncated form of the LTP1 promoter shown in SEQ ID NO:1 (bp 879-1136 of SEQ ID NO:1).
SEQ ID NO:6 is an oligonucleotide primer used in the PCR amplifications of the full length LTP1 promoter in SEQ ID NO:1 when paired with SEQ ID NO:7, and the truncated LTP1 promoters in SEQ ID NOs: 2, 3, 4, or 5 when paired with SEQ ID NOs: 8, 9, 10, or 11, respectively.
SEQ ID NO:7 is an oligonucleotide primer used in the PCR amplification of the full length LTP1 promoter in SEQ ID NO:1 when paired with SEQ ID NO:6.
SEQ ID NO:8 is an oligonucleotide primer used in the PCR amplification of the truncated LTP1 promoter in SEQ ID NO:2 when paired with SEQ ID NO:6.
SEQ ID NO:9 is an oligonucleotide primer used in the PCR amplification of the truncated LTP1 promoter in SEQ ID NO:3 when paired with SEQ ID NO:6.
SEQ ID NO:10 is an oligonucleotide primer used in the PCR amplification of the truncated LTP1 promoter in SEQ ID NO:4 when paired with SEQ ID NO:6.
SEQ ID NO:11 is an oligonucleotide primer used in the PCR amplification of the truncated LTP1 promoter in SEQ ID NO:5 when paired with SEQ ID NO:6.
SEQ ID NO:12 is an oligonucleotide primer specific to the soybean LTP1 gene used in the first nested PCR amplification of the LTP1 promoter when paired with SEQ ID NO:13.
SEQ ID NO:13 is an oligonucleotide primer used in the first nested PCR amplification of the LTP1 promoter when paired with SEQ ID NO:12.
SEQ ID NO:14 is an oligonucleotide primer specific to the soybean LTP1 gene used in the second nested PCR amplification of the LTP1 promoter when paired with SEQ ID NO:15.
SEQ ID NO:15 is an oligonucleotide primer used in the second nested PCR amplification of the LTP1 promoter when paired with SEQ ID NO:14.
SEQ ID NO:16 is the nucleotide sequence of the soybean lipid transfer protein cDNA (LTP1). Nucleotides 1 to 69 are the 5′ untranslated sequence, nucleotides 70 to 72 are the translation initiation codon, nucleotides 70 to 444 are polypeptide coding region, nucleotides 445 to 447 are the termination codon, and nucleotides 448 to 573 are part of the 3′ untranslated sequence.
SEQ ID NO:17 is the 8638 bp sequence of QC267.
SEQ ID NO:18 is the 4794 bp sequence of QC267-1Y.
SEQ ID NO:19 is an oligonucleotide primer used in the diagnostic PCR to check for soybean genomic DNA presence in total RNA or cDNA when paired with SEQ ID NO:20.
SEQ ID NO:20 is an oligonucleotide primer used in the diagnostic PCR to check for soybean genomic DNA presence in total RNA or cDNA when paired with SEQ ID NO:19.
SEQ ID NO:21 is the longer strand sequence of the adaptor supplied in ClonTechTM GenomeWalkerTM kit.
SEQ ID NO:22 is an oligonucleotide primer specific to the soybean LTP1 promoter 5′ end for the amplification of the LTP1 promoter when paired with SEQ ID NO:23. An XmaI restriction site CCCGGG is added for subsequent cloning.
SEQ ID NO:23 is an oligonucleotide primer specific to the soybean LTP1 promoter 3′ end for the amplification of the LTP1 promoter when paired with SEQ ID NO:22. An XmaI restriction site CCCGGG is added for subsequent cloning.
SEQ ID NO:24 is an MPSS tag sequence that is specific to the unique gene PSO330124.
SEQ ID NO:25 is a sense primer used in quantitative PCR analysis of SAMS:ALS transgene.copy numbers.
SEQ ID NO:26 is a FAM labeled fluorescent DNA oligo probe used in quantitative PCR analysis of SAMS:ALS transgene.copy numbers.
SEQ ID NO:27 is an antisense primer used in quantitative PCR analysis of SAMS:ALS transgene.copy numbers.
SEQ ID NO:28 is a sense primer used in quantitative PCR analysis of GM-LTP1:YFP transgene.copy numbers.
SEQ ID NO:29 is a FAM labeled fluorescent DNA oligo probe used in quantitative PCR analysis of GM-LTP1:YFP transgene.copy numbers.
SEQ ID NO:30 is an antisense primer used in quantitative PCR analysis of GM-LTP1:YFP transgene.copy numbers.
SEQ ID NO:31 is a sense primer used as an endogenous control gene primer in quantitative PCR analysis of transgene.copy numbers.
SEQ ID NO:32 is a VIC labeled DNA oligo probe used as an endogenous control gene probe in quantitative PCR analysis of transgene.copy numbers.
SEQ ID NO:33 is an antisense primer used as an endogenous control gene primer in quantitative PCR analysis of transgene.copy numbers.
SEQ ID NO:34 is the recombination site attB1 sequence in the Gateway cloning system (Invitrogen).
SEQ ID NO:35 is the recombination site attB2 sequence in the Gateway cloning system (Invitrogen).
SEQ ID NO:36 is the 125 amino acid long putative PSO330124 translation product LTP1 protein sequence.
SEQ ID NO:37 is the 7499 bp sequence of QC258.
SEQ ID NO:38 is the 2817 bp sequence of pCR8/GW/TOPO.
SEQ ID NO:39 is the 3953 bp sequence of QC267-1.
SEQ ID NO:40 is the 3744 bp sequence of QC267-2.
SEQ ID NO:41 is the 3555 bp sequence of QC267-3.
SEQ ID NO:42 is the 3344 bp sequence of QC267-4.
SEQ ID NO:43 is the 3074 bp sequence of QC267-5.
SEQ ID NO:44 is the 4585 bp sequence of QC267-2Y.
SEQ ID NO:45 is the 4396 bp sequence of QC267-3Y.
SEQ ID NO:46 is the 4185 bp sequence of QC267-4Y.
SEQ ID NO:47 is the 3915 bp sequence of QC267-5Y.
SEQ ID NO:48 is the 5286 bp sequence of QC330.
SEQ ID NO:49 is the 4157 bp sequence of pZSL90.
SEQ ID NO:50 is the 3291 bp sequence of QC299i.
FIG. 1 displays the logarithm of relative quantifications of LTP1 gene expression in 14 different soybean tissues by quantitative RT-PCR. The gene expression profile indicates that the LTP1 gene is highly expressed in flower buds and open flowers.
FIG. 2 displays the LTP1 promoter copy number analysis by Southern hybridization. Also displayed is a schematic of the LTP1 promoter showing relative linear position of a number of restriction sites.
FIG. 3 is a schematic representation of the map of plasmid QC258 (FIG. 3A) and QC267 (FIG. 3B).
FIG. 4 displays a schematic representation of a Gateway cloning ready TA cloning vector pCR8/GW/TOPO (Invitrogen; FIG. 4A) and the vector created by cloning the full length LTP1 promoter into pCR8/GW/TOPO, QC267-1 (FIG. 4B). Also displayed is a schematic representation of a Gateway destination vector QC330 (FIG. 4C), containing a reporter ZS-YELLOW1 N1. The LTP1 promoter fragment is cloned into vector QC330 resulting in the displayed plasmid QC267-1Y (FIG. 4D) containing the full length 1136 bp LTP1 promoter, SEQ ID NO:1. Promoter deletion constructs QC267-2Y, QC267-3Y, QC267-4Y, and QC267-5Y containing the 927, 738, 527, 257 bp truncated LTP1 promoters, respectively, have similar map configurations, the difference being in the length of the promoter.
FIG. 5 is a linear schematic of the LTP1 promoter constructs QC267-1Y, QC267-2Y, QC267-3Y, QC267-4Y, and QC267-5Y. For QC267-1Y, the reporter ZS-YELLOW1 N1 is operably linked to the full-length LTP1 promoter. For the promoter constructs QC267-2Y, QC267-3Y, QC267-4Y, and QC267-5Y, the reporter ZS-YELLOW1 N1 is operably linked to each respective truncation of the LTP1 promoter.
FIG. 6 displays the transient expression of the fluorescent protein reporter gene ZS-YELLOW1 N1 in the cotyledons of germinating soybean seeds. The reporter gene is driven by the LTP1 promoter in the stable transformation construct QC267, or driven by the LTP1 promoter or the progressively truncated LTP1 promoters in the transient expression constructs QC267-1Y to QC267-5Y. Additionally, displayed are the results of QC299i, which represents the negative control (no promoter present) and pZSL90, which represents the positive control (constitutive promoter SCP1 drives the reporter gene).
FIG. 7 displays the stable expression of the fluorescent protein reporter gene ZS-YELLOW1 N1 in the floral tissues of transgenic soybean plants containing a single copy of the transgene construct QC267.



DETAILED DESCRIPTION OF THE INVENTION
The disclosure of all patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants, reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.
In the context of this disclosure, a number of terms shall be utilized.
The term “promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Functional RNA includes, but is not limited to, transfer RNA (tRNA) and ribosomal RNA (rRNA). Numerous examples of promoters may be found in the compilation by Okamuro and Goldberg (Biochemistry of Plants 15:1-82 (1989)). 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 which 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 which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that, since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
An “intron” is an intervening sequence in a gene that is transcribed into RNA and then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences. An “exon” is a portion of the sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, and is not necessarily a part of the sequence that encodes the final gene product.
A “flower” is a complex structure consisting of pedicel, sepal, petal, stamen, and carpel. A stamen comprises an anther, pollen and filament. A carpel comprises a stigma, style and ovary. An ovary comprises an ovule, embryo sac, and egg cell. Soybean pods develop from the pistil. It is likely that a gene expressed in the pistil of a flower continues to express in early pod. A “flower cell” is a cell from any one of these structures. Flower promoters in general include promoters that direct gene expression in any of the above tissues or cell types.
The term “flower crop” or “flowering plants” are plants that produce flowers that are marketable within the floriculture industry. Flower crops include both cut flowers and potted flowering plants. Cut flowers are plants that generate flowers that can be cut from the plant and can be used in fresh flower arrangements. Flower crops include roses, carnations, Gerberas, Chrysanthemums, tulips, Gladiolis, Alstroemerias, Anthuriums, lisianthuses, larkspurs, irises, orchids, snapdragons, African violets, azaleas, in addition to other less popular flower crops.
The terms “flower-specific promoter” or “flower-preferred promoter” may be used interchangeably herein and refer to promoters active in flower, with promoter activity being significantly higher in flower tissue versus non-flower tissue. “Preferentially initiates transcription” when describing a particular cell type, refers to the relative level of transcription in that particular cell type as opposed to other cell types. The described LTP1 promoters are promoters that preferentially initiate transcription in flower cells. Preferably, the promoter activity in terms of expression levels of an operably linked sequence are more than ten-fold higher in flower tissue than in non-flower tissue. More preferably, the promoter activity is present in flower tissue while undetectable in non-flower tissue.
As used herein, an “LTP1 promoter” refers to one type of flower-specific promoter. The native LTP1 promoter (or full-length native LTP1 promoter) is the native promoter of the putative soybean LTP1 polypeptide, which is a protein with significant homology to lipid transfer proteins from different plant species. The “LTP1 promoter”, as used herein, also refers to fragments of the full-length native promoter that retain significant promoter activity. For example, an LTP1 promoter of the present invention can be the full-length promoter (SEQ ID NO:1) or a promoter-functioning fragment thereof, which includes, among others, the polynucleotides of SEQ ID NOs: 2, 3, 4 and 5. An LTP1 promoter also includes variants that are substantially similar and functionally equivalent to any portion of the nucleotide sequence set forth in SEQ ID NOs: 1, 2, 3, 4, or 5, or sequences there between.
An “isolated nucleic acid fragment” or “isolated polynucleotide” refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated polynucleotide in the form of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5′-monophosphate form) are referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
A “heterologous nucleic acid fragment” or “heterologous nucleotide sequence” refers to a nucleotide sequence that is not naturally occurring with the plant promoter sequence of the invention. While this nucleotide sequence is heterologous to the promoter sequence, it may be homologous, or native, or heterologous, or foreign, to the plant host. However, it is recognized that the instant promoters may be used with their native coding sequences to increase or decrease expression resulting in a change in phenotype in the transformed seed.
The terms “fragment (or variant) that is functionally equivalent” and “functionally equivalent fragment (or variant)” are used interchangeably herein. These terms refer to a portion or subsequence or variant of the promoter sequence of the present invention in which the ability to initiate transcription or drive gene expression (such as to produce a certain phenotype) is retained. Fragments and variants can be obtained via methods such as site-directed mutagenesis and synthetic construction. As with the provided promoter sequences described herein, the contemplated fragments and variants operate to promote the flower-preferred expression of an operably linked heterologous nucleic acid sequence, forming a recombinant DNA construct (also, a chimeric gene). For example, the fragment or variant can be used in the design of recombinant DNA constructs to produce the desired phenotype in a transformed plant. Recombinant DNA constructs can be designed for use in co-suppression or antisense by linking a promoter fragment or variant thereof in the appropriate orientation relative to a heterologous nucleotide sequence.
In some aspects of the present invention, the promoter fragments can comprise at least about 20 contiguous nucleotides, or at least about 50 contiguous nucleotides, or at least about 75 contiguous nucleotides, or at least about 100 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In another aspect, a promoter fragment is the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. The nucleotides of such fragments will usually comprise the TATA recognition sequence of the particular promoter sequence. Such fragments may be obtained by use of restriction enzymes to cleave the naturally occurring promoter nucleotide sequences disclosed herein, by synthesizing a nucleotide sequence from the naturally occurring promoter DNA sequence, or may be obtained through the use of PCR technology. See particularly, Mullis et al., Methods Enzymol. 155:335-350 (1987), and Higuchi, R. In PCR Technology: Principles and Applications for DNA Amplifications; Erlich, H. A., Ed.; Stockton Press Inc.: New York, 1989.
The terms “substantially similar” and “corresponding substantially” as used herein refer to nucleic acid sequences, particularly promoter sequences, wherein changes in one or more nucleotide bases do not substantially alter the ability of the promoter to initiate transcription or drive gene expression or produce a certain phenotype. These terms also refer to modifications, including deletions and variants, of the nucleic acid sequences of the instant invention by way of deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting promoter relative to the initial, unmodified promoter. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
In one example of “substantially similar”, substantially similar nucleic acid sequences include those that are also defined by their ability to hybridize to the disclosed nucleic acid sequences, or portions thereof. Substantially similar nucleic acid sequences include those sequences that hybridize, under moderately stringent conditions (for example, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences reported herein and which are functionally equivalent to the promoter of the invention. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds.; In Nucleic Acid Hybridisation; IRL Press: Oxford, U.K., 1985). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes partially determine stringency conditions. One set of conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. Another set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS is increased to 60° C. Another set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.
In some examples, substantially similar nucleic acid sequences are those sequences that are 80% identical to the nucleic acid sequences reported herein or which are 80% identical to any portion of the nucleotide sequences reported herein. In some instances, nucleic acid sequences are those that are 90% identical to the nucleic acid sequences reported herein, or 90% identical to any portion of the nucleotide sequences reported herein. In some examples, nucleic acid sequences are those that are 95% identical to the nucleic acid sequences reported herein, or are 95% identical to any portion of the nucleotide sequences reported herein. It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying related polynucleotide sequences. Useful examples of percent identities are those listed above, or also any integer percentage from 80% to 100%, such as, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. 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 nucleic acid sequence for improved expression in a host cell, it is desirable to design the nucleic acid sequence such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
Sequence alignments and percent similarity calculations may be determined using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences are performed using the Clustal method of alignment (Higgins and Sharp, CABIOS 5:151-153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are GAP PENALTY=10, GAP LENGTH PENALTY=10, KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. A “substantial portion” of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1993)) and Gapped Blast (Altschul, S. F. et al., Nucleic Acids Res. 25:3389-3402 (1997)).
The term “gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” or “recombinant expression construct”, which are used interchangeably, refers to 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, and arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, 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.
“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, and are not limited to, promoters, enhancers, translation leader sequences, introns, and polyadenylation recognition sequences.
The “translation leader sequence” refers to 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 have been described (Turner, R. and Foster, G. D., Molecular Biotechnology 3:225 (1995)).
The “3′ non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized as affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al., Plant Cell 1:671-680 (1989).
“RNA transcript” refers to a product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When an RNA transcript is a perfect complementary copy of a DNA sequence, it is referred to as a primary transcript, or it may be a RNA sequence derived from post transcriptional processing of a primary transcript and is referred to as a mature RNA. “Messenger RNA” (“mRNA”) refers to RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a DNA that is complementary to and synthesized from an mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes mRNA and so can be translated into protein within a cell or in vitro. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks expression or transcript accumulation of a target gene. 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-coding sequence, introns, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated yet has an effect on cellular processes.
The term “operably linked” refers to the association of nucleic acid sequences on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a heterologous nucleotide sequence, e.g., a coding sequence, when it is capable of affecting the expression of that heterologous nucleotide sequence (i.e., for example, the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to promoter sequences in sense or antisense orientation.
The terms “initiate transcription”, “initiate expression”, “drive transcription”, and “drive expression” are used interchangeably herein and all refer to the primary function of a promoter. As detailed throughout this disclosure, a promoter is a non-coding genomic DNA sequence, usually upstream (5′) to the relevant coding sequence, and its primary function is to act as a binding site for RNA polymerase and initiate transcription by the RNA polymerase. Additionally, there is “expression” of RNA, including functional RNA, or the expression of polypeptide for operably linked encoding nucleotide sequences, as the transcribed RNA ultimately is translated into the corresponding polypeptide.
The term “expression”, as used herein, refers to the production of a functional end-product, e.g., an mRNA or a protein (precursor or mature).
The term “recombinant DNA construct” or “recombinant expression construct” is used interchangeably and refers to a discrete polynucleotide into which a nucleic acid sequence or fragment can be moved. Preferably, it is a plasmid vector or a fragment thereof comprising the promoters of the present invention. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the recombinant DNA construct. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by PCR and Southern analysis of DNA, RT-PCR and Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.
Expression or overexpression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression or transcript accumulation of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020). The mechanism of co-suppression may be at the DNA level (such as DNA methylation), at the transcriptional level, or at post-transcriptional level.
Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. Recent work has described the use of “hairpin” structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential “stem-loop” structure for the expressed RNA (PCT Publication Nos. WO99/53050 and WO02/00904). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or “silencing”, of proximal mRNA encoding sequences (PCT Publication No. WO98/36083). Neither of these co-suppressing phenomena has been elucidated mechanistically at the molecular level, although genetic evidence has been obtained that may lead to the identification of potential components (Elmayan et al., Plant Cell 10:1747-1757 (1998)).
As stated herein, “suppression” refers to a reduction of the level of enzyme activity or protein functionality (e.g., a phenotype associated with a protein) detectable in a transgenic plant when compared to the level of enzyme activity or protein functionality detectable in a non-transgenic or wild type plant with the native enzyme or protein. The level of enzyme activity in a plant with the native enzyme is referred to herein as “wild type” activity. The level of protein functionality in a plant with the native protein is referred to herein as “wild type” functionality. The term “suppression” includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. This reduction may be due to a decrease in translation of the native mRNA into an active enzyme or functional protein. It may also be due to the transcription of the native DNA into decreased amounts of mRNA and/or to rapid degradation of the native mRNA. The term “native enzyme” refers to an enzyme that is produced naturally in a non-transgenic or wild type cell. The terms “non-transgenic” and “wild type” are used interchangeably herein.
“Altering expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ significantly from the amount of the gene product(s) produced by the corresponding wild-type organisms (i.e., expression is increased or decreased).
“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Thus, a “transgenic plant cell” as used herein refers to a plant cell containing the transformed nucleic acid fragments. The preferred method of soybean cell transformation is the use of particle-accelerated or “gene gun” transformation technology (Klein, T., Nature (London) 327:70-73 (1987); U.S. Pat. No. 4,945,050).
“Transient expression” refers to the temporary expression of often reporter genes such as β-glucuronidase (GUS), fluorescent protein genes GFP, ZS-YELLOW1 N1, AM-CYAN1, DS-RED in selected certain cell types of the host organism in which the transgenic gene is introduced temporally by a transformation method. The transformed material of the host organism is subsequently discarded after the transient gene expression assay.
A “marketable flower trait” is a characteristic or phenotype of the flower of a plant such as the color, scent or morphology of a flower. The marketable flower trait is a characteristic of a flower that is of high regard to a flower crop consumer in deciding whether to purchase the flower crop.
The phrase “genes involved in anthocyanin biosynthesis” refers to genes that encode proteins that play a role in converting metabolic precursors into the one of a number of anthocyanins. Examples of genes involved in the biosynthesis of anthocyanin are dyhydroflavonol 4-reductase, flavonoid 3,5-hydroxylase, chalcone synthase, chalcone isomerase, flavonoid 3-hydroxylase, anthocyanin synthase, and UDP-glucose 3-O-flavonoid glucosyl transferase (see, e.g., Mori et al., Plant Cell Reports 22:415-421 (2004)).
The phrase “genes involved in the biosynthesis of fragrant fatty acid derivatives” refers to genes that encode proteins that play a role in manipulating the biosynthesis of fragrant fatty acid derivatives such as terpenoids, phenylpropanoids, and benzenoids in flowers (see, e.g., Tanaka et al., Plant Cell, Tissue and Organ Culture 80:1-24 (2005)). Examples of such genes include S-linalool synthase, acetyl CoA:benzylalcohol acetyltransferase, benzyl CoA:benzylalcohol benzoyl transferase, S-adenosyl-L-methionine:benzoic acid carboxyl methyl transferase (BAMT), mycrene synthases, (E)-β-ocimene synthase, orcinol O-methyltransferase, and limonene synthases (see, e.g., Tanaka et al., supra).
The term “flower homeotic genes” or “flower morphology modifying genes” refers to genes that are involved in pathways associated with flower morphology. A modification of flower morphology can lead to a novel form of the respective flower that can enhance its value in the flower crop marketplace. Morphology can include the size, shape, or petal pattern of a flower. Examples of flower homeotic genes include genes involved in cell-fate determination (in ABC combinatorial model of gene expression), including AGAMOUS, which determines carpel fate in the central whorl, APETALA3, which determines the sepal fate in the outer whorl, and PISTILLATA, which determines petal development in the second whorl (Espinosa-Soto et al., Plant Cell 16:2923-2939 (2004)).
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J. et al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook et al., 1989”) or Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; In Current Protocols in Molecular Biology; John Wiley and Sons: New York, 1990 (hereinafter “Ausubel et al., 1990”).
“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis of large quantities of specific DNA segments consisting of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double stranded DNA is heat denatured, the two primers complementary to the 3′ boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps comprises a cycle.
Embodiments of the present invention include isolated polynucleotides comprising a nucleotide sequence that is a promoter. In some instances the nucleotide sequence includes one or more of the following:

    
    
        a) the sequence set forth in SEQ ID NO:1 or a full-length complement thereof;
        b) a nucleotide sequence comprising a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; or
        c) a nucleotide sequence comprising a sequence having at least 90% sequence identity, based on the BLASTN method of alignment, when compared to the sequence set forth in SEQ ID NO:1.

The nucleotide sequences of the present invention can be referred to as a promoter or as having promoter-like activity. In some embodiments the nucleotide sequence is a promoter that preferentially initiates transcription in a plant flower cell. Such promoter is referred to as a flower-specific promoter. Preferably the promoter of the present invention is the soybean “LTP1” promoter.

    
    


In a preferred embodiment, the promoter comprises the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. The present invention also includes nucleic acid fragments, variants, and complements of the aforementioned nucleotide sequences or promoters, provided that they are substantially similar and functionally equivalent to the nucleotide sequence set forth in these nucleotide sequences. A nucleic acid fragment or variant that is functionally equivalent to the present LTP1 promoter is any nucleic acid fragment or variant that is capable of initiating the expression, preferably initiating flower-specific expression, of a coding sequence or functional RNA in a similar manner to the LTP1 promoter. The expression patterns of LTP1 gene and its promoter are set forth in Examples 1, 7, and 8. In one example, the expression pattern of a LTP1 promoter fragment or variant will have expression patterns similar to that of the LTP1 promoter.
In some aspects, a recombinant DNA construct can be formed in part by operably linking at least one of the promoters of the present invention to any heterologous nucleotide sequence. The heterologous nucleotide sequence can be expressed in a cell as either a functional RNA or a polypeptide. The cell for expression includes a plant or bacterial cell, preferably a plant cell. The recombinant DNA construct preferably includes the LTP1 promoter. The recombinant DNA construct preferably includes a heterologous nucleotide sequence that encodes a protein that plays a role in flower color formation, fragrance production, or shape/morphology development of the flower. The color of a flower can be altered transgenically by expressing genes involved in betalain, carotenoid, or flavanoid biosynthesis. In regard to genes involved in the biosynthesis of anthocyanin, dyhydroflavonol 4-reductase, flavonoid 3,5-hydroxylase, chalcone synthase, chalcone isomerase, flavonoid 3-hydroxylase, anthocyanin synthase, and UDP-glucose 3-O-flavonoid glucosyl transferase are some examples. The scent of a flower can be altered transgenically by expressing genes that manipulate the biosynthesis of fragrant fatty acid derivatives such as terpenoids, phenylpropanoids, and benzenoids in flowers. Some embodiments of the invention include a heterologous nucleotide sequence that is selected from S-linalool synthase, acetyl CoA:benzylalcohol acetyltransferase, benzyl CoA:benzylalcohol benzoyl transferase, S-adenosyl-L-methionine:benzoic acid carboxyl methyl transferase, mycrene synthases, (E)-β-ocimene synthase, orcinol O-methyltransferase, or limonene synthases. Flower structures/morphologies can be altered transgenically by expressing flower homeotic genes to create novel ornamental varieties. Some embodiments of the invention include a heterologous nucleotide sequence that is selected from genes such as, for example, AGAMOUS, APETALA3, and PISTILLATA.
It is recognized that the instant promoters may be used with their native coding sequences to increase or decrease expression in flower tissue. The selection of the heterologous nucleic acid fragment depends upon the desired application or phenotype to be achieved. The various nucleic acid sequences can be manipulated so as to provide for the nucleic acid sequences in the proper orientation.
Plasmid vectors comprising the instant recombinant DNA construct can be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host cells. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the recombinant DNA construct.
The described polynucleotide embodiments encompass isolated or substantially purified nucleic acid compositions. An “isolated” or “purified” nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An “isolated” nucleic acid is essentially free of sequences (preferably protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
In another embodiment, the present invention includes host cells comprising either the recombinant DNA constructs or isolated polynucleotides of the present invention. Examples of the host cells of the present invention include, and are not limited to, yeast, bacteria, and plants, including flower crops such as, e.g., rose, carnation, Gerbera, Chrysanthemum, tulip, Gladioli, Alstroemeria, Anthurium, lisianthus, larkspur, irises, orchid, snapdragon, African violet, or azalea. Preferably, the host cells are plant cells, and more preferably, flower crop cells, and more preferably, Gerbera, rose, carnation, Chrysanthemum, or tulip cells.
Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants have been published, among others, for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653-657 (1996); McKently et al., Plant Cell Rep. 14:699-703 (1995)); papaya (Ling et al., Bio/technology 9:752-758 (1991)); and pea (Grant et al., Plant Cell Rep. 15:254-258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C. A., Mol. Biotechnol. 16:53-65 (2000). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F., Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT Publication No. WO 92/17598), electroporation (Chowrira et al., Mol. Biotechnol. 3:17-23 (1995); Christou et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)), microinjection (Neuhaus et al., Physiol. Plant. 79:213-217 (1990)), or particle bombardment (McCabe et al., Biotechnology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).
In another embodiment, the present invention includes transgenic plants comprising the recombinant DNA constructs provided herein. The transgenic plants are selected from, for example, one of a number of various flower crops including roses, carnations, Gerberas, Chrysanthemums, tulips, Gladiolis, Alstroemerias, Anthuriums, lisianthuses, larkspurs, irises, orchids, snapdragons, African violets, azaleas, in addition to other less popular flower crops.
In some embodiments of the invention, there are provided transgenic seeds produced by the transgenic plants provided. Such seeds are able to produce another generation of transgenic plants.
There are a variety of methods for the regeneration of plants from plant tissues. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, Eds.; In Methods for Plant Molecular Biology; Academic Press, Inc.: San Diego, Calif., 1988). This regeneration and growth process typically includes the steps of selection of transformed cells and culturing of those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. 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. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
In addition to the above discussed procedures, there are generally available standard resource materials that describe specific conditions and procedures for the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, and the like), generation of recombinant DNA fragments and recombinant expression constructs, and the screening and isolating of clones (see, for example, Sambrook et al., 1989; Maliga et al., In Methods in Plant Molecular Biology; Cold Spring Harbor Press, 1995; Birren et al., In Genome Analysis: Detecting Genes, 1; Cold Spring Harbor New York, 1998; Birren et al., In Genome Analysis: Analyzing DNA, 2; Cold Spring Harbor: New York, 1998; Clark, Ed., In Plant Molecular Biology: A Laboratory Manual; Springer: New York, 1997).
The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression of the chimeric genes (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)). Thus, multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by northern analysis of mRNA expression, western analysis of protein expression, or phenotypic analysis. Also of interest are seeds obtained from transformed plants displaying the desired expression profile.
The level of activity of the LTP1 promoter in flowers is in some cases comparable to that of many known strong promoters such as the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)), the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)), the Arabidopsis ubiquitin extension protein promoters (Callis et al., J. Biol. Chem. 265(21):12486-12493 (1990)), a tomato ubiquitin gene promoter (Rollfinke et al., Gene 211:267-276 (1998)), a soybean heat shock protein promoter (Raschke et al., J. Mol. Biol. 199(4):549-557 (1988)), and a maize H3 histone gene promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998)).
In some embodiments, the promoters of the present invention are useful when flower-specific expression of a target heterologous nucleic acid fragment is required. In addition, while the promoters of the present invention are most active in developing flower buds and open flowers (See FIG. 1), they still have activity in developing seeds, although the activity is approximately ten times less. Thus, the promoters can be used for gene expression or gene silencing in flowers, especially when gene expression or gene silencing is desired predominantly in flowers along with a lower degree in developing seeds.
In some embodiments, the promoters of the present invention are to construct recombinant DNA constructs that can be used to reduce expression of at least one heterologous nucleic acid sequence in a plant cell. To accomplish this, a recombinant DNA construct can be constructed by linking the heterologous nucleic acid sequence to a promoter of the present invention. (See U.S. Pat. No. 5,231,020 and PCT Publication Nos. WO99/53050, WO02/00904, and WO98/36083 for methodology to block plant gene expression via cosuppression.) Alternatively, recombinant DNA constructs designed to express antisense RNA for a heterologous nucleic acid fragment can be constructed by linking the fragment in reverse orientation to a promoter of the present invention. (See U.S. Pat. No. 5,107,065 for methodology to block plant gene expression via antisense RNA.) Either the cosuppression or antisense chimeric gene can be introduced into plants via transformation. Transformants, wherein expression of the heterologous nucleic acid sequence is decreased or eliminated, are then selected.
There are embodiments of the present invention that include promoters of the present invention being utilized for methods of altering (increasing or decreasing) the expression of at least one heterologous nucleic acid sequence in a plant cell which comprises: transforming a plant cell with a recombinant DNA expression construct described herein; growing fertile mature plants from the transformed plant cell; and selecting plants containing a transformed plant cell wherein the expression of the heterologous nucleotide sequence is altered (increased or decreased).
Transformation and selection can be accomplished using methods well-known to those skilled in the art including, but not limited to, the methods described herein.
There are provided some embodiments that include methods of expressing a coding sequence in a plant that is a flower crop comprising: introducing a recombinant DNA construct disclosed herein into the plant; growing the plant; and selecting a plant displaying expression of the coding sequence; wherein the nucleotide sequence comprises: a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1 or a full-length complement thereof; a nucleotide sequence comprising a fragment of the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or in alternative embodiments, the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; or a nucleotide sequence comprising a sequence having at least 90% sequence identity, based on the BLASTN method of alignment, when compared to the sequence set forth in SEQ ID NO:1; wherein said nucleotide sequence initiates transcription in a flower cell of the plant.
Furthermore, some embodiments of the present invention include methods of transgenically altering a marketable flower trait of a flowering plant, comprising: introducing a recombinant DNA construct disclosed herein into the flowering plant; growing a fertile, mature flowering plant resulting from the introducing step; and selecting a flowering plant expressing the heterologous nucleotide sequence in flower tissue based on the altered marketable flower trait.
As further described in the Examples below, the promoter activity of the soybean genomic DNA fragment upstream of the LTP1 protein coding sequence SEQ ID NO:1 was assessed by linking the fragment to a yellow fluorescence reporter gene, ZS-YELLOW1 N1 (YFP) (Matz et al., Nat. Biotechnol. 17:969-973 (1999)), transforming the promoter::YFP expression cassette into soybean, and analyzing YFP expression in various cell types of the transgenic plants (see Example 7 and 8). All parts of the transgenic plants were analyzed and YFP expression was predominantly detected in flowers. These results indicated that the nucleic acid fragment contained flower-preferred promoter.
Some embodiments of the present invention provide recombinant DNA constructs comprising at least one isopentenyl transferase nucleic acid sequence operably linked to a provide promoter, preferably a LTP1 promoter. The isopentenyl transferase plays a key step in the biosynthesis of plant cytokinin (Kakimoto, J. Plant Res. 116:233-239 (2003)). Elevated levels of cytokinin in plant cells might help to delay floral senescence and abortion which may present a potential way to improve crop yields (Chang et al., Plant Physiol. 132:2174-2183 (2003); Young et al., Plant J. 38:910-922 (2004)).
Utilities for Flower-Specific Promoters
The color, scent or morphology of a flower represents marketable flower traits, or characteristics/phenotypes of a flower that consumers, particularly floriculturalists, consider when determining which flowers are desirable and will be purchased. Hence, it would be beneficial to be able to alter these characteristics in order to satisfy the desires of consumers. Transgenic technologies can be implemented in order to achieve such results.
The phenotype of a flower can be altered transgenically by expressing genes, preferably in flower tissue, that play a role in color formation, fragrance production, or shape/morphology development of the flower. This type of alteration is particularly useful in the floriculture industry, and particularly useful for flowering plants.
The color of a flower is mainly the result of three types of pigment, flavanoids, carotenoids, and betalains. The flavanoids are the most common of the three and they contribute to colors ranging from yellow to red to blue, with anthocyanins being the major flavanoid. Carotenoids are C-40 tetraterpenoids that contribute to the majority of yellow hues and contribute to orange/red, bronze and brown colors, e.g., that seen in roses and chrysanthemums. Betalains are the least abundant and contribute to various hues of ivory, yellow, orange, red and violet. The color of a flower can be altered transgenically by expressing genes involved in, e.g., betalain, carotenoid, or flavanoid biosynthesis. In one example, the color of a flower can be altered transgenically by expressing genes involved in the biosynthesis of anthocyanin, for example, dyhydroflavonol 4-reductase, flavonoid 3,5-hydroxylase, chalcone synthase, chalcone isomerase, flavonoid 3-hydroxylase, anthocyanin synthase, and UDP-glucose 3-O-flavonoid glucosyl transferase. In some aspects of the invention, the gene involved in anthocyanin biosynthesis is the flavonoid 3,5-hydroxylase gene (see, e.g., Mori et al., Plant Cell Reports 22:415-421 (2004)). This type of alteration is particularly useful in the floriculture industry, providing novel flower colors in flower crops.
In addition to color, the scent of a flower can be altered transgenically by expressing genes that manipulate the biosynthesis of fragrant fatty acid derivatives such as terpenoids, phenylpropanoids, and benzenoids in flowers (see, e.g., Tanaka et al., Plant Cell, Tissue and Organ Culture 80:1-24 (2005)). Genes involved in the biosynthesis of fragrant fatty acid derivatives can be operably linked to the flower-specific promoters presently described for preferential expression in flower tissue. The preferential expression in flower tissue can be utilized to generate new and desirable fragrances to enhance the demand for the underlying cut flower. A number of known genes that are involved in the biosynthesis of floral scents are described below. A strong sweet scent can be generated in a flower by introducing or upregulating expression of S-linalool synthase, which was earlier isolated from Clarkia breweri. Two genes that are responsible for the production of benzylacetate and benzylbenzoate are acetyl CoA:benzylalcohol acetyltransferase and benzyl CoA:benzylalcohol benzoyl transferase, respectively. These transferases were also reported to have been isolated from C. breweri. A phenylpropanoid floral scent, methylbenzoate, is synthesized in part by S-adenosyl-L-methionine:benzoic acid carboxyl methyl transferase (BAMT), which catalyzes the final step in the biosynthesis of methyl benzoate. BAMT is known to have a significant role in the emission of methyl benzoate in snapdragon flowers. Two monoterpenes, mycrene and (E)-β-ocimene, from snapdragon are known to be synthesized in part by the terpene synthases: mycrene synthases and (E)-β-ocimene synthases. Other genes involved in biosynthesis of floral scents have been reported and are being newly discovered, many of which are isolated from rose. Some genes involved in scent production in the rose include orcinol O-methyltransferase, for synthesis of S-adenosylmethionine, and limonene synthases (see, e.g., Tanaka et al., supra).
Flower structures/morphologies can be altered transgenically by expressing flower homeotic genes to create novel ornamental varieties. The flower homeotic genes that are determinative of flower morphology include genes such as AGAMOUS, APETALA3, PISTILLATA, and others that are known and/or are being elucidated (see, e.g., Espinosa-Soto et al., Plant Cell 16:2923-2939 (2004)).
EXAMPLES
Aspects of the present invention are exemplified in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
In the discussion below, parts and percentages are by weight and degrees are Celsius, unless otherwise stated. Sequences of promoters, cDNA, adaptors, and primers listed herein are in the 5′ to 3′ orientation unless described otherwise. Techniques in molecular biology were typically performed as described in Ausubel et al., 1990 or Sambrook et al., 1989.
Example 1
Lynx MPSS Profiling of Soybean Genes Preferably Expressed in Flowers
Soybean expression sequence tags (ESTs) were generated by sequencing randomly selected clones from cDNA libraries constructed from different soybean tissues. Multiple EST sequences may have different lengths representing different regions of the same soybean gene. For those EST sequences representing the same gene that are found more frequently in a flower-specific cDNA library, there is a possibility that the representative gene could be a flower preferred gene candidate. Multiple EST sequences representing the same soybean gene were compiled electronically based on their overlapping sequence homology into a full length sequence representing a unique gene. These assembled, unique gene sequences were cumulatively collected and the information was stored in a searchable database. Flower specific candidate genes were identified by searching this database to find gene sequences that are frequently found in flower libraries but are rarely found in other tissue libraries, or not found in other tissue libraries.
One unique gene, PSO330124, was identified in the search as a flower specific gene candidate since all of the ESTs representing PSO330124 were found only in flower tissue. The PSO330124 cDNA sequence (SEQ ID NO:16) and its putative translated protein sequence (SEQ ID NO:36) were used to search National Center for Biotechnology Information (NCBI) databases. PSO330124 was found to be a novel soybean gene having high homology to several lipid transfer protein genes of other species. PSO330124 was subsequently named as GM-LTP1, Glycine max lipid transfer protein 1.
A more sensitive gene expression profiling methodology MPSS (Mass Parallel Signature Sequence) transcript profiling technique (Brenner et al., Proc Natl Acad Sci USA 97:1665-70 (2000)) was used to confirm PSO330124 as a flower specific gene. The MPSS technology involves the generation of 17 base signature tags from mRNA samples that have been reverse transcribed from poly A+ RNA isolated using standard molecular biology techniques (Sambrook et al., 1989). The tags are simultaneously sequenced and assigned to genes or ESTs. The abundance of these tags is given a number value that is normalized to parts per million (PPM) which then allows the tag expression, or tag abundance, to be compared across different tissues. Thus, the MPSS platform can be used to determine the expression pattern of a particular gene and its expression levels in different tissues.
MPSS gene expression profiles were generated from different soybean tissues over time, and the profiles were accumulated in a searchable database. PSO330124 cDNA sequence was used to search the MPSS database to identify a MPSS tag that was identical to a 17 base pair region in the 3′ end of the PSO330124 cDNA sequence: GATCCCACTAGGGAGTA (SEQ ID NO:24). The identified MPSS tag was then used to search the MPSS database to reveal its abundance in different tissues. As illustrated in Table 1, the PSO330124 gene was confirmed to be highly abundant in flowers and pods, a desired expression profile for its promoter to be able to express genes in flowers and in early developing pods.







TABLE 1







Lynx MPSS Expression Profiles of the PSO330124 Gene















TAG_NAME
TAG_SEQ
Anther
Flower
Leaf
Pod
Root
Seed
Stem





PSO330124
GATCCCACTAGGGAGTA
0
4319
2
2619
0
613
4









Example 2
Quantitative RT-PCR Profiles of LTP1 Gene Expression in Soybean
The MPSS profile of the LTP1 gene, PSO330124, was confirmed and extended by analyzing 14 different soybean tissues using the relative quantitative RT-PCR (qRT-PCR) technique with a 7500 real time PCR system (Applied Biosystems, Foster City, Calif.).
Fourteen soybean tissues (somatic embryo, somatic embryo grown one week on charcoal plate, leaf, leaf petiole, root, flower bud, open flower, R3 pod, R4 seed, R4 pod coat, R5 seed, R5 pod coat, R6 seed, R6 pod coat) were collected from cultivar ‘Jack’ and flash frozen in liquid nitrogen. The seed and pod development stages were defined according to descriptions in Fehr and Caviness, IWSRBC 80:1-12 (1977). Total RNA was extracted with Trizol reagents (Invitrogen, Carlsbad, Calif.) and treated with DNase I to remove any trace amount of genomic DNA contamination. The first strand cDNA was synthesized with Superscript III reverse transcriptase (Invitrogen).
PCR analysis was performed to confirm that the cDNA was free of genomic DNA. The PCR analysis used the following primers:








SEQ ID NO: 19
GACCAAGACACACTCGTTCATATATC


 


SEQ ID NO: 20
TCTGCTGCTCAATGTTTACAAGGAC






The primers are specific to the 5′UTR intron/exon junction region of a soybean S-adenosylmethionine synthetase gene promoter (WO00/37662). PCR using this primer set amplifies a 967 bp DNA fragment from any soybean genomic DNA template and a 376 bp DNA fragment from the cDNA template. The cDNA aliquots were used in qRT-PCR analysis in which an endogenous soybean ATP sulfurylase gene was used as an internal control and wild type soybean genomic DNA was used as the calibrator for relative quantification.

The qRT-PCR profiling of the LTP1 gene expression confirmed its predominant flower expression and also showed ongoing expression at levels approximately ten fold lower during early pod and seed development (see FIG. 1).
Example 3
Isolation of Soybean LTP1 Promoter
The soybean genomic DNA fragment corresponding to the LTP1 promoter was isolated using a polymerase chain reaction (PCR) based approach called genome walking using the Universal GenomeWalkerTM kit from ClontechTM (Product User Manual No. PT3042-1).
Soybean genomic DNA was digested to completion with DraI, a DNA restriction enzyme that generates DNA fragments having blunt ends according to standard protocols. This process was repeated three times, separately, using either EcoRV, HpaI, and PmlI, each of which generates DNA fragments having blunt ends.
Double strand adaptors supplied in the GenomeWalkerTM kit were added to the blunt ends of the genomic DNA fragments by DNA ligase. Two rounds of PCR were performed to amplify the LTP1 corresponding genomic DNA fragment using two nested primers supplied in the Universal GenomeWalkerTM kit that are specific for the adaptor sequence (AP1 and AP2, for the first and second adaptor primer, respectively), and two LTP1 gene specific primers (GSP1 and GSP2) designed based on the LTP1 5′ coding sequence PSO330124. The oligonucleotide sequences of the four primers are shown below:









SEQ ID NO: 12
(GSP1)
CTTCATGACAAGCAGTGAGCTAGCC


 


SEQ ID NO: 13
(AP1)
GTAATACGACTCACTATAGGGCACG


 


SEQ ID NO: 14
(GSP2)
CCATGGATTTGGAAGAGTTAGAGGATGAAAT




TG


 


SEQ ID NO: 15
(AP2)
CTATAGGGCACGCGTGGTCGAC






The underlined bases in GSP2 primer are the recognition site for the restriction enzyme NcoI. The AP2 primer from the Universal GenomeWalkerTM kit contains a Sa/I restriction site, also underlined. The 3′ end of the adaptor sequence GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT (SEQ ID NO:21) also contains a XmaI recognition site downstream to the corresponding Sa/I restriction site in AP2 primer.

The AP1 and the GSP1 primers were used in the first round PCR using each of the adaptor ligated genomic DNA populations (DraI, EcoRV, HpaI or Pm/I) under conditions defined in the GenomeWalkerTM protocol. Cycle conditions were 94° C. for 4 minutes; 35 cycles of 94° C. for 30 seconds, 60° C. for 1 minute, and 68° C. for 3 minutes; and a final 68° C. for 5 minutes before holding at 4° C. One microliter from each of the first round PCR products was used as templates for the second round PCR with the AP2 and GSP2 primers. Cycle conditions for second round PCR were 94° C. for 4 minutes; 25 cycles of 94° C. for 30 seconds, 60° C. for 1 minute, and 68° C. for 3 minutes; and a final 68° C. for 5 minutes before holding at 4° C. Agarose gels were run to identify specific PCR product with an optimal fragment length. An approximately 1.2 Kb PCR product was detected and subsequently cloned into pCR2.1-TOPO vector by TOPO TA cloning (Invitrogen). Sequencing of the cloned PCR products revealed that its 3′ end matched the 96 bp 5′ end of the LTP1 cDNA sequence, indicating that the PCR product was indeed the corresponding LTP1 genomic DNA fragment. The 1136 bp sequence upstream of the putative LTP1 start codon ATG is herein designated as soybean LTP1 promoter (SEQ ID NO:1).
Example 4
LTP1 Promoter Copy Number Analysis
Southern hybridization analysis was performed to determine whether there were other sequences in the soybean genome with high similarity to the LTP1 promoter. Soybean ‘Jack’ wild type genomic DNA was digested with nine different restriction enzymes (BamHI, Bg/II, DraI, EcoRI, EcoRV, HindIII, MfeI, NdeI, and SpeI), each separately, and distributed in a 0.7% agarose gel by electrophoresis. Each of the digested DNA samples was blotted onto a Nylon membrane and hybridized with digoxigenin (DIG) labeled LTP1 promoter DNA probe according to the standard protocol (Roche Applied Science, Indianapolis, Ind.). The LTP1 promoter probe was labeled by PCR using the DIG DNA labeling kit (Roche Applied Science) with two gene specific primers to make a 1154 bp probe covering the entire 1136 bp LTP1 promoter sequence. The two gene specific primers used were:








SEQ ID NO: 22
ATAATCCCGGGTCCTACTCCTACTCGACAA


 


SEQ ID NO: 23
GAGCTACCCGGGATTTGGAAGAGTTAGAGGATG






Both primers contain an XmaI restriction site CCCGGG, introducing extra base pairs in the LTP1 probe as subsequent cloning sites. These extra base pairs should not affect Southern hybridization results.

A single band was detected in each of five digestions, BamHI, BglII, EcoRI, EcoRV, and NdeI, suggesting that the LTP1 promoter sequence exists in soybean genome as a single copy unique sequence (FIG. 2A). The fact that no band was detected on the Southern blot of the DraI digestion could be explained by presence of multiple DraI restriction sites in the promoter sequence (FIG. 2B), and another DraI restriction site in the LTP1 coding region resulting in DNA fragments too small to be kept on the blot (any band smaller than 1 Kb would run out of the agarose gel under the experiment conditions). Two bands, one strong and one weak, detected in the MfeI digestion could be due to the presence of an MfeI restriction site in the 3′ end region of the LTP1 promoter. The weak band detected in HindIII digestion and the three faint bands detected in SpeI digestion that could be highlighted by over exposure suggested the likelihood of a sequence with low similarity to the LTP1 promoter sequence in soybean genome (FIG. 2A).
Example 5
LTP1:YFP Reporter Constructs and Soybean Transformation
Two oligonucleotide primers were designed to re-amplify the LTP1 promoter with a XmaI restriction site incorporated in each of the primer sequences (underlined in SEQ ID NO:22 and SEQ ID NO:23, respectively) as shown below:








SEQ ID NO: 22
ATAATCCCGGGTCCTACTCCTACTCGACAA


 


SEQ ID NO: 23
GAGCTACCCGGGATTTGGAAGAGTTAGAGGATG






The re-amplified LTP1 promoter fragment was digested with XmaI, gel purified and cloned into the XmaI site of a vector plasmid QC258 (SEQ ID NO:37; FIG. 3A) containing the soybean transformation selectable marker gene SAMS:ALS (S-adenosyl methionine synthetase:acetolactate synthase) and a promoter-less fluorescent reporter gene ZS-YELLOW1 N1 (YFP) to make the reporter construct QC267 (SEQ ID NO:17) with the soybean LTP1 promoter driving the YFP gene expression (FIG. 3B). The 6124 bp DNA fragment containing the linked LTP1:YFP and SAMS:ALS expression cassettes was cut out of QC267 plasmid by AscI digestion, separated from the vector backbone fragment by agarose gel electrophoresis, and purified from the gel using a DNA gel extraction kit (Qiagen, Valencia, Calif.). The purified DNA fragment was used to transform soybean cultivar Jack using the particle gun bombardment method (Klein et al., Nature 327:70-73 (1987); U.S. Pat. No. 4,945,050) to study the LTP1 promoter activity in stably transformed soybean plants.
Soybean somatic embryos from the Jack cultivar were induced as follows. Cotyledons ( ̃3 mm in length) were dissected from surface-sterilized, immature seeds and were cultured for 6-10 weeks under fluorescent light at 26° C. on a Murashige and Skoog media (“MS media”) containing 0.7% agar and supplemented with 10 mg/ml 2,4-dichlorophenoxyacetic acid (2,4-D). Globular stage somatic embryos, which produced secondary embryos, were then excised and placed into flasks containing liquid MS medium supplemented with 2,4-D (10 mg/ml) and cultured in the light on a rotary shaker. After repeated selection for clusters of somatic embryos that multiplied as early, globular staged embryos, the soybean embryogenic suspension cultures were maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. with fluorescent lights on a 16:8 hour day/night schedule. Cultures were subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of the same fresh liquid MS medium.
Soybean embryogenic suspension cultures were then transformed by the method of particle gun bombardment using a DuPont BiolisticTM PDS1000/HE instrument (helium retrofit) (Bio-Rad Laboratories, Hercules, Calif.). To 50 μl of a 60 mg/ml 1.0 mm gold particle suspension were added (in order): 30 μl of 10 ng/μl LTP1:YFP+SAMS:ALS DNA fragment, 20 μl of 0.1 M spermidine, and 25 μl of 5 M CaCl2. The particle preparation was then agitated for 3 minutes, spun in a centrifuge for 10 seconds and the supernatant removed. The DNA-coated particles were then washed once in 400 μl 100% ethanol and resuspended in 45 μl of 100% ethanol. The DNA/particle suspension was sonicated three times for one second each. 5 μl of the DNA-coated gold particles were then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture was placed in an empty 60×15-mm Petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5 to 10 plates of tissue were bombarded. Membrane rupture pressure was set at 1100 psi and the chamber was evacuated to a vacuum of 28 inches mercury. The tissue was placed approximately 3.5 inches away from the retaining screen and bombarded once. Following bombardment, the tissue was divided in half and placed back into liquid media and cultured as described above.
Five to seven days post bombardment, the liquid media was exchanged with fresh media containing 100 ng/ml chlorsulfuron as selection agent. This selective media was refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue was observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue was removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each clonally propagated culture was treated as an independent transformation event and subcultured in the same liquid MS media supplemented with 2,4-D (10 mg/ml) and 100 ng/ml chlorsulfuron selection agent to increase mass. The embryogenic suspension cultures were then transferred to agar, solid MS media plates without 2,4-D supplement to allow somatic embryos to develop. A sample of each event was collected at this stage for PCR and quantitative PCR analysis.
Cotyledon stage somatic embryos were dried-down (by transferring them into an empty small Petri dish that was seated on top of a 10 cm Petri dish to allow slow dry down) to mimic the last stages of soybean seed development. Dried-down embryos were placed on germination solid media, and transgenic soybean plantlets were regenerated. The transgenic plants were then transferred to soil and maintained in growth chambers for seed production.
Genomic DNA was extracted from somatic embryo samples and analyzed by quantitative PCR using the 7500 real time PCR system (Applied Biosystems) with gene-specific primers and 6-carboxyfluorescein (FAM)-labeled fluorescence probes to check copy numbers of both the SAMS:ALS expression cassette and the LTP1:YFP expression cassette. The qPCR analysis was done in duplex reactions with a heat shock protein (HSP) gene as the endogenous control and a transgenic DNA sample with a known single copy of SAMS:ALS or YFP transgene as the calibrator using the relative quantification methodology. The endogenous control HSP probe was labeled with VIC (Applera Corporation, Norwalk, Conn.) and the target gene SAMS or YFP probe was labeled with FAM for the simultaneous detection of both fluorescent probes in the same duplex reactions. The primers and probes used in the qPCR analysis are listed in Table 2 below.







TABLE 2







Primers and Probes used in qPCR Analysis









SEQ




ID




NO:
Description
Sequence












25
SAMS forward primer
GGAAGAAGAGAATCGGGTGGTT


 


26
FAM labeled SAMS probe
ATTGTGTTGTGTGGCATGGTTAT


 


27
SAMS reverse primer
GGCTTGTTGTGCAGTTTTTGAAG


 


28
YFP forward primer
AACGGCCACAAGTTCGTGAT


 


29
FAM labeled YFP probe
ACCGGCGAGGGCATCGGCTA


 


30
YFP reverse primer
CTTCAAGGGCAAGCAGACCA


 


31
HSP forward primer
CAAACTTGACAAAGCCACAACTCT


 


32
VIC labeled HSP probe
CTCTCATCTCATATAAATAC


 


33
HSP reverse primer
GGAGAAATTGGTGTCGTGGAA









FAM labeled DNA oligo probes and VIC labeled oligo probes were obtained from Sigma Genosys (The Woodlands, Tex.).

Only transgenic soybean events containing 1 or 2 copies of both the SAMS:ALS expression cassette and the LTP1:YFP expression cassette were selected for further gene expression evaluation and seed production (see Table 3). Events negative for YFP qPCR or with more than 2 copies for the SAMS qPCR were terminated. YFP expressions in flowers as described in EXAMPLE 8 are also recorded in the same table.







TABLE 3







Relative Transgene Copy Numbers and YFP Expression













SAMS
YFP
YFP



Event ID
qPCR
qPCR
Expression







4708.1.1
1.07
3.00
+



4708.3.1
0.94
1.25
+



4708.3.2
1.01
1.16
+



4708.3.3
1.13
1.01
−



4708.3.4
0.90
1.69
+



4708.4.1
1.22
1.23
+



4708.4.2
1.13
1.37
−



4708.5.1
3.28
1.33
Terminated



4708.5.2
1.27
0.00
Terminated



4708.5.3
1.74
2.06
+



4708.5.4
0.98
1.09
+



4708.5.5
2.98
2.26
Terminated



4708.6.1
1.47
1.24
+



4708.8.1
0.99
1.68
+



4708.8.2
1.13
0.00
Terminated



4708.8.3
1.02
1.06
+



4708.8.4
3.68
3.09
Terminated



4708.8.5
0.99
0.93
−










Example 6
Construction of LTP1 Promoter Deletion Constructs
To define the transcriptional elements controlling the LTP1 promoter activity, the 1136 bp full length and four 5′ unidirectional deletion fragments (SEQ ID NO:1 of 1136 bp, SEQ ID NO:2 of 927 bp, SEQ ID NO:3 of 738 bp, SEQ ID NO:4 of 527 bp, SEQ ID NO:5 of 257 bp) were made by utilizing PCR amplification and the full length soybean LTP1 promoter contained in the original construct QC267 (FIG. 3B). The same antisense primer CAATTTCATCCTCTAACTCTTCCAAATCC (SEQ ID NO:6) was used in the amplification of all the five LTP1 promoter fragments by pairing with different sense primers SEQ ID NOs: 7, 8, 9, 10, 11, respectively, to produce the promoter fragments represented by SEQ ID NOs: 1, 2, 3, 4, 5.
Each of the PCR amplified promoter DNA fragments was cloned into the Gateway cloning ready TA cloning vector pCR8/GW/TOPO (Invitrogen; SEQ ID NO:38; FIG. 4A) and clones with the correct orientation, relative to the Gateway recombination sites attL1 and attL2 (Invitrogen, Carlsbad, Calif.), were selected by MfeI+XbaI double restriction enzyme digestion analysis or sequence confirmation (see FIG. 4B for the example map QC267-1 (SEQ ID NO:39)). The maps of constructs QC267-2 (SEQ ID NO:40), QC267-3 (SEQ ID NO:41), QC2674 (SEQ ID NO:42), and QC267-5 (SEQ ID NO:43) containing the LTP1 promoter fragments SEQ ID NOs: 2, 3, 4, 5 were similar. The promoter fragment in the right orientation was subsequently cloned into the Gateway destination vector QC330 (SEQ ID NO:48; FIG. 4C) by Gateway LR clonase reaction (Invitrogen) to place the promoter fragment in front of the reporter gene YFP (see the example map QC267-1Y in FIG. 4D (SEQ ID NO:18)). A 21 bp Gateway recombination site attB2 CAGCTTTCTTGTACAAAGTGG (SEQ ID NO:35) was inserted between the promoter and the YFP reporter gene coding region as a result of the Gateway cloning process. The maps of constructs QC267-2Y (SEQ ID NO:44), QC267-3Y (SEQ ID NO:45), QC267-4Y (SEQ ID NO:46), and QC267-5Y (SEQ ID NO:47) containing the LTP1 promoter fragments SEQ ID NOs: 2, 3, 4, 5 were similar.
The LTP1:YFP promoter deletion constructs were ready to be transformed into germinating soybean cotyledons by gene gun bombardment method for transient gene expression study. The 1136 bp full length LTP1 promoter was cloned similarly as a positive control for transient expression analysis. A simple schematic description of the five LTP1 promoter deletions can be found in FIG. 5.
Example 7
Transient Expression Analysis of LTP1:YFP Constructs
The full length LTP1 promoter and a series of deletion constructs QC267-1Y, 2Y, 3Y, 4Y, and 5Y were tested by transiently expressing the ZS-YELLOW1 N1 (YFP) reporter gene in germinating soybean cotyledons. Germinating soybean cotyledons were used as the target tissue for transient expression assays. Soybean seeds were rinsed with 10% Tween 20 in sterile water, surface-sterilized with 70% ethanol for 2 minutes and then by 6% sodium hypochloride for 15 minutes. After rinsing, the seeds were placed on wet filter paper in a Petri dish to germinate for 4-6 days under fluorescent light at 26° C. Green cotyledons were excised and placed inner side up on a 0.7% agar plate containing MS media for particle gun bombardment.
The DNA and gold particle mixtures were prepared similarly as described in EXAMPLE 5 except with more DNA (100 ng/μl). The bombardments were also carried out under similar parameters as described in EXAMPLE 5. YFP expression was checked under a Leica MZFLIII stereo microscope equipped with UV light source and appropriate light filters (Leica Microsystems Inc., Bannockburn, Ill.) and pictures were taken with the same settings. Pictures were taken approximately 24 hours after bombardment with 8× magnification and camera settings: 1.06 gamma, 0.0% gain, and 0.58 seconds exposure.
The stable transformation constructs QC267 containing the linked LTP1:YFP and SAMS:ALS expressed well in transient expression assay as shown by the large green dots (FIG. 6A). Each dot represented a single cotyledon cell which appeared larger if the fluorescence was strong or smaller if the fluorescence was weak, even under the same magnification. The QC267-1 Y construct containing the same full length 1136 bp LTP1 promoter with an attB2 Gateway recombination site (Invitrogen) inserted between the LTP1 promoter and YFP and without the SAMS:ALS cassette had seemingly stronger expression with some dots glowing yellow (FIG. 6B).
The four promoter deletion constructs QC267-2Y, 3Y, 4Y, 5Y had the same structure as QC267-1Y with shorter, truncated LTP1 promoter, as described in EXAMPLE 6. The 927 bp truncated LTP1 promoter construct QC267-2Y had the same expression level as the full length LTP1 promoter construct QC267-1Y (FIG. 6C). The 738 bp truncated LTP1 promoter construct QC267-3Y had lower YFP expression as indicated by the smaller fluorescence dots (FIG. 6D). Further truncation of the LTP1 promoter to 527 bp in construct QC267-4Y further reduced the promoter strength (FIG. 6E). When the LTP1 promoter was truncated to the 257 bp minimal size in construct QC267-5Y, the promoter still retained activity at a minimal level marginally detected by the transient assay (FIG. 6F). Construct pZSL90 (SEQ ID NO:49) with a constitutive promoter SCP1 to drive the YFP expression and construct QC299i (SEQ ID NO:50) without any promoter to drive the YFP expression were used in the transient assays as positive and negative controls, respectively (FIG. 6G, H). No fluorescence was detected in the negative control.
Example 8
LTP1:YFP Expression in Stable Transgenic Soybean Plants
YFP gene expression was checked at different stages of transgenic plant development for yellow fluorescence emission under a Leica MZFLIII stereo microscope equipped with UV light source and appropriate light filters (Leica Microsystems Inc., Bannockburn, Ill.). No specific yellow fluorescence was detected during somatic embryo development or in vegetative tissues such as leaf, petiole, stem, or root. Fluorescence was only detected in flowers.
A soybean flower consists of five sepals, five petals including one standard large upper petal, two large side petals, and two small fused lower petals called kneel to enclose ten stamens and one carpel. The carpel consists of a stigma, a style, and an ovary in which there are 2-4 ovules. Specific fluorescence signal (green color) was first detected in the distal part of petals in young flower bud when the petals were still mostly enclosed by sepals (FIG. 7A), and clearly in petals of flower bud (FIG. 7E) and of open flower (FIG. 7J). No fluorescence was detected in sepals or in flower pedicle. No expression was detected in very young flower bud when the petals were completely enclosed by sepals even when the bud was cut open to expose all the inner structures.
When a young flower bud in which petals were still mostly enclosed by sepals was dissected, fluorescence was only detected in petals, often as patches indicating the start of YFP expression (FIG. 7B). No fluorescence was detected in the developing anthers, filaments (FIG. 7C), stigma, style, ovary wall, and ovules (FIG. 7D). When an older flower bud in which the petals were no longer enclosed by sepals was dissected, strong fluorescence was detected in petals (FIG. 7F) and in the fused base of filaments, but not in the separated part of filaments or in the anthers (FIG. 7G). Strong fluorescence was also detected in the style but not in the ovary part of the pistil (FIG. 7H) or in ovules. The seemingly glowing stigma in FIG. 7H glowed stronger under a non-specific cyan filter, suggesting that the fluorescence in stigma was non-specific auto fluorescence (FIG. 7I). When an open flower post to pollination was dissected, fluorescence was detected in the same tissues, i.e., petals, the stigma, and filaments but still not in sepals, carpel wall, or ovules (FIG. 7K). Fluorescence was detected in the entire filaments including the separated part but still not in anthers (FIG. 7L). No fluorescence was detected in pollen grains scattered on the stigma and carpel hairs (FIG. 7M). Fluorescence remained strong in the lower fused petals and faded away in the side petals and in the upper petal (FIG. 7N, O, P). When a young pod ( ̃10 mm long) was cut longitudinally to expose the pod wall and the inside developing seed, no fluorescence was detected (FIG. 7P).
In conclusion, the LTP1:YFP expression was only detected in petals, filaments, and was strongest in the style of a soybean flower. The expression was first detectable in the lower petals in young flower bud and faded away first in the upper petal as the flower aged. No expression was detectable in other parts of the flower or other tissues of transgenic soybean plants.
Ten out of 13 transgenic events expressed YFP in the same manner as described in details above (Table 3). The other three events though also contained the transgene as revealed by qPCR and regular PCR but failed to express YFP.

## Claims

1. An isolated polynucleotide comprising:
a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1;
b) a nucleotide sequence comprising a fragment of SEQ ID NO:1 selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; or
c) the full length complement of (a) or (b);

wherein said nucleotide sequence is a promoter.

2. A recombinant DNA construct comprising the isolated polynucleotide of claim 1 operably linked to at least one heterologous sequence.

3. The recombinant DNA construct of claim 2 wherein the heterologous nucleotide sequence encodes a gene involved in anthocyanin biosynthesis, a gene involved in the synthesis of fragrant fatty acid derivatives, a gene that is determinative of flower morphology, or a gene involved in biosynthesis of plant cytokinin.

4. The recombinant DNA construct of claim 3, wherein the gene involved in anthocyanin biosynthesis is dyhydroflavonol 4-reductase, flavonoid 3,5-hydroxylase, chalcone synthase, chalcone isomerase, flavonoid 3-hydroxylase, anthocyanin synthase, or UDP-glucose 3-O-flavonoid glucosyl transferase.

5. The recombinant DNA construct of claim 3, wherein the gene involved in the synthesis of fragrant fatty acid derivatives is S-linalool synthase, acetyl CoA:benzylalcohol acetyltransferase, benzyl CoA:benzylalcohol benzoyl transferase, S-adenosyl-L-methionine:benzoic acid carboxyl methyl transferase, mycrene synthase, (E)-β-ocimene synthase, orcinol O-methyltransferase, or limonene synthase.

6. The recombinant DNA construct of claim 3, wherein the gene that is determinative of flower morphology is AGAMOUS, APETALA, or PISTILLATA.

7. The recombinant DNA construct of claim 3, wherein the gene involved in biosynthesis of plant cytokinin is isopentenyl transferase.

8. A vector comprising the recombinant DNA construct of claim 2.

9. A cell comprising the recombinant DNA construct of claim 2.

10. The cell of claim 9, wherein the cell is a plant cell.

11. A transgenic plant having stably incorporated into its genome the recombinant DNA construct of claim 2.

12. The transgenic plant of claim 11, wherein the plant is a flowering plant.

13. The transgenic plant of claim 12, wherein the flowering plant is rose, carnation, Gerbera, Chrysanthemum, tulip, Gladioli, Alstroemeria, Anthurium, lisianthus, larkspur, irises, orchid, snapdragon, African violet, or azalea.

14. A transgenic seed produced by the transgenic plant of claim 11.

15. A method of expressing a coding sequence or an RNA in a flowering plant comprising:
a) introducing the recombinant DNA construct of claim 2 into the plant, wherein the at least one heterologous sequence comprises a coding sequence;
b) growing the plant of step a); and
c) selecting a plant displaying expression of the coding sequence or the RNA of the recombinant DNA construct.

16. A method of transgenically altering a flower trait of a flowering plant, comprising:
a) introducing a recombinant DNA construct of claim 2 into the flowering plant;
b) growing a fertile, mature flowering plant resulting from step a); and
c) selecting a flowering plant expressing the at least one heterologous nucleotide sequence in flower tissue based on the altered flower trait.

17. The method of claim 16 wherein the flower trait is color, morphology, or fragrance.

18. The isolated polynucleotide of claim 1, wherein the nucleotide sequence of b) is SEQ ID NO:2.

19. The isolated polynucleotide of claim 1, wherein the nucleotide sequence of b) is SEQ ID NO:3.

20. The isolated polynucleotide of claim 1, wherein the nucleotide sequence of b) is SEQ ID NO:4.

21. The isolated polynucleotide of claim 1, wherein the nucleotide sequence of b) is SEQ ID NO:5.