DROUGHT RESISTANCE MULTIGENE CONSTRUCT

Abstract

The present invention relates to a polygenic DNA construct consisting of three abiotic stress tolerance genes, separated by nucleic acids encoding FMDV 2A peptides under the control of a stress inducible promoter. The stable insertion of the construct into plants confers drought tolerance to the plants. The invention also provides for vectors, host cells, transgenic plants and transgenic seeds containing the construct.

Description

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a polygenic DNA construct consisting of three abiotic stress tolerance genes, separated by nucleic acids encoding FMDV 2A peptides under the control of a stress inducible promoter. The stable insertion of the construct into plants confers drought tolerance to the plants. The invention also provides for vectors, host cells, transgenic plants and transgenic seeds containing the construct.

[0002] The invention specifically relates to a polygenic DNA construct consisting of three genes from the plant Xerophyta viscosa controlled by a stress inducible promoter. The stable insertion of the construct into plants has shown that transgenic plants containing the construct are more drought tolerant compared to the wild type plants which do not contain the construct.

[0003] Plant promoters play an important role in the process of plant gene expression and regulation. The use of constitutive promoters to drive gene expression in transgenic plants often results in stunted growth and reduction of yield. Accordingly, in order to prevent over-expression of genes of interest inducible promoters have significant advantages. Proteins expressed under the control of stress inducible promoters are only expressed when the plant is exposed to a stress.

[0004] Abiotic stresses include inter alia drought, salinity, cold and extreme temperatures. Drought has been the major cause of crop losses in agriculture. It is widely known that genes act together rather than in isolation in order to counteract the effects of dehydration due to water deficit in a plant. A need therefore exists for a number of genes to be switched on by a stress inducible promoter to counteract the effects of abiotic stress.

[0005] The stacking of multiple genes in plants has become an increasing area of study of modern plant research and biotechnology. Several methods have been used to stack genes into various plant genomes and then coordinate their expression. However, many of these strategies are unreliable because of the co-expression of the heterologous proteins.

[0006] The use of a self-processing viral 2A peptide bridge such as the Foot and Mouth Disease Virus 2A (FMDV 2A) polyprotein manipulates the ribosome to skip the synthesis of the glycyl-prolyl peptide bond at its C terminus leading to the release of the nascent protein and allowing translation of the downstream sequence. The FMDV 2A oligopeptide is only 23 amino acids (aa) long. The FMDV 2A oligopeptide comprises the following amino acid sequence GSGQLLNFDLLKLAGDVESNPGP (SEQ ID NO:12) and has a co-translational cleavage at its C terminus and a post translational cleavage at its N terminus mediated by the virus encoded proteinase.

SUMMARY OF THE INVENTION

[0007] The present invention relates to a polygenic DNA construct consisting of three abiotic stress tolerance genes, separated by nucleic acids encoding FMDV 2A peptides under the control of a stress inducible promoter. The stable insertion of the construct into plants confers drought tolerance to the plants. The invention also provides for vectors, host cells, transgenic plants and transgenic seeds containing the construct.

[0008] According to a first aspect of the invention there is provided for a recombinant nucleic acid molecule comprising a polygenic nucleic acid construct operably linked to an abiotic stress inducible promoter. The polygenic nucleic acid construct comprises: a nucleotide sequence encoding a Xvsap1 polypeptide, a nucleotide sequence encoding a first 2A element peptide, a nucleotide sequence encoding a XvAld polypeptide, a nucleotide sequence encoding a second 2A element peptide, a nucleotide sequence encoding a XvPrx2 polypeptide, and a terminator.

[0009] In one embodiment of the invention the first and second 2A element peptides are Foot-and-Mouth disease virus 2A element peptides.

[0010] In another embodiment of the invention the abiotic stress may be selected from the group consisting of osmotic stress, dehydration stress, temperature stress, drought, salinity and desiccation.

[0011] In a second aspect of the invention the polygenic nucleic acid construct comprises a nucleotide sequence encoding a first polypeptide of interest, a nucleotide sequence encoding a first 2A element peptide, nucleotide sequence encoding a second polypeptide of interest, a nucleotide sequence encoding a second 2A element peptide, a nucleotide sequence encoding a third polypeptide of interest, and a terminator. In this aspect of the invention the abiotic stress inducible promoter comprises a sequence of SEQ ID NO:2.

[0012] In one embodiment of the invention the polygenic nucleic acid construct comprises a sequence of SEQ ID NO:1.

[0013] In another embodiment of the invention the first, second and third polypeptides of interest are abiotic stress tolerance polypeptides. The abiotic stress tolerance peptides may be selected from the group consisting of XvSap1, XvPrx2 and XvAld.

[0014] In a third aspect of the invention there is provided for a vector comprising a recombinant nucleic acid molecule of the invention.

[0015] A forth aspect of the invention provides for a host cell transformed with the vector of the present invention. Preferably the host cell is a plant cell.

[0016] In one embodiment of the invention the host cell is stably transformed with the recombinant nucleic acid molecule of the invention. Those of skill in the art will however appreciate that it will be possible to also transiently express the recombinant nucleic acid molecule of the invention.

[0017] In yet another aspect of the invention there is provided for a transgenic plant comprising the recombinant nucleic acid molecule of the invention or the host cell as described herein.

[0018] The transgenic plant may be selected from the group consisting of alfalfa, barley, canola, cassava, cotton, maize, oats, rye, sorghum, soybean, sunflower, sweet potato, tobacco and wheat.

[0019] The invention also provides for a transgenic seed comprising the recombinant nucleic acid molecule the invention.

[0020] A further aspect of the invention provides for a method of producing an abiotic stress tolerant transgenic plant, the method comprising obtaining a recombinant nucleic acid molecule as described herein and stably transforming a plant with the recombinant nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

[0021] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0022] FIG. 1: Schematic illustration of the polygenic DNA construct in the plant transformation vector pTF101.1.

[0023] FIG. 2: Photos of 4 replicates of the wild-type (upper set) and transgenic plants (lowers set; MG2), respectively. These were dehydrated for seven days and then tested for recovery by rehydrating the plants for five days.

[0024] FIG. 3: The effect of dehydration on seed pod formation of WT and transgenic plants. The bars reflect a shift in pod formation over a 11 day period in mature plants.

[0025] FIG. 4: The effect of dehydration on flower formation of WT and transgenic plants. The bars reflect a shift in pod formation over a 11 day period in mature plants.

SEQUENCE LISTING

[0026] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing:

[0027] SEQ ID NO:1--Complete nucleotide sequence of polygenic DNA construct (3792 bp)

[0028] SEQ ID NO:2--Nucleotide sequence of truncated promoter Psap1D (1120 bp)

[0029] SEQ ID NO:3--Nucleotide sequence of XvSap1 gene (798 bp)

[0030] SEQ ID NO:4--Nucleotide sequence of XvPrx2 gene (489 bp)

[0031] SEQ ID NO:5--Nucleotide sequence of XvAldmut2 gene (960 bp)

[0032] SEQ ID NO:6--Nucleotide sequence of FMDV 2A region (69 bp)

[0033] SEQ ID NO:7--Nucleotide sequence of nosT terminator (257 bp)

[0034] SEQ ID NO:8--Nucleotide sequence of the pTF101.1 vector

[0035] SEQ ID NO:9--Amino acid sequence of XvSap1 polypeptide

[0036] SEQ ID NO:10--Amino acid sequence of XvPrx2 polypeptide

[0037] SEQ ID NO:11--Amino acid sequence of XvAldmut2 polypeptide

[0038] SEQ ID NO:12--Amino acid sequence of the FMDV 2A linker

[0039] SEQ ID NO:13--Nucleotide sequence of the Psap1-RB F5 oligonucleotide primer

[0040] SEQ ID NO:14--Nucleotide sequence of the Psap1-RB R5 oligonucleotide primer

[0041] SEQ ID NO:15--Nucleotide sequence of the Bar I-F oligonucleotide primer

[0042] SEQ ID NO:16--Nucleotide sequence of the Bar I-R oligonucleotide primer

[0043] SEQ ID NO:17--Nucleotide sequence of the M13F oligonucleotide primer

[0044] SEQ ID NO:18--Nucleotide sequence of the M13R oligonucleotide primer

[0045] SEQ ID NO:19--Nucleotide sequence of the XvSap1 Bcl F oligonucleotide primer

[0046] SEQ ID NO:20--Nucleotide sequence of the XvPrx2 ClaI R oligonucleotide primer

[0047] SEQ ID NO:21--Nucleotide sequence of the XvSap1 ClaI R oligonucleotide primer

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0049] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0050] As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

[0051] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0052] This invention relates to the production of a transgenic plant incorporating three genes separated by a nucleic acid sequence encoding the FMDV 2A peptide and regulated by a stress-inducible promoter, hereinafter referred to as the polygenic DNA construct (FIG. 1, (SEQ ID NO:1)). The FMDV 2A sequence contains a cleavage site so that upon translation each protein is expressed individually and not as one continuous fusion polypeptide. This is advantageous as a single polygenic nucleic acid construct is provided which encodes each of the three polypeptides of interest. Accordingly, each protein encoded by the polygenic DNA construct will be expressed as an individual polypeptide and functions separately and provides the desired protection to the cells upon exposure to an abiotic stress. The transgenic plant displays enhanced tolerance to drought stress due to the concerted expression of the transgenes under water limiting conditions. Furthermore, the transgenic plant does not demonstrate any adverse growth characteristics due to the fact that the promoter is stress-inducible, which ensures that the heterologous polypeptides are only produced when required.

[0053] It will be appreciated that the polynucleotides for minimal promoter (Psap1D), the stacked genes namely, XvSapI, XvPrx2, XvAldmut2, the FMDV 2A element and the nos terminator (nosT) may be artificially synthesised.

[0054] The polynucleotide of the present invention comprises a polygenic expression cassette which includes an abiotic stress-inducible promoter, a first gene of interest, a 2A element, a second gene of interest, a second 2A element, a third gene of interest and a terminator.

[0055] The terms "nucleic acid", "nucleic acid molecule" or "polynucleotide" encompass both ribonucleotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.

[0056] A "polycistronic expression cassette" as used herein refers to a polycistronic expression unit comprising nucleic acid molecules encoding more than one polypeptide of interest for the simultaneous and coordinated expression of the more than one polypeptide of interest in response to an abiotic stress. The abiotic-stress inducible promoter allows for the simultaneous transcription of the more than one gene of interest, as well as the FMDV 2A peptide. The cleavage of the more than one polypeptide of interest occurs at the 2A peptide sequence during translation.

[0057] The promoter of the polycistronic expression cassette includes signals for DNA or RNA dependent RNA polymerase binding and transcription initiation. The promoter is an abiotic stress inducible promoter. Those of skill in the art will appreciate that the activity on an inducible promoter increases or decreases in response to a signal, which in the present invention is an abiotic stress, such as, osmotic stress, dehydration stress, temperature stress, drought, salinity or desiccation.

[0058] The term "gene of interest" refers to a polynucleotide sequence, of any, length that encodes a gene product of interest, i.e. the protein, polypeptide or polynucleotide of interest. The gene of interest is a heterologous gene with respect to the host cell. The selected sequence can be a full length or a truncated gene, a fusion or tagged gene and can be a cDNA, a genomic DNA, or a DNA fragment, preferably, a cDNA.

[0059] A "protein," "peptide" or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation).

[0060] A "host cell" refers to a cell into which the polycistronic expression cassette and its promoter are introduced. The term host cell includes both prokaryotic cells used for propagation of the construct to prepare vector stocks, and eukaryotic cells for expression of the polypeptides of interest, such as plant cells.

[0061] The term "complementary" refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus "complementary" to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

[0062] As used herein a "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

[0063] Alternatively, or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridisation conditions would be hybridisation carried out for 18 hours at 65.degree. C. with gentle shaking, a first wash for 12 min at 65.degree. C. in Wash Buffer A (0.5% SDS; 2.times.SSC), and a second wash for 10 min at 65.degree. C. in Wash Buffer B (0.1% SDS; 0.5% SSC).

[0064] In some embodiments, the nucleic acid molecules of the invention are operably linked to other sequences. By "operably linked" is meant that the nucleic acid molecules encoding the XvSap1 (SEQ ID NO:9), XvPrx2 (SEQ ID NO:10) and XvAldmut2 (SEQ ID NO:11) polypeptides or FMDV 2A (SEQ 1D NO:12) peptides described herein and regulatory sequences are connected in such a way as to permit expression of the proteins of interest when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that expression of the proteins of interest will occur in response to an abiotic stress.

[0065] The nucleic acid molecules of the invention include the XvSapI gene (SEQ ID NO:3), the XvPrx2 gene (SEQ ID NO:4) and the XvAldmut2 gene (SEQ ID NO:5), the aforementioned genes are separated by a FMDV 2A linker (SEQ ID NO:6).

[0066] XvSap1 is a stress associated protein isolated from a cDNA library synthesized from dehydration stressed Xerophytha viscosa leaves. The XvSap1 protein (SEQ ID NO:9) is highly hydrophobic and possesses two membrane lipoprotein lipid attachment sites. These features suggest that XvSap1 is anchored within the plasma membrane. It displays high sequence similarity with G-protein coupled receptors and consequently is postulated to play a signalling role during abiotic stress. The amino acid sequence of XvSap1 displayed 49% identity to WCOR413 from wheat (renamed TacCOR413-PM). This family of stress inducible proteins consist of two distinct groups based on their intracellular localisation, COR413 plasma membrane (COR413-PM) proteins and COR413 thylakoid (COR413-TM) proteins. The COR413 protein family has not been identified in other eukaryotic or prokaryotic databases suggesting that it is unique to the plant kingdom. Expression of XvSapI in Escherichia coli cells conferred osmotic stress tolerance when the cells were grown in 1 M sorbitol. Transgenic Arabidopsis thaliana and Nicotiana tabacum plants constitutively expressing XvSapI showed increase tolerance to osmotic, salt, heat and dehydration stress. These features suggest that XvSapI could play an important role in repair of damage to the cell membrane.

[0067] XvPrx2, encodes a type II Prx, the XvPrx2 protein (SEQ ID NO:10) was isolated from Xerophyta viscosa from a low temperature stress library. The Prx's are a family of multifunctional antioxidant thiol-dependent peroxidases that have been identified to be ubiquitous in most organisms. This diversity is reflected in slight evolutionary modifications in sequence and structure built around a common peroxidatic active site. The major functions of Prx's comprise cellular protection against oxidative stress, modulation of intracellular signalling cascades that apply H.sub.2O.sub.2 as a second messenger molecule and regulation of cell proliferation. The accumulation of these toxic compounds in plant cells especially reactive oxygen/nitrogen species can cause cell death which is detrimental to the plant. The XvPrx2 protein was determined to be a cytosol localised, stress inducible antioxidant enzyme involved in the protection of nucleic acids by scavenging reactive oxygen species. Besides these characteristics, two further characteristics of this protein are of significance. The first is the discovery that multiple XvPrx2 homologues exist in X. viscosa. The second is that the XvPrx2 protein is atypical in that it possesses a single cysteine only.

[0068] The XvAldmut2 gene was isolated from a Xerophyta viscosa dehydration library and encodes for an aldose reductase, which catalyses the reduction of sugars to their analogous alcohol. It has also been demonstrated that plant aldose reductase can detoxify cytotoxic aldehydes, such as 4-hydroxynon-2-enal that is a product of ROS-induced lipid peroxidation. Transcript and protein levels of XvAldmut2 (SEQ ID NO:11) have been shown to increase within leaves in response to water deficit.

[0069] The term "recombinant" means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may be introduced into a host cell by transformation.

[0070] The term "2A" or "2A element" refers to an about 18-24 amino acid sequence, which can be found in picornaviruses, such as Foot-and-Mouth Disease Virus. A highly conserved consensus motif at the C-terminus of the 2A element mediates cleavage between the C-terminal glycine and the N-terminal proline. This "cleavage" does not require any additional factors like proteases (Szymczak et al (2005)) and allows for polycistronic transcription of genes of interest with resultant cleavage of the translated polypeptides into separate proteins. Those of skill in the art will appreciate that when a FMDV 2A element is operably linked to a first gene and a second gene is operably linked to the 2A element, then the 2A element facilitates the co-translational "cleavage" of the expressed polypeptides into separate proteins. The 2A element thus allows multiple proteins to be encoded as a single polyprotein, which dissociate into component proteins on translation. The 2A peptide sequence impairs normal peptide bond formation through a mechanism of ribosomal skipping.

[0071] The term "vector" refers to a means by which polynucleotides or gene sequences can be introduced into a cell. There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term "cassette" refers to a polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequence encoding the XvSap1, XvPrx2 and XvAld polypeptides and the FMDV 2A peptides. It will be appreciated in the present invention that the cassette provides regulatory sequences in the form of an abiotic stress inducible promoter and a terminator. "Regulatory sequences" include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.

[0072] The current invention provides a specific combination of a stress-inducible promoter and three stress inducible genes isolated from Xerophyta viscosa, where each gene is separated by a 69 bp FMDV 2A nucleotide sequence.

[0073] Various combinations of stress inducible genes, operably linked to the XvPsap1D promoter (SEQ ID NO:2) and nosT terminator (SEQ ID NO:7) and individually separated by the FMDV 2A linker sequence were evaluated under drought stress conditions. The combination XvSap1::FMDV2A::XvPrx2::FMDV2A::XvAldmut2::nosT under control of the minimal stress inducible promoter (XvPsap1D (SEQ ID NO:2); as described in WO 2014/037919) displayed the highest levels of tolerance to drought.

[0074] The following example is offered by way of illustration and not by way of limitation.

Example

[0075] Cloning of the polygenic DNA construct into pTF101.1::Psap1D recombinant vector

[0076] The XvSapI::FMDV2A::XvPrx2::FMDV2A::XvAldmut2::nosT construct was synthesised de nova incorporating a BclI site at the 5' end as well as a HindIII site at the 3' site of the construct. Plasmid isolation was carried out on the synthesised construct from the pUC57 plasmid. The synthesised product was then digested, electrophoresed and excised from the gel and purified.

[0077] The polygenic DNA construct (SEQ ID NO:1) without the minimal promoter was cloned into the pDrive (Qiagen, USA) vector by digesting both the vector as well as the polygenic DNA construct with BclI and HindIII restriction enzymes at 37.degree. C. for 1 hour. The digested products where ligated to form the recombinant pDrive plasmid (pDrive::XvSapI::FMDV2A::XvPtx2::FMDV2A::XvAldmut2::nosT). The recombinant plasmid was transformed into E. coli DH5a cells and colony PCR was performed to identify positively transformed clones. Colony PCR was performed using two primer sets M13 F (-20) having the sequence GTA AAA CGA CGG CCA GT (SEQ ID NO:17) and M13 R (-20) having the sequence AAC AGC TAT GAC CAT G (SEQ ID NO:18) as well as XvSap1 BclI F having the sequence ATG ATC AAT GAG GM CGA GGG TTT TCT G (SEQ ID NO:19) and XvPrx2 ClaI R having the sequence TAT CGA TGA CTG CCT TCA AGA TCT C (SEQ ID NO:20). These primer sets amplified a 2500 bp as well as 1401 bp fragment, respectively. The following PCR conditions were used for the amplifications for both primer sets: 94.degree. C. for 5 min; 30 cycles of 94.degree. C. for 30 s, 54.degree. C. for 45 s, 72.degree. C. for 90 s; and a final extension of 72.degree. C. for 10 min.

[0078] The polygenic DNA construct was then cloned into pTF101.1 vector (SEQ ID NO:8) containing the minimal Psap1 D promoter by digesting the pDrive polygenic DNA construct with BclI and HindIII restriction enzymes and the pTF101.1 vector containing the minimal Psap1 D promoter with BamHI and HindIII. It should be noted that BclI and BamHI have compatible cohesive ends and can therefore ligate. The polygenic DNA construct was ligated to pTF101.1::Psap1 D digested ends to form the recombinant pTF101.1 plasmid (pTF101.1::Psap1D::XvSapI::FMDV2A::XvPrx2::FMDV2A::XvAldmut2::nosT) (FIG. 1). The recombinant plasmid was transformed into E. coli DH5a cells and colony PCR was performed using XvSap1 specific primers (XvSap1 BclI F (SEQ ID NO:19) and XvSapI ClaI R having the sequence TAT CGA TAA ACT CAG CCT CAT AGA TGA AGA C (SEQ ID NO:21). under the following conditions: 95.degree. C. for 10 min; 30 cycles of 95.degree. C. for 30 s, 54.degree. C. for 45 s, 72.degree. C. for 60 s and a final extension of 72.degree. C. for 10 min in order to identify positively transformed clones. An EcoRI and HindIII restriction enzyme digest was performed to release the entire insert (Psap1D::XvSapI::FMDV2A::XvPrx2::FMDV2A::XvAldmut::nosT) and the size was verified on an ethidium bromide stained agarose gel.

[0079] Transformation of Agrobacterium tumefaciens

[0080] The recombinant pTF101.1 construct was transformed into competent A. tumefaciens EHA101 cells. Following transformation and growth of A. tumefaciens cells, colony PCR was performed to identify positively transformed clones. The presence of the polygenic DNA construct was determined by PCR amplification of part of the minimal Psap1D promoter fragment as well as part of the T-DNA RB region using a promoter-specific forward primer: pSap1-RB F5 having the sequence TOG AAT GCT ATT GAT CCT GTC GT (SEQ ID NO:13) and a T-DNA right border specific reverse primer: pSap1-RB R5 having the sequence AGO TCA AGC TCC AAT ACG CAA (SEQ ID NO:14). Amplification of the 449 bp fragment was carried out under the following PCR conditions: 95.degree. C. for 5 min; 35 cycles of 95.degree. C. for 30 s, 54.degree. C. for 30 s, 72.degree. C. for 30 s; and a final extension of 72.degree. C. for 10 min.

[0081] Plant Transformation and Selection

[0082] Wild type N. tabacum (SR1 ecotype) seed was sterilised and germinated on sterile potting soil mix. Plants were cultured in the growth room with set conditions (24.degree. C.; 16 h light, 8 h dark).

[0083] Four to six inch leaves were selected from 2 month old plants. Leaves were soaked in sterile water for 30 min and sterilised. Sterile leaves were sliced into uniform segments of 5 mm avoiding the leaf margins and mid vein. Leaf explants were placed adaxial side up onto pre-culture media.

[0084] One millilitre of a 16 h culture of A. tumefaciens carrying the polycistronic multigene construct was inoculated into 200 ml of YEP media supplemented with the appropriate antibiotics. The cultures were incubated at 30.degree. C. until an absorbance reading of approximately 0.8 at 600 nm was obtained. The cultures were centrifuged for 20 min at 6000.times.g at 4.degree. C. The supernatant was discarded and the bacterial pellets re-suspended in 50 ml of liquid co-cultivation media.

[0085] Leaf discs were infected for 30 min in the dark with the Agrobacterium inoculum containing the polygenic DNA construct in sterile petri dishes. The petri dish was agitated once every 10 min. Thereafter, infected leaf discs were blot dried on sterile filter paper. Each infected explant was transferred to co-cultivation medium and incubated for 3 days at 23.degree. C. (18 h light, 6 h dark; light intensity of 140 .mu.mol/m.sup.2/s). The adaxial part of the leaf was kept in contact with the medium.

[0086] Following the 3 day co-cultivation period, leaf discs were selected on shooting medium. Leaf explants were placed under an 18 h light regime with light intensity of 140 .mu.mol/m.sup.2/s at 28.degree. C. Putative transformants were sub-cultured fortnightly onto fresh media until sizable shoots were formed. BASTA resistant shoots were selected, excised and transferred to rooting media.

[0087] Putative transformants with well-established root systems were transferred to pots containing sterile potting soil and cultured with set conditions. The plants were covered with Saran Wrap.RTM. for 8 days to assist acclimatisation and minimise dehydration. Once acclimatised, the putative transformants were transferred to 6 inch pots containing potting soil under normal growth conditions. Mature plants were self-pollinated and seed was harvested from mature dry pods.

[0088] Putative transgenic tobacco seed was sterilised and germinated on MS media supplemented with 8 g/l agar and 3 mg/ml BASTA. Plants were cultured in the growth room with set conditions (24.degree. C.; 16 h day, 8 h night). The surviving BASTA resistant plants with well-established root systems were transferred to trays containing 0.1 g/l Gaucho SW treated potting soil and covered with Saran Wrap.RTM. for 1 week. Three weeks later, BASTA resistant transgenic plants were transferred to pots containing potting soil.

[0089] Leaves were sampled from putative transgenic plants and were flash frozen in liquid nitrogen. Genomic DNA was extracted using the Dellaporta extraction protocol. The presence of the polygenic DNA construct was determined by PCR amplification with the Psap1-RB F5 (SEQ ID NO:13) and Psap1-RB R5 (SEQ ID NO:14) primer pair. The bar gene (421 bp) was also amplified using the following PCR conditions (95.degree. C. for 10 min; 35 cycles of 95.degree. C. for 30 s, 54.degree. C. for 30 s, 72.degree. C. for 30 s; and a final extension of 72.degree. C. for 5 min) and using the bar gene specific primers: Bar I-F GGT CTG CAC CAT CGT CAA CC (SEQ ID NO:15) and Bar I-R GTC ATG CCA GTT CCC GTG CT (SEQ ID NO:16).

[0090] Dehydration and Rehydration of Transgenic Plants

[0091] The first dehydration/rehydration treatments were carried out in a Percival.RTM. chamber and the second dehydration/rehydration treatment was carried out in a Conviron.RTM. chamber.

[0092] Prior to the first dehydration/rehydration treatment, plants were transferred to pots containing set amounts of soil and water. Plants were moved to the Percival.RTM. chamber (Percival.RTM. Intellus control system) and incubated under set conditions (26.degree. C.; 16 h day, 8 h night; 60% humidity; light intensity of 100 .mu.mol/m.sup.2/s) for 1 week to acclimatise. Dehydration stress was carried out on whole plants and achieved by withholding water for a period of seven days and then rehydrating the plants for a further five days.

[0093] Prior to the second dehydration/rehydration treatment, whole mature plants were transferred to pots containing set amounts of soil and water. Plants were moved to the Conviron.RTM. chamber and incubated under set conditions (26.degree. C.; 16 h day, 8 h night; 60% humidity; light intensity of 100 .mu.mol/m.sup.2/s) for 1 week to acclimatise. Dehydration stress was carried out on whole plants and achieved by withholding water for a period of ten days and rehydrating the plants for a further four days. Throughout the dehydration period, flowers and seed pods were counted on day one and day ten of dehydration, prior to rehydration. Rehydration was initiated on day ten after the flowers and pods were counted. Rehydration lasted four days and the total biomass of the transgenic plants was measured on day four of the rehydration.

[0094] Dehydration and Rehydration Treatment of Transgenic N. tabacum Plants

[0095] The first dehydration and rehydration treatments on wild type (WT) tobacco had a major impact on the overall morphology of the plant. In the absence of the polygenic DNA construct the leaves look withered and close to dying when compared to the transgenic lines containing the polygenic DNA construct (FIG. 2). The transgenic plants also had leaves that looked fuller and bigger and appeared to have a minor response to the stress. On the other hand the wild type control plants were severely affected following the stress treatment.

[0096] The Effect of Dehydration on Seed Pods and Flowers of Wild Type Control and Transgenic Tobacco Plants

[0097] Transgenic plants (fully hydrated) reached maturity significantly earlier than the wild type counterparts as evidenced by the higher number of pods (FIG. 3) and flowers (FIG. 4) observed. For those transgenic plants that were dehydrated there was also an observable difference in the number of pods and flowers compared to wild type plants (FIGS. 3 and 4),

REFERENCES

[0098] Szymczak A. L. et al (2005) Expert Opin. Biol. Ther. 5: 627-638

Sequence CWU 1

1

2113792DNAArtificial SequenceMultigene Construct 1actgtctggg tagctggcaa tatagagacg taaataattg tctgtaaata gggagaaatt 60catggatcat caccctaatt cggtctttca ctcattttat catagacctg actaaagaac 120ttggtcagag tttttactta tttaaaataa agaggacttc atggcatcca tgtgcaggta 180cagctcccag aaaaaaaagc atgaaacacg agaggatcaa tagcattcga tctgaaacaa 240aaggttgcag ctcaagactt tctccaaaat attaagatga tccaaagaat taccccaaga 300tatccaacgt ataccaatgt gtataccgaa agtaagaaag ttcacgtgca ttctttgatt 360tttctcccga gtgttctttt ctgaaatgag taaataagac tagaataaga gctaatgtat 420tttttttcta aaaaaagttg aatgtggata caatatgatt atacattcat tagctatttt 480aagtatattc tatttttttt ccccccaaaa gaacacaaat gtgttccgtc actttccatg 540gagatcagat ctatcttaga attggacagg gtgcttatga tacaacttgt tcctatcaac 600aactgcatgt tagacagcgc cgaatttaca gtcctactgg gcgccacttt tcaacccaca 660tcatcaagat gaacaccacg ttatcttcat ccgctccaac cacatggtcc agcgccactg 720gccaagaccg ccagccagcc aggccatcca acgtggtgca ttttctaaca ctccacgttc 780gctgtacggc attatttctc cagccagaaa gaccgagaca gcgacgctgt tgggcgggcc 840cgcggcctgc tctctctgct tccccatgag attcacgggc atcgctcctc gctcgtgcct 900acgcgaccgc gccgatccac gtgacgtggc gcagcaatcg ttcttactag gcgcttgcac 960gtgtcgttcg catgcgaagc gtccacactg ccaacgacct ccttaaatat ccttgtgata 1020ttcgccttac gatctcacac ttcgcacgca aaggccagtc gcagatttgg gttgaatttg 1080ctgcgttttg gcagattttg agcgagagat attagggaag ggatcaatga ggaacgaggg 1140ttttctgaaa atgaagaccg acgttggagt cgccgacgag gtgatctccg gagatctcaa 1200gcagcttggt gacgctgcaa agcggctagc taaacatgcg atcaagctcg gcgccagctt 1260cggggttggc tctaccatag tccaggctat tgcttcgatc gctgctatct atttgttgat 1320attggaccgg acaaactggc gtacaaatat cttgacatca cttctaattc catatgttta 1380cttgagtctt ccttcagtga tattcaacct attcaggggc gacctgggca gatggctttc 1440attcattggc gtagtaatga agctcttctt ccaccgacac ttcccagtta ccttggaact 1500gcttgtgtct ctcattctcc tgattgtggt ttcccccact ttcattgccc acacaatcag 1560aggcagtctc attggagtct tcatcttcct tgtcatcgcc tgctacctcc tccaagagca 1620cattagatca gctggtggct tcaaaaacgc gttcacaaag agcaatggga tttcaaacag 1680cgtcgggatc atcattctac tgatccaccc gatctggagc ttggtggtgt atttcctcta 1740cacgtctttg ctgcaacttc ttgcatactc tccttcccct tgttgttgca tattatacaa 1800taagtggttt aatttcatgc atgtttgtaa atgtgtaagc cttcatatgt attctcagtc 1860aattgggtca tgcgtgtcca tatttttcgt gcagtttgta ttcatctatg aagctgaatt 1920tatcgatggc agtggacagc tgttgaattt tgaccttctt aaacttgcgg gagacgtcga 1980gtccaaccct gggcccgtcg agatggcacc gatcgcagtc ggtgaaacga tcccagacgg 2040aacgctcgga tggttcgacg agaaggacga gttgaagcag atctcgatcc actcgctcgc 2100cgccggaaag aagatcgtgc tcatcggtgt ccccggcgca ttcactccta cttgcagtat 2160gcaacacgtt ccaagtttca ttgagaaagc agaggagctg aaagctaagg gcgttgatga 2220gttccttgtt attagtgtta atgatccctt cgtgatgaag gcttggtcga aaacatatcc 2280tgagaacaag catgtgaagt tcctagccga tggatcgggg aagtacaccc aagctcttgg 2340cgtggaactc gatctgtccg agaaggggct cgggctccgt tcacggaggt ttgctatcct 2400tgtagacgac ttgaaggtta aggttgcaaa tgtcgaggag ggcggagcat ttaccatttc 2460aggtgccgat gagatcttga aggcagtcat cgatggcagt ggacagctgt tgaattttga 2520ccttcttaaa cttgcgggag acgtcgagtc caaccctggg cccgtcgaga tggcgcatgc 2580accgtgtttt gctgatgcga agacacagag cttcaagctc ctcagcgggc actcaatccc 2640cgcagttggg ctcggcacgt ggaagtctgg tgacaaggcc ggcaacgccg tatacactgc 2700catcactgag ggaggataca ggcacattga taccgcagca caatatggag tccatgaaga 2760ggtaggcaat gctcttcaat ctgctttgaa agcagggatc aataggaagg ctttgttcgt 2820cacatcgaaa gtatggtgcg aagatttatc acctgaaaga gttcgacctg cattgaaaaa 2880tacacttgag gagctacaac tggattacct tgatctctac ctgattcact ggcctatcca 2940ccttaaaaag ggcgcacaca tgcctcctga ggctggtgag gtgctagagt tcgacatagg 3000aggagtgtgg agggaaatgg agaagctcgt caaagtaggg cttgttagag atattggtat 3060ctctaacttc actgtgaaga aactcgaaaa acttctaaat tttgctgaaa taaagccctc 3120ggtgtgccag atggagatgc acccgggttg gagaaacgac aagatgtttg agatttgcag 3180gaaatatggt attcatacaa ctgcttattc acctctcgga tcttccgagc gtgatctcct 3240cagtgatcca actgttttga agatagcaaa caagctcaac aagagcccag gtaaacttct 3300ggtgagatgg gctgttcaaa gaggaactag tgtcatccca aaatcgacca acccggagag 3360gataaaggag aacatccagg tcttcgggtg ggagattcct gcagaggatt tccagatttt 3420gagcagcctt agtgaacaga agagagtctt ggatggtgaa gatctcttcg tcaacaaaac 3480ccatggcccg ttcaggagcg ctgctgaact ctgggacggt gaagtctaag tcgacacccg 3540atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga 3600tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca 3660tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg 3720cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta 3780tgttactaga tc 379221120DNAArtificial SequenceTruncated pSap1D Promoter 2actgtctggg tagctggcaa tatagagacg taaataattg tctgtaaata gggagaaatt 60catggatcat caccctaatt cggtctttca ctcattttat catagacctg actaaagaac 120ttggtcagag tttttactta tttaaaataa agaggacttc atggcatcca tgtgcaggta 180cagctcccag aaaaaaaagc atgaaacacg agaggatcaa tagcattcga tctgaaacaa 240aaggttgcag ctcaagactt tctccaaaat attaagatga tccaaagaat taccccaaga 300tatccaacgt ataccaatgt gtataccgaa agtaagaaag ttcacgtgca ttctttgatt 360tttctcccga gtgttctttt ctgaaatgag taaataagac tagaataaga gctaatgtat 420tttttttcta aaaaaagttg aatgtggata caatatgatt atacattcat tagctatttt 480aagtatattc tatttttttt ccccccaaaa gaacacaaat gtgttccgtc actttccatg 540gagatcagat ctatcttaga attggacagg gtgcttatga tacaacttgt tcctatcaac 600aactgcatgt tagacagcgc cgaatttaca gtcctactgg gcgccacttt tcaacccaca 660tcatcaagat gaacaccacg ttatcttcat ccgctccaac cacatggtcc agcgccactg 720gccaagaccg ccagccagcc aggccatcca acgtggtgca ttttctaaca ctccacgttc 780gctgtacggc attatttctc cagccagaaa gaccgagaca gcgacgctgt tgggcgggcc 840cgcggcctgc tctctctgct tccccatgag attcacgggc atcgctcctc gctcgtgcct 900acgcgaccgc gccgatccac gtgacgtggc gcagcaatcg ttcttactag gcgcttgcac 960gtgtcgttcg catgcgaagc gtccacactg ccaacgacct ccttaaatat ccttgtgata 1020ttcgccttac gatctcacac ttcgcacgca aaggccagtc gcagatttgg gttgaatttg 1080ctgcgttttg gcagattttg agcgagagat attagggaag 11203798DNAXerophyta viscosa 3atgaggaacg agggttttct gaaaatgaag accgacgttg gagtcgccga cgaggtgatc 60tccggagatc tcaagcagct tggtgacgct gcaaagcggc tagctaaaca tgcgatcaag 120ctcggcgcca gcttcggggt tggctctacc atagtccagg ctattgcttc gatcgctgct 180atctatttgt tgatattgga ccggacaaac tggcgtacaa atatcttgac atcacttcta 240attccatatg tttacttgag tcttccttca gtgatattca acctattcag gggcgacctg 300ggcagatggc tttcattcat tggcgtagta atgaagctct tcttccaccg acacttccca 360gttaccttgg aactgcttgt gtctctcatt ctcctgattg tggtttcccc cactttcatt 420gcccacacaa tcagaggcag tctcattgga gtcttcatct tccttgtcat cgcctgctac 480ctcctccaag agcacattag atcagctggt ggcttcaaaa acgcgttcac aaagagcaat 540gggatttcaa acagcgtcgg gatcatcatt ctactgatcc acccgatctg gagcttggtg 600gtgtatttcc tctacacgtc tttgctgcaa cttcttgcat actctccttc cccttgttgt 660tgcatattat acaataagtg gtttaatttc atgcatgttt gtaaatgtgt aagccttcat 720atgtattctc agtcaattgg gtcatgcgtg tccatatttt tcgtgcagtt tgtattcatc 780tatgaagctg aattttaa 7984489DNAXerophyta viscosa 4atggctccga tcgcagtcgg tgaaacgatc ccagacggaa cgctcggatg gttcgacgag 60aaggacgagt tgaagcagat ctcgatccac tcgctcgccg ccggaaagaa gatcgtgctc 120atcggtgtcc ccggcgcatt cactcctact tgcagtatgc aacacgttcc aagtttcatt 180gagaaagcag aggagctgaa agctaagggc gttgatgagt tccttgttat tagtgttaat 240gatcccttcg tgatgaaggc ttggtcgaaa acatatcctg agaacaagca tgtgaagttc 300ctagccgatg gatcggggaa gtacacccaa gctcttggcg tggaactcga tctgtccgag 360aaggggctcg ggctccgttc acggaggttt gctatccttg tagacgactt gaaggttaag 420gttgcaaatg tcgaggaggg cggagcattt accatttcag gtgccgatga gatcttgaag 480gcagtctag 4895960DNAXerophyta viscosa 5atggcgcatg caccgtgttt tgctgatgcg aagacacaga gcttcaagct cctcagcggg 60cactcaatcc ccgcagttgg gctcggcacg tggaagtctg gtgacaaggc cggcaacgcc 120gtatacactg ccatcactga gggaggatac aggcacattg ataccgcagc acaatatgga 180gtccatgaag aggtaggcaa tgctcttcaa tctgctttga aagcagggat caataggaag 240gctttgttcg tcacatcgaa agtatggtgc gaagatttat cacctgaaag agttcgacct 300gcattgaaaa atacacttga ggagctacaa ctggattacc ttgatctcta cctgattcac 360tggcctatcc accttaaaaa gggcgcacac atgcctcctg aggctggtga ggtgctagag 420ttcgacatag gaggagtgtg gagggaaatg gagaagctcg tcaaagtagg gcttgttaga 480gatattggta tctctaactt cactgtgaag aaactcgaaa aacttctaaa ttttgctgaa 540ataaagccct cggtgtgcca gatggagatg cacccgggtt ggagaaacga caagatgttt 600gagatttgca ggaaatatgg tattcataca actgcttatt cacctctcgg atcttccgag 660cgtgatctcc tcagtgatcc aactgttttg aagatagcaa acaagctcaa caagagccca 720ggtcaacttc tggtgagatg ggctgttcaa agaggaacta gtgtcatccc aaaatcgacc 780aacccggaga ggataaagga gaacatccag gtcttcgggt gggagattcc tgcagaggat 840ttccagattt tgagcagcct tagtgaacag aagagagtct tggatggtga agatctcttc 900gtcaacaaaa cccatggccc gttcaggagc gctgctgaac tctgggacgg tgaagtctaa 960669DNAFoot and Mouth Disease Virus 6ggcagtggac agctgttgaa ttttgacctt cttaaacttg cgggagacgt cgagtccaac 60cctgggccc 697257DNAAgrobacterium tumefaciens 7acccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 60tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 120atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 180atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 240atctatgtta ctagatc 25789189DNAArtificial SequencepTF101.1 vector 8agtactttaa agtactttaa agtactttaa agtactttga tccaacccct ccgctgctat 60agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag 120tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt 180tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag 240agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc 300aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc 360ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac 420gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt 480gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac 540accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag 600cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg 660aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc 720gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc 780ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt 840gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc 900caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg 960aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct 1020caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc 1080cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg 1140agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa 1200tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa 1260gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt 1320agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc 1380gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg 1440gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat 1500caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac 1560cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc 1620ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc 1680gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc 1740aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg 1800cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa 1860gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc 1920aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag 1980ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt 2040accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat 2100gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac 2160cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg 2220ggttgtctgc cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgagc 2280ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag 2340aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt 2400gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc 2460ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg 2520atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt 2580ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac 2640gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg 2700atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag 2760cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat 2820ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt 2880gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa 2940gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag 3000atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg 3060acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg 3120gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc 3180agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca 3240aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc 3300atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag 3360atgctagggc aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat 3420agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac 3480ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa 3540ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc 3600tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg 3660tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca 3720aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca 3780ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga 3840aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 3900gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 3960gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag 4020attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 4080taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 4140ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 4200gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 4260gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 4320cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 4380ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 4440tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 4500gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4560tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4620ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4680ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 4740ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4800accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4860tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4920cgttaaggga ttttggtcat gcatgatata tctcccaatt tgtgtagggc ttattatgca 4980cgcttaaaaa taataaaagc agacttgacc tgatagtttg gctgtgagca attatgtgct 5040tagtgcatct aacgcttgag ttaagccgcg ccgcgaagcg gcgtcggctt gaacgaattt 5100ctagctagac attatttgcc gactaccttg gtgatctcgc ctttcacgta gtggacaaat 5160tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag ataagcctgt 5220ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc ccagtcggca 5280gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg ggacaacgta 5340agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag cgttaaggtt 5400tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc ctccgccgct 5460ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc cagatcaatg 5520tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat tgcgctgcca ttctccaaat 5580tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg 5640acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg 5700ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac cagcaaatca 5760atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg tacggccagc 5820aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg agtcgatact 5880tcggcgatca ccgcttcccc catgatgttt aactttgttt tagggcgact gccctgctgc 5940gtaacatcgt tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc 6000ttggatgccc gaggcataga ctgtacccca aaaaaacagt cataacaagc catgaaaacc 6060gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt gcgtgacggc 6120agttacgcta cttgcattac agcttacgaa ccgaacgagg cttatgtcca ctgggttcgt 6180gcccgaattg atcacaggca gcaacgctct gtcatcgtta caatcaacat gctaccctcc 6240gcgagatcat ccgtgtttca aacccggcag cttagttgcc gttcttccga atagcatcgg 6300taacatgagc aaagtctgcc gccttacaac ggctctcccg ctgacgccgt cccggactga 6360tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct 6420ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac 6480attgcggacg tttttaatgt actgaattaa cgccgaattg ctctagcatt cgccattcag 6540gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 6600gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 6660acgttgtaaa acgacggcca gtgccaagct aattcgcttc aagacgtgct caaatcacta 6720tttccacacc cctatatttc tattgcactc ccttttaact gttttttatt acaaaaatgc 6780cctggaaaat gcactccctt tttgtgtttg tttttttgtg aaacgatgtt gtcaggtaat 6840ttatttgtca gtctactatg gtggcccatt atattaatag caactgtcgg tccaatagac 6900gacgtcgatt ttctgcattt gtttaaccac gtggatttta tgacatttta tattagttaa 6960tttgtaaaac ctacccaatt aaagacctca tatgttctaa agactaatac ttaatgataa 7020caattttctt ttagtgaaga aagggataat tagtaaatat

ggaacaaggg cagaagattt 7080attaaagccg cggtaagaga caacaagtag gtacgtggag tgtcttaggt gacttaccca 7140cataacataa agtgacatta acaaacatag ctaatgctcc tatttgaata gtgcatatca 7200gcatacctta ttacatatag ataggagcaa actctagcta gattgttgag cagatctcgg 7260tgacgggcag gaccggacgg ggcggtaccg gcaggctgaa gtccagctgc cagaaaccca 7320cgtcatgcca gttcccgtgc ttgaagccgg ccgcccgcag catgccgcgg ggggcatatc 7380cgagcgcctc gtgcatgcgc acgctcgggt cgttgggcag cccgatgaca gcgaccacgc 7440tcttgaagcc ctgtgcctcc agggacttca gcaggtgggt gtagagcgtg gagcccagtc 7500ccgtccgctg gtggcggggg gagacgtaca cggtcgactc ggccgtccag tcgtaggcgt 7560tgcgtgcctt ccaggggccc gcgtaggcga tgccggcgac ctcgccgtcc acctcggcga 7620cgagccaggg atagcgctcc cgcagacgga cgaggtcgtc cgtccactcc tgcggttcct 7680gcggctcggt acggaagttg accgtgcttg tctcgatgta gtggttgacg atggtgcaga 7740ccgccggcat gtccgcctcg gtggcacggc ggatgtcggc cgggcgtcgt tctgggctca 7800tggtagatcc cccgttcgta aatggtgaaa attttcagaa aattgctttt gctttaaaag 7860aaatgattta aattgctgca atagaagtag aatgcttgat tgcttgagat tcgtttgttt 7920tgtatatgtt gtgttgagaa ttaattctcg aggtcctctc caaatgaaat gaacttcctt 7980atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg 8040agatatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 8100tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggtagaggca tcttgaacga 8160tagcctttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt tccactatct 8220tcacaataaa gtgacagata gctgggcaat ggaatccgag gaggtttccg gatattaccc 8280tttgttgaaa agtctcaatt gccctttggt cttctgagac tgtatctttg atatttttgg 8340agtagacaag tgtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg 8400tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 8460cactgtcggc agaggcatct tcaacgatgg cctttccttt atcgcaatga tggcatttgt 8520aggagccacc ttccttttcc actatcttca caataaagtg acagatagct gggcaatgga 8580atccgaggag gtttccggat attacccttt gttgaaaagt ctcaattgcc ctttggtctt 8640ctgagactgt atctttgata tttttggagt agacaagtgt gtcgtgctcc accatgttga 8700cctgcaggca tgcaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga 8760gctcgaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat 8820tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 8880ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 8940ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggagcttg 9000agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgacaggata 9060tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat atttaaaagg 9120gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct 9180cgggatcaa 91899265PRTXerophyta viscosa 9Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala1 5 10 15Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu65 70 75 80Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr145 150 155 160Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His225 230 235 240Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 26510162PRTXerophyta viscosa 10Met Ala Pro Ile Ala Val Gly Glu Thr Ile Pro Asp Gly Thr Leu Gly1 5 10 15Trp Phe Asp Glu Lys Asp Glu Leu Lys Gln Ile Ser Ile His Ser Leu 20 25 30Ala Ala Gly Lys Lys Ile Val Leu Ile Gly Val Pro Gly Ala Phe Thr 35 40 45Pro Thr Cys Ser Met Gln His Val Pro Ser Phe Ile Glu Lys Ala Glu 50 55 60Glu Leu Lys Ala Lys Gly Val Asp Glu Phe Leu Val Ile Ser Val Asn65 70 75 80Asp Pro Phe Val Met Lys Ala Trp Ser Lys Thr Tyr Pro Glu Asn Lys 85 90 95His Val Lys Phe Leu Ala Asp Gly Ser Gly Lys Tyr Thr Gln Ala Leu 100 105 110Gly Val Glu Leu Asp Leu Ser Glu Lys Gly Leu Gly Leu Arg Ser Arg 115 120 125Arg Phe Ala Ile Leu Val Asp Asp Leu Lys Val Lys Val Ala Asn Val 130 135 140Glu Glu Gly Gly Ala Phe Thr Ile Ser Gly Ala Asp Glu Ile Leu Lys145 150 155 160Ala Val11319PRTXerophyta viscosa 11Met Ala His Ala Pro Cys Phe Ala Asp Ala Lys Thr Gln Ser Phe Lys1 5 10 15Leu Leu Ser Gly His Ser Ile Pro Ala Val Gly Leu Gly Thr Trp Lys 20 25 30Ser Gly Asp Lys Ala Gly Asn Ala Val Tyr Thr Ala Ile Thr Glu Gly 35 40 45Gly Tyr Arg His Ile Asp Thr Ala Ala Gln Tyr Gly Val His Glu Glu 50 55 60Val Gly Asn Ala Leu Gln Ser Ala Leu Lys Ala Gly Ile Asn Arg Lys65 70 75 80Ala Leu Phe Val Thr Ser Lys Val Trp Cys Glu Asp Leu Ser Pro Glu 85 90 95Arg Val Arg Pro Ala Leu Lys Asn Thr Leu Glu Glu Leu Gln Leu Asp 100 105 110Tyr Leu Asp Leu Tyr Leu Ile His Trp Pro Ile His Leu Lys Lys Gly 115 120 125Ala His Met Pro Pro Glu Ala Gly Glu Val Leu Glu Phe Asp Ile Gly 130 135 140Gly Val Trp Arg Glu Met Glu Lys Leu Val Lys Val Gly Leu Val Arg145 150 155 160Asp Ile Gly Ile Ser Asn Phe Thr Val Lys Lys Leu Glu Lys Leu Leu 165 170 175Asn Phe Ala Glu Ile Lys Pro Ser Val Cys Gln Met Glu Met His Pro 180 185 190Gly Trp Arg Asn Asp Lys Met Phe Glu Ile Cys Arg Lys Tyr Gly Ile 195 200 205His Thr Thr Ala Tyr Ser Pro Leu Gly Ser Ser Glu Arg Asp Leu Leu 210 215 220Ser Asp Pro Thr Val Leu Lys Ile Ala Asn Lys Leu Asn Lys Ser Pro225 230 235 240Gly Gln Leu Leu Val Arg Trp Ala Val Gln Arg Gly Thr Ser Val Ile 245 250 255Pro Lys Ser Thr Asn Pro Glu Arg Ile Lys Glu Asn Ile Gln Val Phe 260 265 270Gly Trp Glu Ile Pro Ala Glu Asp Phe Gln Ile Leu Ser Ser Leu Ser 275 280 285Glu Gln Lys Arg Val Leu Asp Gly Glu Asp Leu Phe Val Asn Lys Thr 290 295 300His Gly Pro Phe Arg Ser Ala Ala Glu Leu Trp Asp Gly Glu Val305 310 3151223PRTFoot and Mouth Disease Virus 12Gly Ser Gly Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp1 5 10 15Val Glu Ser Asn Pro Gly Pro 201323DNAArtificial SequencepSap1-RB F5 Oligonucleotide 13tcgaatgcta ttgatcctgt cgt 231421DNAArtificial SequencepSap1-RB R5 Oligonucleotide 14agctcaagct ccaatacgca a 211520DNAArtificial SequenceBar I-F Oligonucleotide 15ggtctgcacc atcgtcaacc 201620DNAArtificial SequenceBar I-R Oligonucleotide 16gtcatgccag ttcccgtgct 201717DNAArtificial sequenceM13F Oligonucleotide 17gtaaaacgac ggccagt 171816DNAArtificial sequenceM13R Oligonucleotide 18aacagctatg accatg 161928DNAArtificial sequenceXvSapI BclI F Oligonucleotide 19atgatcaatg aggaacgagg gttttctg 282025DNAArtificial sequenceXvPrx2 ClaI R Oligonucleotide 20tatcgatgac tgccttcaag atctc 252131DNAArtificial sequenceXvSapI ClaI R Oligonucleotide 21tatcgataaa ctcagcctca tagatgaaga c 31

Claims

1. A recombinant nucleic acid molecule comprising a polygenic nucleic acid construct operably linked to an abiotic stress inducible promoter, wherein the polygenic nucleic acid construct comprises: a. a nucleotide sequence encoding a Xvsap1 polypeptide, b. a nucleotide sequence encoding a first 2A element peptide, c. a nucleotide sequence encoding a XvAld polypeptide, d. a nucleotide sequence encoding a second 2A element peptide, e. a nucleotide sequence encoding a XvPrx2 polypeptide, and f. a terminator.

2. The recombinant nucleic acid molecule of claim 1, wherein the 2A element peptide is a Foot-and-Mouth disease virus 2A element peptide.

3. The recombinant nucleic acid molecule of claim 1, wherein the abiotic stress is selected from the group consisting of osmotic stress, dehydration stress, temperature stress, drought, salinity and desiccation.

4. A recombinant nucleic acid molecule comprising a polygenic nucleic acid construct operably linked to an abiotic stress inducible promoter, wherein the polygenic nucleic acid construct comprises: a. a nucleotide sequence encoding a first polypeptide of interest, b. a nucleotide sequence encoding a first 2A element peptide, c. a nucleotide sequence encoding a second polypeptide of interest, d. a nucleotide sequence encoding a second 2A element peptide, e. a nucleotide sequence encoding a third polypeptide of interest, and f. a terminator, and further wherein the abiotic stress inducible promoter comprises a sequence of SEQ ID NO: 2.

5. The recombinant nucleic acid molecule of claim 4 wherein the polygenic nucleic acid construct comprises a sequence of SEQ ID NO: 1.

6. The recombinant nucleic acid molecule of claim 4, wherein the first, second, and third polypeptides of interest are abiotic stress tolerance polypeptides.

7. The recombinant nucleic acid molecule of claim 4, wherein the first, second and third polypeptides of interest are selected from the group consisting of XvSap1, XvPrx2 and XvAld.

8. The recombinant nucleic acid molecule of claim 4, wherein the 2A element peptide is a Foot-and-Mouth disease virus 2A element peptide.

9. The recombinant nucleic acid molecule of claim 4, wherein the abiotic stress is selected from the group consisting of osmotic stress, dehydration stress, temperature stress, drought, salinity, and desiccation.

10. A vector comprising a recombinant nucleic acid molecule of claim 1.

11. A host cell transformed with the vector of claim 10.

12. The host cell of claim 11 wherein the host cell is a plant cell.

13. The host cell of claim 11 wherein the host cell is stably transformed with the recombinant nucleic acid molecule.

14. A transgenic plant comprising the recombinant nucleic acid molecule of claim 1.

15. The transgenic plant of claim 14, wherein the plant is selected from the group consisting of alfalfa, barley, canola, cassava, cotton, maize, oats, rye, sorghum, soybean, sunflower, sweet potato, tobacco and wheat.

16. A transgenic seed comprising the recombinant nucleic acid molecule of claim 1.

17. A method of producing an abiotic stress tolerant transgenic plant, the method comprising: obtaining a recombinant nucleic acid molecule of claim 1; and stably transforming the plant with the recombinant nucleic acid molecule.

18. A transgenic plant comprising the host cell of claim 11.

19. The transgenic plant of claim 18, wherein the plant is selected from the group consisting of alfalfa, barley, canola, cassava, cotton, maize, oats, rye, sorghum, soybean, sunflower, sweet potato, tobacco and wheat.