Isolated eIF-5A and Polynucleotides Encoding Same

Abstract

The present invention relates to unique isoforms of eukaryotic initiation Factor 5A ("eIF-5A"): senescence-induced eIF-5A; wounding-induced eIF-5A; and growth eIF-5A, as well as polynucleotides that encode these three factors. The present invention also relates to methods involving modulating the expression of these factors. The present invention also relates to deoxyhypusine synthase ("DHS"), polynucleotides that encode DHS, and methods involving modulating the expression of DHS.

Description

RELATED APPLICATIONS

[0001] This application is a divisional application of Ser. No. 09/725,019, filed Nov. 29, 2000, which is a continuation-in-part of Ser. No. 09/597,771, filed Jun. 19, 2000, now U.S. Pat. No. 6,538,182, which is a continuation in part of Ser. No. 09/348,675, filed Jul. 6, 1999, now abandoned. This application claims priority to and herein incorporates by reference U.S. provisional applications 60/479,968 and 60/479,969 both filed Jun. 20, 2003, and U.S. provisional applications 60/(awaited) docket number 10799/120 and 60/(awaited) docket number 10799/120, both filed on May 14, 2004.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable text file, entitled "061945-5020-02-SequenceListing.txt" created on or about Jul. 30, 2012 with a file size of about 182 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to unique isoforms of eukaryotic initiation Factor 5A ("eIF-5A") and polynucleotides that encode eIF-5A and deoxyhypusine synthase ("DHS"), and polynucleotides that encode DHS, and methods involving modulating the expression of the isoforms eIF-5A and DHS.

DESCRIPTION OF THE PRIOR ART

[0004] Senescence is the terminal phase of biological development in the life of a plant. It presages death and occurs at various levels of biological organization including the whole plant, organs, flowers and fruit, tissues and individual cells.

[0005] The onset of senescence can be induced by different factors both internal and external. Senescence is a complex, highly regulated developmental stage in the life of a plant or plant tissue, such as fruit, flowers and leaves. Senescence results in the coordinated breakdown of cell membranes and macromolecules and the subsequent mobilization of metabolites to other parts of the plant.

[0006] In addition to the programmed senescence which takes place during normal plant development, death of cells and tissues and ensuing remobilization of metabolites occurs as a coordinated response to external, environmental factors. External factors that induce premature initiation of senescence, which is also referred to as necrosis or apoptosis, include environmental stresses such as temperature, drought, poor light or nutrient supply, as well as pathogen attack. Plant tissues exposed to environmental stress also produce ethylene, commonly known as stress ethylene (Buchanan-Wollaston, V., 1997, J. Exp. Botany, 48:181-199; Wright, M., 1974, Plant, 120:63-69). Ethylene is known to cause senescence in some plants.

[0007] Senescence is not a passive process, but, rather, is an actively regulated process that involves coordinated expression of specific genes. During senescence, the levels of total RNA decrease and the expression of many genes is switched off (Bate et al., 1991, J. Exper. Botany, 42, 801-11; Hensel et al., 1993, The Plant Cell, 5, 553-64). However, there is increasing evidence that the senescence process depends on de novo transcription of nuclear genes. For example, senescence is blocked by inhibitors of mRNA and protein synthesis and enucleation. Molecular studies using mRNA from senescing leaves and green leaves for in vitro translation experiments show a changed pattern of leaf protein products in senescing leaves (Thomas et al., 1992, J. Plant Physiol., 139, 403-12). With the use of differential screening and subtractive hybridization techniques, many cDNA clones representing senescence-induced genes have been identified from a range of different plants, including both monocots and dicots, such as Arabidopsis, maize, cucumber, asparagus, tomato, rice and potato. Identification of genes that are expressed specifically during senescence is hard evidence of the requirement for de novo transcription for senescence to proceed.

[0008] The events that take place during senescence appear to be highly coordinated to allow maximum use of the cellular components before necrosis and death occur. Complex interactions involving the perception of specific signals and the induction of cascades of gene expression must occur to regulate this process. Expression of genes encoding senescence related proteins is probably regulated via common activator proteins that are, in turn, activated directly or indirectly by hormonal signals. Little is known about the mechanisms involved in the initial signaling or subsequent co-ordination of the process.

[0009] Coordinated gene expression requires factors involved in transcription and translation, including initiation factors. Translation initiation factor genes have been isolated and characterized in a variety of organisms, including plants. Translation initiation factors can control the rate at which mRNA populations are moved out of the nucleus, the rate at which they are associated with a ribosome and to some extent can affect the stability of specific mRNAs. (Zuk, et al., EMBO J. 17:2914-2925 (1998). Indeed, one such translation initiation factor, which is not required for global translation activity, is believed to shuttle specific subsets of mRNAs from the nucleus to the cytoplasm for translation. Jao, et al., J. Cell. Biochem. 86:590-600, (2002); Wang et al., J Biol Chem 276:17541-17549 (2001); Rosorius et al., J. Cell Sci., 112, 2369-2380 (1999). This translation factor is known as the eukaryotic initiation factor 5A (eIF-5A), and is the only protein known to contain the amino acid hypusine. Park, et al., J Biol Chem 263:15264-15269 (1988).

[0010] Eukaryotic translation initiation factor 5A (eIF-5A) is an essential protein factor approximately 17 KDa in size, which is involved in the initiation of eukaryotic cellular protein synthesis. It is characterized by the presence of hypusine [N-(4-amino-2-hydroxybutyl)lysine], a unique modified amino acid, known to be present only in eIF-5A. Hypusine is formed post-translationally via the transfer and hydroxylation of the butylamino group from the polyamine, spermidine, to the side chain amino group of a specific lysine residue in eIF-5A. Activation of eIF-5A involves transfer of the butylamine residue of spermidine to the lysine of eIF-5A, forming hypusine and activating eIF-5A. In eukaryotes, deoxyhypusine synthase (DHS) mediates the post-translational synthesis of hypusine in eIF-5A. The hypusine modification has been shown to be essential for eIF-5A activity in vitro using a methionyl-puromycin assay.

[0011] Hypusine is formed on eIF-5A post-translationally through the conversion of a conserved lysine residue by the action of deoxyhypusine synthase (DHS; EC 1.1.1.249) and deoxyhypusine hydroxylase (DHH; EC 1.14.99.29). DHS has been isolated from several plant species, including tomato (GenBank Accession Number AF296077), Arabidopsis thaliana (AT-DHS; GenBank Accession Number AF296078), tobacco (Ober and Hartmann, 1999), carnation (GenBank Accession Number AF296079) and banana (GenBank Accession Number AF296080), whereas the gene for DHH has not been recognized.

[0012] DHS converts a conserved lysine residue of eIF-5A to deoxyhypusine through the addition of a butylamine group derived from spermidine. This intermediate form of eIF-5A is then hydroxylated by DHH to become hypusine. Park et al., Biol. Signals 6, 115-123 (1997). Both the deoxyhypusine and the hypusine form of eIF-5A are able to bind mRNA in vitro. Liu et al., Biol Signals 6:166-174 (1997). Although the function of eIF-5A is not fully understood, there is some evidence that it may regulate cell division (Park et al., J Biol Chem 263:15264-15269 (1998); Tome et al., Biol Signals 6:150-156, (1997)) and senescence. (Wang et al., J. Biol. Chem. 276(20): 17541-17549 (2001)). See also U.S. Pat. No. 6,538,182 and U.S. application Ser. No. 09/725,019, which are herein incorporated by reference in their entirety. It appears that several organisms are known to have more than one isoform of eIF-5A, which would suit the premise that each isoform is a specific shuttle to specific suites of mRNAs that are involved in such processes as cell division and senescence.

[0013] Wang et al. demonstrated that an increased level of DHS mRNA correlates with fruit softening and natural and stress-induced leaf senescence of tomato. Wang et al., J. Biol. Chem. 276(20):17541-17549 (2001). Furthermore when the expression of DHS was suppressed in transgenic tomato plants by introducing a DHS antisense cDNA fragment under the regulation of a constitutive promoter, the tomato fruit from these transgenic plants exhibited dramatically delayed senescence as evidenced by delayed fruit softening and spoilage. See U.S. Pat. No. 6,538,182 and U.S. application Ser. No. 09/725,019, filed Nov. 29, 2003, incorporated herein by reference in their entirety. Since DHS is known to activate eIF-5A, these data suggest that the hypusine-modified eIF-5A (active eIF-5A) may regulate senescence through selective translation of mRNA species required for senescence. This is further demonstrated through the down-regulation of DHS in Arabidopsis thaliana ("AT") by antisense of the full length or 3'UTR cDNA under the control of a constitutive promoter. By down regulating Arabidopsis thaliana DHS ("AT-DHS") expression and making it less available for eIF-5A activation, senescence was delayed by approximately 2 weeks (See U.S. Pat. No. 6,538,182). Not only was senescence delayed, but also an increase in seed yield, an increase in stress tolerance and an increase in biomass were observed in the transgenic plants, where the extent of each phenotype was determined by the extent of the down-regulation of DHS. Since tomato and Arabidopsis thaliana only have one copy of DHS in their genome, as shown by Southern blot (Wang et al., 2001) and BLAST analysis, in order to target the specific eIF-5A isoform responsible for shuttling of senescence transcripts out of the nucleus, the senescence specific isoform of eIF-5A must be identified and specifically down-regulated through the antisense constructs of senescence-induced eIF-5A (of the 3'UTR) or by taking advantage of the plant's natural ability for down-regulation of an over expressed gene (i.e. creating over-expression through the use of sense polynucleotides).

[0014] Plants lack immune systems and thus, have a unique way of dealing with viruses--called co-suppression, which results in sequence-specific degradation of the viral RNA. When a transgene is under a strong constitutive promoter, like the cauliflower mosaic virus double 35S promoter, it appears as a viral transcript to the plant and sequence-specific degradation occurs, but not just of the transgene, but also the endogenous gene. (reviewed in Fagard and Vaucheret, Annual Review. Plant Physiol. Plant Mol. Biol., June; 51:167-194 (2000). There is some evidence that co-suppression may be as effective, if not more effective, than antisense suppression of expression for the down-regulation of an endogenous gene.

SUMMARY OF THE INVENTION

[0015] The present invention provides isoforms of eukaryotic initiation Factor 5A ("eIF-5A"): senescence-induced eIF-5A; wounding-induced eIF-5A; and growth eIF-5A as well as polynucleotides that encode these three factors. The present invention provides antisense polynucleotides of the three eIF-5A isoforms. The invention also provide expression vectors comprising sense and antisense polynucleotides of the three eIF-5A isoforms. The present invention also relates to methods involving modulating (increasing/up-regulating or inhibiting) the expression of these factors.

[0016] The present invention also relates to deoxyhypusine synthase ("DHS") and polynucleotides that encode DHS. The present invention also provides antisense polynucleotides of DHS. The invention also provide expression vectors comprising sense and antisense polynucleotides of DHS. The present invention also relates to methods involving modulating (increasing/up-regulating or inhibiting) the expression of DHS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 shows the alignment of three isoforms of eIF-5A isolated Arabidopsis thaliana senescence-induced eIF-5A (line 1) (SEQ ID NO: 58) (previously described in U.S. Pat. No. 6,538,182 and pending application Ser. No. 09/725,019); wounding-induced eIF-5A (line 2) (SEQ ID NO: 59); and growth eIF-5A (line 3) (SEQ ID NO: 60). Identical amino acids are highlighted by dashed lines (----) and the regions that were used for peptide design are indicated by the solid lines. Each peptide contains eleven amino acids from eIF-5A sequences as well as additional cysteine residue at the N-terminus, for conjugation with KLH.

[0018] FIG. 2 shows the alignment of the coding regions of these three Arabidopsis thaliana isoforms. Line 1 is senescence-induced eIF-5A (SEQ ID NO: 61). Line 2 is wounding-induced eIF-5A (SEQ ID NO: 62). Line 3 is growth eIF-5A (SEQ ID NO: 63). Base pairs that are identical in all three isoforms are indicated in boxes. The sequences only include the coding region from the methionine (ATG) to the stop codon.

[0019] FIG. 3 provides the genomic sequence (SEQ ID NO: 78) of the senescence-induced eIF-5A of Arabidopsis thaliana. The dashed underscore (----) indicates the areas in which the primers were designed against. The 5' end primer also contained a HindIII restriction site and the 3' end primer contained a SacI restriction site to ensure proper orientation when ligated into the binary vector. The boxed area indicates the 3' end used as probe for Northern blots.

[0020] FIG. 4 provides the genomic sequence (SEQ ID NO: 79) of the wounding-induced eIF5A of Arabidopsis thaliana. The dashed underscore (----) indicates the areas in which the primers were designed against. The 5' end primer also contained a XhoI restriction site and the 3' end primer contained a SacI restriction site to ensure proper orientation when ligated into binary vector. The boxed area indicates the 3' end used as probe for Northern blots.

[0021] FIG. 5 provides the genomic sequence (SEQ ID NO: 52) of the growth eIF5A of Arabidopsis thaliana. The dashed underscore (----) indicates the areas in which the primers were designed against. The 5' end primer also contained a XhoI restriction site and the 3' end primer contained a SacI restriction site to ensure proper orientation when ligated into the binary vector. The boxed area indicates the 3' end used as probe for Northern blots.

[0022] FIG. 6 is a map of binary vector pKYLX71-355² (SEQ ID NO: 80). The binary vector pKYLX71-355² contains tetracycline resistance for transformant selection in E. coli, and kanamycin resistance for seed transformant selection on MS plates containing kanamycin. The promoter is a duplicate 35S promoter, which serves to give higher levels of expression than a single 35S. RbcS 3' is the UTR of ribulose-1,5-bisphosphate carboxylase.

[0023] FIG. 7 is a map of binary vector pGEM®-T Easy Vector. The T overhangs in the middle of the multiple cloning sites provide the insertion site of PCR products. The Amp^r gene is useful for screening transformants based on growth in the presence of ampicillin.

[0024] FIG. 8 shows Western blots of all three isoforms of eIF-5A in different tissues of Arabidopsis thaliana wild type of the Columbia ecotype. The lane descriptions are a follows: lanes labelled 2, 3, 4, 5, 6, 7 are the total rosette leaves collected at 2, 3, 4, 5, 6, 7 weeks of age, Tr are leaves from plants treated with 5% PEG, U are leaves from the PEG control plants watered with water, B are closed unopened flower buds, Fl are flowers of all ages ranging from closed buds to senescent flowers, Si are siliques that were collected at 6 weeks, Se are seeds that were imbibed for 1 day and St are stems collected at 6 week.

[0025] FIG. 9 are Western blots for the senescence-induced eIF-5A and the wounding-induced eIF-5A of infected leaves after 72 hours of Arabidopsis thaliana wild type of the Columbia ecotype. The expression level of senescence-induced AteIF-5A remains constant as these plants are all 4 weeks old. The expression of wounding-induced AteIF-5A increases in the virulent treated plants. The expression of growth AteIF-5A was not detectable and thus not included in the figure.

[0026] FIG. 10 are Northern blots for the three isoforms of eIF-5A in wounded leaves after 72 hours of Arabidopsis thaliana wild type of the Columbia ecotype. Leaves were wounded with a hemostat and collected at 0 hours, immediately after treatment, 1 hour after wounding and 9 hours after wounding. The expression of growth AteIF-5A3 though low to begin with decreases in the event of wounding.

[0027] FIG. 11 depicts PCR products from genomic DNA of senescence-induced AteIF-5A, wounding-induced AteIF-5A, and growth AteIF-5A in lanes 1, 2 and 3 respectively. The single top band was excised and purified for ligation into pGEM.

[0028] FIG. 12 shows an agarose gel with senescence-induced AteIF-5A, wounding-induced AteIF-5A, and growth AteIF-5A genomic sequences in pGEM. The pGEM: senescence-induced AteIF5A, pGEM: wounding-induced AteIF5A, and pGEM: growth AteIF5A were digested with EcoRI for to identify positive transformant colonies that contain inserts of the proper size. These clones were then sent for sequencing to confirm sequence suitability for over expression in planta.

[0029] FIG. 13 shows an agarose gel with wounding-induced AteIF-5A, growth AteIF-5A, genomic sequences in pKYLX71. The colonies that were able to grow on tetracycline containing plates were screened for either the wounding-induced AteIF-5A insert or the growth AteIF-5A insert through both double digestion (D) with appropriate enzymes and PCR (P) with the corresponding primers.

[0030] FIG. 14 is a picture of a T1 plate for plants transformed with a construct having sense wounding-induced AteIF-5A. Two transformants on this plate are circled in black and correspond to lines 13 and 14. The wild type controls are circled in white.

[0031] FIG. 15 is a picture of T1 plants transformed with Sense wounding-induced AteIF-5A at 4 weeks of age. The transgenic lines are indicated by the P tags, the wildtype plants are indicated by the W tags and the binary vector control plants are indicated by the Y tags. Lines 6, 8, 10, 13 and 14 did not produce seeds.

[0032] FIG. 16 is a picture of T1 plants transformed with Sense wounding-induced AteIF-5A at 5.5 weeks of age. Just the lines that were very small are included in this figure. Lines 1, 4, and 12 all produced seed and the rest died eventually without producing seed.

[0033] FIG. 17 is a picture of T2 plants transformed with Sense wounding-induced AteIF-5A at 10 days post seeding. All the T2 lines remain heterozygous as indicated by the mix of kanamycin resistant (dark plants) and non-transformants lacking kanamycin resistance (light plants). Wild type control plants are indicated in the white circles. Line 12 in not included in the figure as it only had one transformant grow and has yet to be transplanted.

[0034] FIG. 18 is a picture of T1 plants transformed with Sense growth AteIF-5A at 10 days post seeding. Three transformants are indicated in black circles for this plate and correspond to lines 6, 7 and 8. Wild type control plants are indicated in the white circle.

[0035] FIG. 19 is a picture of TI plants transformed with Sense growth AteIF-5A at 4 weeks of age. The transformant lines are indicated by the B tags and wild type control by the W tags or the lack of tags. The empty binary control (Y tags) in included at the bottom of the figure showing that it looks no different than wild type.

[0036] FIG. 20 is a Western blot of T2 plants transformed with Sense growth AteIF-5A lines. A representative of each mother line was used to determine the general level of expression in each line.

[0037] FIG. 21 are T2 plants transformed with Sense growth AteIF-5A (Lines 1A-1D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags and wild type control by the W tags. The empty binary control are indicated by Y tags. Line 1A (indicated in the black box) will be carried through to T3.

[0038] FIG. 22 are T2 plants transformed with Sense growth AteIF-5A (Lines 2A-1D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags (grey circles) and wild type control by the W tags (white ellipse). The empty binary control are indicated by Y tags (black circles). Line 2D (indicated in the black box) will be carried through to T3.

[0039] FIG. 23 are T2 plants transformed with Sense growth AteIF-5A (Lines 4A-D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags (grey Circles) and wild type control by the W tags (white ellipse). The empty binary control are indicated by Y tags (black circle). Line 4D (indicated in the black box) will be carried through to T3.

[0040] FIG. 24 are T2 plants transformed with Sense growth AteIF-5A (Lines 15A-D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags and wild type control by the W tags. The empty binary control are indicated by Y tags. Line 15A (indicated in the black box) will be carried through to T3.

[0041] FIG. 25 are T2 plants transformed with Sense growth AteIF-5A (Lines 8A-D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags and wild type control by the W tags. The empty binary control are indicated by Y tags. Line 8D (indicated in the black box) will be carried through to T3.

[0042] FIG. 26 are T2 plants transformed with Sense growth AteIF-5A (Lines 9E-H) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags and wild type control by the W tags. The empty binary control are indicated by Y tags. Line 9H (indicated in the black box) will be carried through to T3.

[0043] FIG. 27 are T2 plants transformed with Sense growth AteIF-5A (Lines 11A-D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags and wild type control by the W tags. The empty binary control are indicated by Y tags. Line 11C (indicated in the black box) will be carried through to T3.

[0044] FIG. 28 are T2 plants transformed with Sense growth AteIF-5A (Lines 16A-D) at 4 weeks of age (top), 5 weeks of age (bottom left) and 6 weeks of age (bottom right). The transformant lines are indicated by the B tags and wild type control by the W tags. The empty binary control are indicated by Y tags. Line 16C (indicated in the black box) will be carried through to T3.

[0045] FIG. 29 are photographs of Arabidopsis thaliana seeds from various plant lines (including wild type control and plant lines having been transformed with sense growth AteIF-5A. Lines 11C and 16C are only 88 and 87% of the average wild type seed size, whereas lines 2D and 2H are 273 and 299% larger than wild type, respectively.

[0046] FIG. 30 is a bar graph of average seed size for each plant subline having been transformed with sense growth AteIF-5A. Each line has sublines A-H not labeled separately in the figure. The binary control and the wild type controls correspond to the last two bars. The standard errors as represented by the error bars were calculated with n=10.

[0047] FIG. 31 is a bar graph of individual seed weight for each plant subline having been transformed with sense growth AteIF-5A. Each line has sublines A-H. The binary control and the wild type controls correspond to the last two bars.

[0048] FIG. 32 is a graph showing the proportional relationship between the weight of the individual seeds versus the volume of individual seeds.

[0049] FIG. 33 is a bar graph showing seed yield per plant for each plant subline having been transformed with sense growth AteIF-5A. Each line has sublines A-H. The binary control and the wild type controls correspond to the last two bars.

[0050] FIG. 34 is a summary of phenotypes displayed in sense growth AteIF-5A plants. The phenotypes are categorized based on the level of expression as determined by Western blotting. The lines that demonstrate high level of expression are blocked in cross-hashing, the lines that demonstrate medium level of expression are blocked in hashing, and the lines that demonstrate low levels of expression or no expression, probably by cosuppresion, are blocked in white.

[0051] FIG. 35 shows a comparison of transgenic arabidopsis plant (transformed with antisense full length senescence-induced eIF-5A) with a wild type plant. The transgenic plant is dwarfed, has an increased number of small rosette leaves, and exhibits delayed senescence.

[0052] FIGS. 36-38 show photographs of a plant (transformed with antisense growth eIF-5A).

[0053] FIG. 39 shows the primers (SEQ ID NOS: 81-82, respectively) used to construct the vector for generating antisense arabidopsis thaliana 3' DHS. Amplified Arabidopsis sequences are shown in SEQ ID NOS: 83-84, respectively.

[0054] FIG. 40 shows the vector construct.

[0055] FIG. 41 shows the sequence for wounding factor eIF-5A (DNA shown in SEQ ID NO: 54, Amino acid sequence shown in SEQ ID NO: 55) isolated from arabidopsis and the location of the antisense construct. The primer sequences are shown in SEQ ID NOS: 85-86, respectively.

[0056] FIG. 42 shows the vector construct (nucleotide sequences shown in SEQ ID NOS: 87-89, respectively).

[0057] FIG. 43 shows plate counts of leaf discs inoculated with pseudomonas. Table 1: shows standard plate counts of A. thaliana leaf discs inoculated with virulent or avirulent Pseudomonas syringae.

[0058] FIG. 44 shows a graph of CFUs in antisense transgenic plants versus wild-type.

[0059] FIG. 45 depict the nucleotide sequence of the tomato leaf DHS cDNA sequence (SEQ ID NO:1) and the derived amino acid sequence (SEQ ID NO. 2) obtained from a tomato leaf cDNA library.

[0060] FIG. 46 depicts the nucleotide sequence of an Arabidopsis DHS gene obtained by aligning the tomato DHS sequence with unidentified genomic sequences in the Arabidopsis gene bank (SEQ ID NO: 5). The gaps between amino acid sequences are predicted introns. FIG. 46B depicts the derived Arabidopsis DHS amino acid sequence (SEQ ID NO: 6). FIG. 46C depicts the nucleotide sequence (SEQ ID NO: 26) of a 600 base pair Arabidopsis DHS cDNA obtained by PCR. FIG. 46D depicts the derived amino acid sequence (SEQ ID NO: 92) of the Arabidopsis DHS cDNA fragment.

[0061] FIG. 47 is an alignment of the derived full length tomato leaf DHS amino acid sequence (SEQ ID NO. 2) and the derived full length (SEQ ID NO: 6) Arabidopsis senescence-induced DHS amino acid sequence with sequence of DHS proteins of human (SEQ ID NO: 3), yeast (SEQ ID NO: 45), fungi (SEQ ID NO: 44), and Archaeobacteria (SEQ ID NO: 46). Identical amino acids among three or four of the sequences are boxed.

[0062] FIG. 48 is a restriction map of the tomato DHS cDNA.

[0063] FIG. 49 is a Southern blot of genomic DNA isolated from tomato leaves and probed with ³²P-dCTP-labeled full length tomato DHS cDNA.

[0064] FIG. 50 is a Northern blot of RNA isolated from tomato flowers at different stages of development. The top panel is the ethidium bromide stained gel of total RNA. Each lane contains 10 μg RNA. The bottom panel is an autoradiograph of the Northern blot probed with ³²P-dCTP-labeled full length tomato DHS cDNA.

[0065] FIG. 51 is a Northern blot of RNA isolated from tomato fruit at various stages of ripening that was probed with ³²P-dCTP-labeled full length tomato DHS cDNA. Each lane contains 10 μg RNA.

[0066] FIG. 52 is a Northern blot of RNA isolated from tomato leaves that had been drought-stressed by treatment with 2 M sorbitol for six hours. Each lane contains 10 μg RNA. The blot was probed with ³²P-dCTP-labeled full length tomato DHS cDNA.

[0067] FIG. 53 is a Northern blot of RNA isolated from tomato leaves that had been exposed to chilling temperature. FIG. 53A is the ethidium bromide stained gel of total RNA. Each lane contained 10 μg RNA. FIG. 53B is an autoradiograph of the Northern blot probed with ³²P-dCTP-labeled full length tomato DHS cDNA. FIG. 53C shows corresponding leakage data measured as conductivity of leaf diffusates.

[0068] FIG. 54 is the carnation DHS full-length (1384 base pairs) cDNA clone nucleotide sequence (SEQ ID NO: 9) not including the PolyA tail and 5' end non-coding region. The derived amino acid sequence is shown below the nucleotide sequence (373 amino acids). (SEQ ID NO:10)

[0069] FIG. 55 is a Northern blot of total RNA from senescing Arabidopsis leaves probed with ³²P-dCTP-labeled full-length Arabidopsis DHS cDNA. The autoradiograph is at the top, the ethidium stained gel below.

[0070] FIG. 56 is a Northern blot of total RNA isolated from petals of carnation flowers at various stages. The blot was probed with ³²P-dCTP-labeled full-length carnation DHS cDNA. The autoradiograph is at the top, the ethidium stained gel below.

[0071] FIG. 57 is the nucleotide (top) (SEQ ID NO:11) and derived amino acid (bottom) (SEQ ID NO:12) sequence of the tomato fruit senescence-induced eIF-5A gene.

[0072] FIG. 58 is the nucleotide (top) (SEQ ID NO:13) and derived amino acid (bottom) (SEQ ID NO:14) sequence of the carnation senescence-induced eIF-5A gene.

[0073] FIG. 59 is the nucleotide (top) (SEQ ID NO:15) and derived amino acid (bottom) (SEQ ID NO:16) sequence of the Arabidopsis senescence-induced eIF-5A gene.

[0074] FIG. 60 is a Northern blot of total RNA isolated from leaves of Arabidopsis plants at various developmental stages. The blot was probed with ³²P-dCTP-labeled full-length Arabidopsis DHS cDNA and full-length senescence-induced eIF-5A. The autoradiograph is at the top, the ethidium stained gel below.

[0075] FIG. 61 is a Northern blot of total RNA isolated from tomato fruit at breaker (BK), red-firm (RF) and red-soft (RS) stages of development. The blot was probed with ³²P-dCTP-labeled full-length DHS cDNA and full-length senescence-induced eIF-5A. DHS and eIF-5A are up-regulated in parallel in red-soft fruit coincident with fruit ripening. The autoradiograph is at the top, the ethidium stained gel below.

[0076] FIG. 62 is a Northern blot of total RNA isolated from leaves of tomato that were treated with sorbitol to induce drought stress. C is control; S is sorbitol treated. The blot was probed with ³²P-dCTP-labeled full-length DHS cDNA and full-length senescence-induced eIF-5A. Both eIF-5A and DHS are up-regulated in response to drought stress. The autoradiograph is at the top, the ethidium stained gel below.

[0077] FIG. 63 is a Northern blot of total RNA isolated from flower buds and open senescing flowers of tomato plants. The blot was probed with ³²P-dCTP-labeled full-length senescence-induced DHS cDNA and full-length senescence-induced eIF-5A. Both eIF-5A and DHS are up-regulated in open/senescing flowers. The autoradiograph is at the top, the ethidium stained gel below.

[0078] FIG. 64 is a Northern blot of total RNA isolated from chill-injured tomato leaves. The blot was probed with ³²P-dCTP-labeled full-length DHS cDNA and full-length senescence-induced eIF-5A. Both eIF-5A and DHS are up-regulated with the development of chilling injury during rewarming. The autoradiograph is at the top, the ethidium stained gel below.

[0079] FIG. 65 is a photograph of 3.1 week old Arabidopsis wild-type (left) and transgenic plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 80) in antisense orientation showing increased leaf size in the transgenic plants.

[0080] FIG. 66 is a photograph of 4.6 week old Arabidopsis wild-type (left) and transgenic plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 80) in antisense orientation showing increased leaf size in the transgenic plants.

[0081] FIG. 67 is a photograph of 5.6 week old Arabidopsis wild-type (left) and transgenic plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 80) in antisense orientation showing increased leaf size in the transgenic plants.

[0082] FIG. 68 is a photograph of 6.1 week old Arabidopsis wild-type (left) and transgenic plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 80) in antisense orientation showing increased size of transgenic plants.

[0083] FIG. 69 is a graph showing the increase in seed yield from three T₁ transgenic Arabidopsis plant lines expressing the DHS gene in antisense orientation. Seed yield is expressed as volume of seed. SE for n=30 is shown for wild-type plants.

[0084] FIG. 70 is a photograph of transgenic tomato plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 80) in antisense orientation (left) and wild-type plants (right) showing increased leaf size and increased plant size in the transgenic plants. The photograph was taken 18 days after transfer of the plantlets to soil.

[0085] FIG. 71 is a photograph of transgenic tomato plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 36) in antisense orientation (left) and wild-type plants (right) showing increased leaf size and increased plant size in the transgenic plants. The photograph was taken 32 days after transfer of the plantlets to soil.

[0086] FIGS. 72 through 79 are photographs of tomato fruit from wild-type (top panels) and transgenic plants expressing the full-length DHS gene in antisense orientation (bottom panels). Fruit were harvested at the breaker stage of development and ripened in a growth chamber. Days after harvest are indicated in the upper left corner of each panel.

[0087] FIG. 80 is the nucleotide (top) (SEQ ID NO:30) and derived amino acid (bottom) sequence (SEQ ID NO: 90) of the 3'-end of the Arabidopsis senescence-induced DHS gene used in antisense orientation to transform plants.

[0088] FIG. 81 is the nucleotide (top) (SEQ ID NO:31) and derived amino acid (bottom) sequence (SEQ ID NO: 91) of the 3'-end of the tomato DHS gene used in antisense orientation to transform plants.

[0089] FIG. 82 is the nucleotide (top) (SEQ ID NO:26) and derived amino acid (bottom) sequence (SEQ ID NO: 92) of a 600 base pair Arabidopsis DHS probe used to isolate the full-length Arabidopsis gene.

[0090] FIG. 83 is the nucleotide (top) (SEQ ID NO:27) and derived amino acid (bottom) sequence (SEQ ID NO: 93) of the 483 base pair carnation DHS probe used to isolate the full-length carnation gene.

[0091] FIGS. 84 (a) and (b) are photographs of tomato fruits from transgenic tomato plants expressing the 3'-end of the DHS gene (sequence shown in FIG. 81) in antisense orientation (right) and tomato fruits from wild-type plants (left). While the wild-type fruit exhibits blossom end rot, the transgenic fruit does not.

[0092] FIG. 85 shows the alignment of various isoforms of eIF-5A from several plant species. It also provides alignment of the hypusine conserved region. See SEQ ID NOS 4 and 94-125, respectively, in order of appearance.

[0093] FIG. 86 provides tomato senescence-induced eIF-5A polynucleotide (SEQ ID NO: 126) and amino acid (SEQ ID NO: 127) sequences.

[0094] FIG. 87 provides Arabidopsis senescence-induced eIF-5A and the construction of pKYLX71-sense senescence-induced eIF-5A. The primer sequences are shown in SEQ ID NOS 128-129, respectively, while the vector sequences are shown in SEQ ID NOS 130-132, respectively.

[0095] FIG. 88 provides tomato senescence-induced eIF-5A and the construction of pKYLX71-sense senescence-induced eIF-5A. The primer sequences are shown in SEQ ID NOS 133-134, respectively, while the vector sequences are shown in SEQ ID NOS 135-137, respectively.

[0096] FIG. 89 provides photographs of a comparison of Arabidopsis thaliana control and transgenic plants comprising a sense polynucleotide senescence-induced eIF-5A. The transgenic plant has thicker inflorescence stems over that of the control plant.

[0097] FIGS. 90 and 91 shows that transgenic plants comprising an sense polynucleotide senescence-induced eIF-5A (FIG. 90--arabidopsis and FIG. 91--tomato) have increased xylogenesis as indicated by the increased xylem in the transgenic plant. The xylem zones were stained grey with phlorogucinol-HCl, bar=100 μm.

[0098] FIG. 92 provides photographs of a comparison of Arabidopsis thaliana control and Arabidopsis thaliana transgenic plants comprising a sense polynucleotide senescence-induced eIF-5A. A tomato sense polynucleotide senescence-induced eIF-5A was used in Arabidopsis thaliana. The transgenic plant has thicker inflorescence stems over that of the control plant.

[0099] FIGS. 93 and 94 are bar graphs that show increased xylogenesis in transgenic plants comprising a sense polynucleotide senescence-induced eIF-5A. FIG. 94 concerned tomato sense polynucleotide senescence-induced eIF-5A.

[0100] FIG. 95 provides canola growth eIF-5A amino acid (SEQ ID NO: 67) and polynucleotide (SEQ ID NO: 66) sequences.

[0101] FIG. 96 provides canola growth eIF-5A and the construction of pKYLX71-sense growth eIF-5A. The primer sequence is shown in SEQ ID NO: 138, while the vector sequences are shown in SEQ ID NOS 139-141, respectively.

[0102] FIG. 97 provides canola DHS amino acid (SEQ ID NO: 71) and polynucleotide (SEQ ID NO: 70) sequences.

[0103] FIG. 98 provides canola DHS and the construction of pKYLX71-sense DHS. The 3'-UTR sequence is shown in SEQ ID NO: 142, while the vector sequences are shown in SEQ ID NOS 143-145, respectively.

[0104] FIG. 99 shows in bar graph form that inhibition of DHS expression increases seed yield in canola.

[0105] FIG. 100 shows in bar graph form that up regulation of growth isoforms of eIF-5A from left to right arabidopsis, canola, tomato, and up regulation of tomato DHS.

[0106] FIG. 101 provides tomato growth eIF-5A amino acid (SEQ ID NO: 65) and polynucleotide (SEQ ID NO: 64) sequences.

[0107] FIG. 102 provides tomato growth eIF-5A and the construction of pKYLX71-sense tomato growth eIF-5A. The primer sequences are shown in SEQ ID NOS 146-147, respectively, while the vector sequences are shown in SEQ ID NOS 148-150, respectively.

[0108] FIG. 103 provides tomato wounding-induced eIF-5A amino acid (SEQ ID NO: 57) and polynucleotide (SEQ ID NO: 56) sequences.

[0109] FIGS. 104a and b provides tomato wounding-induced eIF-5A and the construction of pKYLX71-sense tomato wounding-induced eIF-5A. The primer sequences are shown in SEQ ID NOS 151-152, respectively, while the vector sequences are shown in SEQ ID NOS 153-155, respectively.

[0110] FIG. 105 provides portions of lettuce DHS polynucleotide sequences. The primer sequences are shown in SEQ ID NOS 156-157, respectively, while the Lettuce sequences are shown in SEQ ID NOS 158-159, respectively.

[0111] FIG. 106 provides the construct of pTA7001-3'UTR antisense lettuce DHS.

[0112] FIGS. 107A and B provides alfalfa DHS amino acid (SEQ ID NO: 73) and polynucleotide (SEQ ID NO: 72) sequences.

[0113] FIGS. 108A and B provides banana DHS amino acid (SEQ ID NO: 75) and polynucleotide (SEQ ID NO: 74) sequences.

[0114] FIGS. 109A and B provides cottonwood DHS amino acid (SEQ ID NO: 77) and polynucleotide (SEQ ID NO: 76) sequences.

[0115] FIG. 110 provides partial mycosphaerella fijiensis DHS amino acid and polynucleotide sequences. (see SEQ ID NOS 68, 160, 69, 161-164, 163 and 165, 47, 163 and 53, respectively, in order of appearance).

DETAILED DESCRIPTION

[0116] As used herein, the term "plant" refers to either a whole plant, a plant part, a plant cell or a group of plant cells. The type of plant which can be used in the methods of the invention is not limited and includes, for example, ethylene-sensitive and ethylene-insensitive plants; fruit bearing plants such as apricots, apples, oranges, bananas, grapefruit, pears, tomatoes, strawberries, avocados, etc.; vegetables such as carrots, peas, lettuce, cabbage, turnips, potatoes, broccoli, asparagus, etc.; flowers such as carnations, roses, mums, etc.; agronomic crop plants such as corn, rice, soybean, alfalfa and the like, and forest species such as deciduous trees, conifers, evergreens, etc., and in general, any plant that can take up and express the DNA molecules of the present invention. It may include plants of a variety of ploidy levels, including haploid, diploid, tetraploid and polyploid. The plant may be either a monocotyledon or dicotyledon.

[0117] A transgenic plant is defined herein as a plant which is genetically modified in some way, including but not limited to a plant which has incorporated heterologous or homologous senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS into its genome. The altered genetic material may encode a protein, comprise a regulatory or control sequence, or may be or include an antisense sequence or sense sequence or encode an antisense RNA or sense RNA which is antisense or sense to senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS DNA or mRNA sequence or portion thereof of the plant. A "transgene" or "transgenic sequence" is defined as a foreign gene or partial sequence that has been incorporated into a transgenic plant.

[0118] The term "hybridization" as used herein is generally used to mean hybridization of nucleic acids at appropriate conditions of stringency as would be readily evident to those skilled in the art depending upon the nature of the probe sequence and target sequences. Conditions of hybridization and washing are well known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature and/or ionic strength of the solution are readily accomplished. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989. The choice of conditions is dictated by the length of the sequences being hybridized, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted. Low stringency conditions are preferred when partial hybridization between strands that have lesser degrees of complementarity is desired. When perfect or near perfect complementarity is desired, high stringency conditions are preferred. What is meant herein as high stringency conditions is as follows: the hybridization solution contains 6×S.S.C., 0.01 M EDTA, 1×Denhardt's solution and 0.5% SDS. Hybridization is carried out at about 68° C. for about 3 to 4 hours for fragments of cloned DNA and for about 12 to about 16 hours for total eukaryotic DNA. For lower stringencies the temperature of hybridization is reduced to about 42° C. below the melting temperature (T_M) of the duplex. The T_M is known to be a function of the G-C content and duplex length as well as the ionic strength of the solution.

[0119] As used herein, the term "substantial sequence identity" or "substantial homology" is used to indicate that a nucleotide sequence or an amino acid sequence exhibits substantial structural or functional equivalence with another nucleotide or amino acid sequence. Any structural or functional differences between sequences having substantial sequence identity or substantial homology will be de minimis; that is, they will not affect the ability of the sequence to function as indicated in the desired application. Differences may be due to inherent variations in codon usage among different species, for example. Structural differences are considered de minimis if there is a significant amount of sequence overlap or similarity between two or more different sequences or if the different sequences exhibit similar physical characteristics even if the sequences differ in length or structure. Such characteristics include, for example, ability to hybridize under defined conditions, or in the case of proteins, immunological crossreactivity, similar enzymatic activity, etc. Each of these characteristics can readily be determined by the skilled practitioner by art known methods.

[0120] Additionally, two nucleotide sequences are "substantially complementary" if the sequences have at least about 70 percent, more preferably, 80 percent and most preferably about 90 percent sequence similarity between them. Two amino acid sequences are substantially homologous if they have at least 70% similarity between the active portions of the polypeptides.

[0121] As used herein, the phrase "hybridizes to a corresponding portion" of a DNA or RNA molecule means that the molecule that hybridizes, e.g., oligonucleotide, polynucleotide, or any nucleotide sequence (in sense or antisense orientation) recognizes and hybridizes to a sequence in another nucleic acid molecule that is of approximately the same size and has enough sequence similarity thereto to effect hybridization under appropriate conditions. For example, a 100 nucleotide long antisense molecule from the 3' coding or non-coding region of tomato wounding-induced eIF-5A will recognize and hybridize to an approximately 100 nucleotide portion of a nucleotide sequence within the 3' coding or non-coding region, respectively of AT wounding-induced eIF-5A gene or any other plant wounding-induced eIF-5A gene so long as there is about 70% or more sequence similarity between the two sequences. It is to be understood that the size of the "corresponding portion" will allow for some mismatches in hybridization such that the "corresponding portion" may be smaller or larger than the molecule which hybridizes to it, for example 20-30% larger or smaller, preferably no more than about 12-15% larger or smaller.

[0122] The term "functional derivative" of a nucleic acid (or polynucleotide) as used herein means a fragment, variant, homolog, or analog of the gene or nucleotide sequence encoding senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS. A functional derivative retains at least a portion of the function of the senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS encoding DNA, which permits its utility in accordance with the invention. Such function may include the ability to hybridize under high stringency conditions with native isolated senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS or substantially homologous DNA from another plant or an mRNA transcript thereof, and which senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS in antisense orientation inhibits expression of senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS.

[0123] A "fragment" of the gene or DNA sequence refers to any subset of the molecule, e.g., a shorter polynucleotide or oligonucleotide. A "variant" refers to a molecule substantially similar to either the entire gene or a fragment thereof, such as a nucleotide substitution variant having one or more substituted nucleotides, but which maintains the ability to hybridize with the particular gene or to encode mRNA transcript which hybridizes with the native DNA. A "homolog" refers to a fragment or variant sequence from a different plant genus or species. An "analog" refers to a non-natural molecule substantially similar to or functioning in relation to either the entire molecule, a variant or a fragment thereof.

[0124] By "modulating expression" it is meant that either the expression is inhibited or up-regulated "Inhibition of expression" refers to the absence or detectable decrease in the level of protein and/or mRNA product from a target gene, such as senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS. "Up-regulation" or "over expression" refers to a detectable increase in the level of protein and/or mRNA product from a target gene, such as senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS.

[0125] Isolated polynucleotides of the present invention include those isolated from natural sources, recombinantly produced or synthesized.

[0126] Isolated peptides of the present invention include those isolated from natural sources, recombinantly produced or synthesized. Isolated proteins of the present invention include senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS expressed as a fusion protein, preferably comprising eIF-5A or DHS fused with maltose binding protein.

[0127] "Functional derivatives" of the senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A, or DHS peptides of the present invention include fragments, variants, analogs, or chemical derivatives of senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A or DHS, which retain at least a portion of the activity or immunological cross reactivity with an antibody specific for the eIF-5A isoform or DHS. A fragment of eIF-5A or DHS peptide refers to any subset of the molecule. Variant peptides may be made by direct chemical synthesis, for example, using methods well known in the art. An analog of eIF-5A or DHS peptide refers to a non-natural protein substantially similar to either the entire protein or a fragment thereof. Chemical derivatives of eIF-5A or DHS contain additional chemical moieties not normally a part of the peptide or peptide fragment. Modifications may be introduced into peptides or fragments thereof by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

[0128] A eIF-5A or DHS peptide according to the invention may be produced by culturing a cell transformed with a nucleotide sequence of this invention (in the sense orientation), allowing the cell to synthesize the protein and then isolating the protein, either as a free protein or as a fusion protein, depending on the cloning protocol used, from either the culture medium or from cell extracts. Alternatively, the protein can be produced in a cell-free system. Ranu, et al., Meth. Enzymol., 60:459-484, (1979).

[0129] Preparation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis of DNA, polyacrylamide gel electrophoresis of protein, PCR, RT-PCR, Southern blots, Northern blots, DNA ligation and bacterial transformation were carried out using conventional methods well-known in the art. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989. Techniques of nucleic acid hybridization are disclosed by Sambrook.

[0130] Procedures for constructing recombinant nucleotide molecules in accordance with the present invention are disclosed in Sambrook, et al., In: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Maniatis, T. et al., Molecular mechanisms in the Control of Gene expression, eds., Nierlich, et al., eds., Acad. Press, N.Y. (1976), which are both incorporated herein in its entirety.

[0131] Transgenic plants made in accordance with the present invention may be prepared by DNA transformation using any method of plant transformation known in the art. Plant transformation methods include direct co-cultivation of plants, tissues or cells with Agrobacterium tumefaciens or direct infection (Miki, et al., Meth. in Plant Mol. Biol. and Biotechnology, (1993), p. 67-88); direct gene transfer into protoplasts or protoplast uptake (Paszkowski, et al., EMBO J., 12:2717 (1984); electroporation (Fromm, et al., Nature, 319:719 (1986); particle bombardment (Klein et al., BioTechnology, 6:559-563 (1988); injection into meristematic tissues of seedlings and plants (De LaPena, et al., Nature, 325:274-276 (1987); injection into protoplasts of cultured cells and tissues (Reich, et al., BioTechnology, 4:1001-1004 (1986)).

[0132] Generally a complete plant is obtained from the transformation process. Plants are regenerated from protoplasts, callus, tissue parts or explants, etc. Plant parts obtained from the regenerated transgenic plants in which the expression of the eIF-5A isoform or DHS is altered, such as leaves, flowers, fruit, seeds and the like are included in the definition of "plant" as used herein. Progeny, variants and mutants of the regenerated plants are also included in the definition of "plant."

eIF-5A Generally

[0133] The present invention relates to three different isoforms of eIF-5A: senescence-induced eIF-5A; wounding induced eIF-5A; and growth eIF-5A. The present invention provides various isoforms of eIF-5A isolated from various plant species and methods of isolating the various isoforms eIF-5A. The present invention also provides polynucleotides that encode these various isoforms of eIF-5A of the present invention. The invention also provides antisense polynucleotides of the isoforms of eIF-5A and expression vectors containing such polynucleotides or antisense polynucleotides. In some embodiments, there are provided methods of inhibiting expression of endogenous eIF-5As through the use of expression vectors containing antisense polynucleotides of the isoforms of eIF-5A to transform plants. In some embodiments, there are provided methods of up-regulating endogenous eIF-5A isoforms by providing expression vectors containing polynucleotides of the isoforms of eIF-5A in the sense orientation.

[0134] The different isoforms are naturally up or down-regulated depending upon the life stage of the plant or the plant's condition. For example in senescing tissues, the senescence-induced eIF-5A isoform is up-regulated. The senescence-induced eIF-5A is thought to participate in further senescence of the plant or plant tissues by shuttling specific subsets of mRNAs (those involved in the senescence pathway) from the nucleus to the cytoplasm for translation. By down regulating or inhibiting the expression of senescence-induced eIF-5A, senescence can be delayed in the plant and/or plant tissues. Delayed senescence is manifested in the transformed/transgenic plants by having a larger bio-mass, increased shelf life for fruit, increased shelf life of flowers, increased seed size and increased seed yield as compared to non-transformed or wild type plants.

[0135] When a plant and/or plant tissues are exposed to a wounding event, such as chilling, dehydration, or mechanical forces, wounding-induced eIF-5A isoform is up-regulated. By down regulating the expression of wounding-induced eIF-5A, an increased resistance to virulent damage arising from pathogen ingression is conferred on the plants as compared to resistance to virulent damage in non-transformed or wild type plants.

[0136] When a plant is in the growth phase, growth eIF-5A isoform is up-regulated. By up-regulating growth eIF-5A, the resulting transgenic plants have an increased seed size, increased biomass and increased seed yield.

[0137] FIG. 1 shows the alignment of three isoforms of eIF-5A isolated from Arabidopsis thaliana ("At"). FIG. 2 shows the alignment of the coding regions of these three isoforms. FIGS. 3-5 provide the genomic sequence of the three isoforms.

[0138] Western blots (see FIG. 8) show the expression in these three isoforms at different plant life stages. FIG. 8 reveals that the amount of the senescence-induced factor eIF-5A isoform increases as the ages of the leaves increases. It is not seen in the unopened flower buds, siliques or stems but it is seen in the imbibed seeds. In the imbibed seeds there is cotyledon tissue as well as growing embryo. Thus, senescence-induced eIF-5A is present in the imbibed seeds because the cotyledon tissue is senescing as the embryo is growing. Growth eIF-5A is seen in the imbibed seeds because there the embryo is actively growing. The wounding-induced eIF-5A is seen in the siliques, seeds and stems as the harvesting of these tissues induces some wounding.

[0139] Although there is a high degree of homology (about 85%) between the different isoforms and between the isoforms in different plant species, the different isoforms vary from each other in the 3'UTR. One region that is highly conserved between the isoforms and between species as well, is the area that is believed to be the hypusine site. The hypusine site is believed to be the following amino acids: 5'-CKVVEVSTSKTGKHGHAKCHFV-3' (SEQ ID NO:32). See FIG. 85 for alignment of various eIF-5A isoforms and of several plant species.

Senescence-Induced eIF-5A

[0140] Senescence-induced eIF-5A is expressed in senescing tissues. The present invention relates to the discovery of senescence-induced eIF-5A in Arabidopsis thaliana, tomato, and carnation plants. Senescence-induced eIF-5A is up-regulated in senescing tissues and is involved in the induction of senescence related morphological changes in plants and plant tissues. Inhibiting expression of senescence-induced eIF-5A in plants can be used to alter senescence and senescence-related processes in plants. Down-regulation may occur through either the use of antisense constructs or through use of sense constructs to achieve co-suppression Inhibiting expression of senescence-induced eIF-5A results in various morphological changes in the transgenic plants, including increased plant bio-mass, delayed fruit softening or spoilage, delayed browning of cut flowers or plant tissues, such as lettuce leaves, increased seed yield and increased seed size.

[0141] Thus, one embodiment of the present invention is isolated senescence-induced eIF-5A from Arabidopsis thaliana. The amino acid sequence is provided in FIG. 59 and is SEQ ID NO: 16. The polynucleotide encoding the amino acid is provided in FIG. 59 and is SEQ ID NO: 15.

[0142] Another embodiment of the present invention is isolated senescence-induced eIF-5A from tomato. The amino acid sequence is provided in FIG. 57 and is SEQ ID NO: 12. The polynucleotide encoding the amino acid is provided in FIG. 57 and is SEQ ID NO: 11.

[0143] Another embodiment of the present invention is isolated senescence-induced eIF-5A from carnation. The amino acid sequence is provided in FIG. 58 and is SEQ ID NO: 14. The polynucleotide encoding the amino acid is provided in FIG. 58 and is SEQ ID NO: 13.

[0144] The present invention also provides isolated polynucleotides of senescence-induced eIF-5A that have 90% sequence homology to the above enumerated SEQ ID NOs, and hybridize under high stringency conditions to the complement of the enumerated SEQ ID NOs and which encode senescence-induced eIF-5A.

[0145] The present invention also provides antisense polynucleotides of the senescence-induced eIF-5As. The antisense polynucleotides may be of any length as long as they are able to inhibit expression. In some embodiments the antisense polynucleotides comprise the full length coding sequence and in other particularly preferred embodiments the antisense polynucleotides are directed at the 3'UTR since the different isoforms of eIF-5A have a higher degree of variation in the isoforms at the 3'UTR. In some embodiments the antisense polynucleotides are directed at the 5'-non-coding sequence Antisense polynucleotides primarily complementary to 5'-non-coding sequences are known to be effective inhibitors of expression of genes encoding transcription factors. Branch, M. A., Molec. Cell Biol., 13:4284-4290 (1993).

[0146] The term "antisense polynucleotide of senescence-induced eIF5A" as used herein and in the claims encompasses not only those antisense polynucleotides that share 100% homology of the complement of an enumerated SEQ ID NO but also includes those antisense polynucleotides that are a functional variants. Functional variants are those variants, either natural or man made, that have at least 80% sequence homology to and hybridizes under high stringency conditions with the corresponding portion of the senescence-induced eIF-5A. Further the variant must have the function as intended by the present invention, that is it is capable of modulating expression of endogenous senescence-induced eIF-5A when introduced into an expression vector and wherein such vector is incorporated into the genome of at least one plant cell. One skilled in the art can appreciate that insubstantial changes can be made in the sequence that would not effect detrimentally the ability of the antisense polynucleotide to bind to the transcript and reduce or inhibition expression of the gene. Thus, the term "antisense polynucleotide" encompasses those polynucleotides that are substantially complementary to the transcript and that still maintain the ability to specifically bind to the transcript and inhibit or reduce gene expression. For a general discussion of antisense see Alberts, et al., Molecular Biology of the Cell, 2nd ed., Garland Publishing, Inc. New York, N.Y., 1989 (in particular pages 195-196, incorporated herein by reference).

[0147] One embodiment of the present invention provides expression vectors comprising either the senescence-induced eIF-5A polynucleotides (of the present invention as described above) or antisense polynucleotides of senescence-induced eIF-5A (of the present invention as described above). Vectors can be plasmids, preferably, or may be viral or other vectors known in the art to replicate and express genes encoded thereon in plant cells or bacterial cells. The vector becomes chromosomally integrated such that it can be transcribed to produce the desired antisense polynucleotide of senescence-induced eIF-5A RNA. Such plasmid or viral vectors can be constructed by recombinant DNA technology methods that are standard in the art. For example, the vector may be a plasmid vector containing a replication system functional in a prokaryotic host and an antisense polynucleotide according to the invention. Alternatively, the vector may be a plasmid containing a replication system functional in Agrobacterium and an antisense polynucleotide according to the invention. Plasmids that are capable of replicating in Agrobacterium are well known in the art. See, Miki, et al., Procedures for Introducing Foreign DNA Into Plants, Methods in Plant Molecular Biology and Biotechnology, Eds. B. R. Glick and J. E. Thompson. CRC Press (1993), PP. 67-83.

[0148] The vector further comprises regulatory sequences operatively linked to the polynucleotides to allow expression of such polynucleotides. The regulatory sequences may include a promoter functional in the transformed plant cell. The promoter may be inducible, constitutive, or tissue specific. Such promoters are known by those skilled in the art.

[0149] Promoter regulatory elements that are useful in combination with the various isoforms of eIF-5A and DHS of the present invention to generate sense or antisense transcripts of the gene include any plant promoter in general, and more particularly, a constitutive promoter such as the fig wart mosaic virus 35S promoter, the cauliflower mosaic virus promoter, CaMV35S promoter, or the MAS promoter, or a tissue-specific or senescence-induced promoter, such as the carnation petal GST1 promoter or the Arabidopsis SAG12 promoter (See, for example, J. C. Palaqui et al., Plant Physiol., 112:1447-1456 (1996); Morton et al., Molecular Breeding, 1: 123-132 (1995); Fobert et al., Plant Journal, 6:567-577 (1994); and Gan et al., Plant Physiol., 113:313 (1997), incorporated herein by reference). Preferably, the promoter used in the present invention is a constitutive promoter. The SAG12 promoter is preferably preferred when using antisense polynucleotides of senescence-induced eIF-5A. See example 23.

[0150] Expression levels from a promoter which is useful for the present invention can be tested using conventional expression systems, for example by measuring levels of a reporter gene product, e.g., protein or mRNA in extracts of the leaves, flowers, fruit or other tissues of a transgenic plant into which the promoter/reporter gene have been introduced. An exemplary reporter gene is GUS.

[0151] Optionally, the regulatory sequences include a 5' non-translated leader sequence or a polyadenylation signal or enhancers. The present invention further contemplates other regulatory sequences as known by those skilled in the art.

[0152] The invention also provides a transgenic plant cell transformed with a vector or combination of vectors of the present invention comprising polynucleotides of senescence-induced eIF-5A in sense or antisense orientation, a transgenic plantlet or mature transgenic plant generated from such a cell, or a plant part, such as a flower, fruit, leaves, seeds, etc. of the transgenic plant.

[0153] The present invention also provides methods of inhibiting expression of endogenous senescence-induced eIF-5A. These methods comprise integrating into the genome of at least one cell of a plant, expression vectors of the present invention comprising antisense polynucleotides of senescence-induced eIF-5A. The antisense polynucleotides of senescence-induced eIF-5A are transcribed and inhibit expression of endogenous senescence-induced eIF-5A.

[0154] In another method of inhibiting expression of endogenous senescence-induced eIF-5A, an expression vector containing a senescence-induced eIF-5A polynucleotide of the present invention in a sense orientation is integrated into the genome of at least one cell of a plant. The polynucleotide of senescence-induced eIF-5A is transcribed and the resulting co-expression of exogenous senescence-induced eIF-5A causes a down-regulation or inhibition of expression of endogenous senescence-induced eIF-5A.

Wounding-Induced eIF-5A

[0155] Wounding-induced eIF-5A is expressed in wounded tissues. The present invention relates to the discovery of wounding-induced eIF-5A in Arabidopsis thaliana and tomato. The present inventors have discovered that this isoform is upregulated during a wounding event to the plant. The up-regulation occurs at the transcriptional level. Further, it is up-regulated exclusively at the protein level following virulent infection, which then gives rise to cell death, leading to the inference that wounding-induced eIF-5A is driving cell death in the event of ingression by pathogens. FIG. 9 shows that senescence-induced eIF-5A remains constant in the control plant, the mock treated plant, the Avr treated plant and the Vir treated plant (it is detected as the plants were 4 weeks old). But wounding-induced eIF-5A is up-regulated in the Vir treated plant.

[0156] FIG. 10 shows the results of an experiment where leaves of a plant were wounded with a hemostat. Levels of senescence-induced eIF-5A, wounding-induced eIF-5A and growth eIF-5A in arabidopsis thaliana ("At") were measured immediately after the wounding, 1 hour, and 9 hours after the wounding. The Northern Blots show that senescence-induced eIF-5A remained constant, but there was a noticeable increase in the levels expression of the wounding-induced eIF-5A. The levels of expression of the growth eIF-5A began to decrease in the event of wounding.

[0157] The present inventors have demonstrated that when wounding-induced eIF-5A is up-regulated and a wounding event is imposed upon the plants (such as occurs when the seedlings are transplanted), this wounding results in a very strong suppression of growth eIF-5A. See FIGS. 14-17. The resulting plants have very stunted growth. But when the seeds are soaked in kanomycin and are planted directly into the soil (no need to transplant and thus no transplant wounding), the seeds develop into normal sized plants.

[0158] The differences seen between the various test plants all having a sense wounding-induced eIF-5A construct (FIG. 15) incorporated is due to varying degrees of expression of the wounding-induced eIF-5A. One skilled in the art will appreciate that when a gene is introduced (either sense or antisense) one gets varying degrees of either gene up-regulation or down-regulation. The degree of differences depends on where the gene gets incorporated and how many copies get incorporated. By having varying degrees of expression, one can correlate the various phenotypes to the gene expression. Once the desired phenotype is produced, that plant can be picked and used to create the desired progeny. Thus in FIG. 15, the plants that were strongly up-regulated for wounding-induced eIF-5A barely grew after the wounding event (plant tag 10), but the plants that grew a little better (but not as good as wild type) (plant tag 4) were not as strongly up-regulated.

[0159] One embodiment of the present invention is isolated wounding-induced eIF-5A from Arabidopsis thaliana. The amino acid sequence is provided in FIG. 41 and is SEQ ID NO: 55. The polynucleotide encoding the amino acid is provided in FIG. 41 and is SEQ ID NO: 54.

[0160] Another embodiment of the present invention is isolated wounding-induced eIF-5A from tomato. The amino acid sequence is provided in FIG. 103 and is SEQ ID NO: 57. The polynucleotide encoding the amino acid is provided in FIG. 103 and is SEQ ID NO: 56.

[0161] The present invention also provides isolated polynucleotides of wounding-induced eIF-5A that have 90% sequence homology to the above enumerated SEQ ID NOs, and hybridize under high stringency conditions to the complement of the enumerated SEQ ID NOs and which encode wounding-induced eIF-5A.

[0162] The present invention also provides antisense polynucleotides of the wounding-induced eIF-5As. The antisense polynucleotides may be of any length as long as they are able to inhibit expression. In some embodiments the antisense polynucleotides comprise the full length coding sequence and in other particularly preferred embodiments the antisense polynucleotides are directed at the 3'UTR since the different isoforms of eIF-5A have a higher degree of variation in isoforms at the 3'UTR. In some embodiments the antisense polynucleotides are directed at the 5'-non-coding sequence Antisense polynucleotides primarily complementary to 5'-non-coding sequences are known to be effective inhibitors of expression of genes encoding transcription factors. Branch, M. A., Molec. Cell Biol., 13:4284-4290 (1993).

[0163] The term "antisense polynucleotide of wounding-induced eIF5A" as used herein and in the claims encompasses not only those antisense polynucleotides that share 100% homology of the complement of an enumerated SEQ ID NO but also includes those antisense polynucleotides that are a functional variants. Functional variants are those as described above. The variant functions as intended by the present invention, that is it is capable of modulating expression of endogenous wounding-induced eIF-5A when introduced into an expression vector and wherein such vector is incorporated into the genome of at least one plant cell.

[0164] One embodiment of the present invention provides expression vectors comprising either wounding-induced eIF-5A polynucleotides (of the present invention as described above) or antisense polynucleotides of wounding-induced eIF-5A (of the present invention as described above). Vectors are as described above.

[0165] The invention also provides a transgenic plant cell transformed with a vector or combination of vectors of the present invention comprising polynucleotides of wounding-induced eIF-5A in sense or antisense orientation, a transgenic plantlet or mature transgenic plant generated from such a cell, or a plant part, such as a flower, fruit, leaves, seeds, etc. of the transgenic plant.

[0166] The present invention also provides methods of inhibiting expression of endogenous wounding-induced eIF-5A. These methods comprise integrating into the genome of at least one cell of a plant, expression vectors of the present invention comprising antisense polynucleotides of wounding-induced eIF-5A. The antisense polynucleotides of wounding-induced eIF-5A are transcribed and inhibit expression of endogenous wounding-induced eIF-5A.

[0167] In another method of inhibiting expression of endogenous wounding-induced eIF-5A, an expression vector containing a wounding-induced eIF-5A polynucleotide of the present invention in a sense orientation is integrated into the genome of at least one cell of a plant. The polynucleotide of wounding-induced eIF-5A is transcribed and the resulting co expression of exogenous wounding-induced eIF-5A causes a down-regulation or inhibition of expression of endogenous wounding-induced eIF-5A.

[0168] By inhibiting expression of endogenous eIF-5A, resulting transgenic plants have an increased resistance to virulent damage arising from pathogen ingression. See example 16 and FIGS. 43 and 44.

Growth eIF-5A

[0169] The present invention also relates to growth eIF-5A. Growth eIF-5A is expressed in growing tissues. When eIF-5A is up-regulated with polynucleotides of growth eIF-5A in sense orientation, three phenotypic changes are noticed: increased seed size, increased biomass, and increased seed yield.

[0170] One embodiment of the present invention is isolated growth eIF-5A from Arabidopsis thaliana. The amino acid sequences are provided in FIG. 1 and are SEQ ID NOS: 58-60, respectively. The polynucleotides encoding the amino acid sequences are provided in FIG. 2 and are SEQ ID NOS: 61-63, respectively.

[0171] Another embodiment of the present invention is isolated growth eIF-5A from tomato. The amino acid sequence is provided in FIG. 101 and is SEQ ID NO: 65. The polynucleotide encoding the amino acid is provided in FIG. 101 and is SEQ ID NO: 64.

[0172] Another embodiment of the present invention is isolated growth eIF-5A from canola. The amino acid sequence is provided in FIG. 95 and is SEQ ID NO: 67. The polynucleotide encoding the amino acid is provided in FIG. 95 and is SEQ ID NO: 66.

[0173] The present invention also provides isolated polynucleotides of growth eIF-5A that have 90% sequence homology to the above enumerated SEQ ID NOs, and hybridize under high stringency conditions to the complement of the enumerated SEQ ID NOs and which encode growth eIF-5A.

[0174] The present invention also provides antisense polynucleotides of the growth eIF-5As. The antisense polynucleotides may be of any length as long as they are able to inhibit expression. In some embodiments the antisense polynucleotides comprise the full length coding sequence and in other particularly preferred embodiments the antisense polynucleotides are directed at the 3'UTR since the different isoforms of eIF-5A have a higher degree of variation in isoforms at the 3'UTR. In some embodiments the antisense polynucleotides are directed at the 5'-non-coding sequence. Antisense polynucleotides primarily complementary to 5'-non-coding sequences are known to be effective inhibitors of expression of genes encoding transcription factors. Branch, M. A., Molec. Cell Biol., 13:4284-4290 (1993).

[0175] The term "antisense polynucleotide of growth eIF5A" as used herein and in the claims encompasses not only those antisense polynucleotides that share 100% homology of the complement of an enumerated SEQ ID NO but also includes those antisense polynucleotides that are a functional variants. Functional variants are those as described above. The variant functions as intended by the present invention, that is it is capable of modulating expression of endogenous growth eIF-5A when introduced into an expression vector and wherein such vector is incorporated into the genome of at least one plant cell.

[0176] One embodiment of the present invention provides expression vectors comprising either growth eIF-5A polynucleotides (of the present invention as described above) or antisense polynucleotides of growth eIF-5A (of the present invention as described above). Vectors are as described above.

[0177] The invention also provides a transgenic plant cell transformed with a vector or combination of vectors of the present invention comprising polynucleotides of growth eIF-5A either in sense or antisense orientation, a transgenic plantlet or mature transgenic plant generated from such a cell, or a plant part, such as a flower, fruit, leaves, seeds, etc. of the transgenic plant.

[0178] The present invention also provides methods of inhibiting expression of endogenous growth eIF-5A. These methods comprise integrating into the genome of at least one cell of a plant, expression vectors of the present invention comprising antisense polynucleotides of growth eIF-5A. The antisense polynucleotides of growth eIF-5A are transcribed and inhibit expression of endogenous growth eIF-5A.

[0179] In another method of inhibiting expression of endogenous growth eIF-5A, an expression vector containing a growth eIF-5A polynucleotide of the present invention in a sense orientation is integrated into the genome of at least one cell of a plant. The polynucleotide of growth eIF-5A is transcribed and the resulting co-expression of exogenous growth eIF-5A causes a down-regulation or inhibition of expression of endogenous growth eIF-5A.

[0180] In another embodiment of the present invention there is provided a method of up-regulating expression of growth eIF-5A. An expression vector containing a growth eIF-5A polynucleotide of the present invention in a sense orientation is integrated into the genome of at least one cell of a plant. The polynucleotide of growth eIF-5A is transcribed and the resulting co-expression of exogenous growth eIF-5A causes the cells to express more growth eIF-5A than non-transgenic cells.

[0181] FIG. 19 shows that plants that were up-regulated for growth eIF-5A had an increased biomass over that of the control plants. Growth eIF-5A was inserted into Arabidopsis thaliana plants in a sense orientation to up-regulate the expression of growth eIF-5A. Sixteen mother lines (1-16) were assayed to determine the general level of growth eIF-5A expression. From each mother line, 8 sister lines were produced (A-H). The level of expression of growth eIF-5A in each mother line was tested and the results shown in FIG. 20. Various degrees of expression are noticed throughout the mother lines. For example, lines 2 and 10 have very high levels of expression whereas lines 11 and 16 have very low or no expression.

[0182] FIGS. 21 and 22 show the plants from lines 1 and 2. These plants are bigger than the control plants. Because the growth eIF-5A is a cell-division isoform and because it is constitutively expressed, there is increased cell division. A reduction in senescence occurs because the plant is locked into a growth mode and can not make the switch to the senescence pathway.

[0183] FIGS. 23 and 24 are from lines that had medium level of expression of growth eIF-5A. They appear to have bigger leaves and delayed senescence.

[0184] FIGS. 25 and 26 are from lines that had low levels of up-regulation. They have large leaves and large rosettes.

[0185] FIGS. 27 and 28 are from lines that have no up-regulation (which may be due to co-suppression of the gene). Since the plant is kanomycin resistant, the gene must be present in order for the plants to grow on the media. It appears that the senescence-induced eIF-5A is also co-suppressed as well thus giving rise to an increase in size.

[0186] In addition to increased biomass, there is also increased seed size in plants having growth eIF-5A up-regulated. The seed size of all of the lines was measured. In the lines having the highest levels of growth eIF-5A expression, a greater than 3× increase in seed size is seen. This occurs because up-regulation of growth eIF-5A, increases cell division and thus increases seed size.

[0187] The growth eIF-5A (from Arabidopsis thaliana) in the above examples was being constitutively expressed, i.e. is being expressed everywhere in the plant through the use of a universal promoter. In contrast, by using a tissue specific promoter, one may direct the up-regulation in particular tissues. For example, by using a seed specific promoter, the growth eIF-5A would only be up-regulated in the seed, allowing the leaves to grow normally, but produce an increase in the amount of seeds. Thus, using a specific promoter, the growth eIF-5A can be up-regulated in the desired plant part to get a desired phenotype.

[0188] By up-regulating growth eIF-5A, three phenotypes result--increased biomass, increased seed yield, or increased seed size, but not all three phenotypes are present at the same time (or in the same plant). For example, if a plant exhibits an increase in seed size, a smaller plant will be present. In the plant lines that had the highest up-regulation of growth eIF-5A, the biggest seeds were produced, but the plants were smaller because there was massive cell division going on throughout the whole plant, which was at the expense of cell enlargement (needed for bigger leaves). At lower levels of up-regulation of expression of growth AteIF-5A, one sees an impact on the leaves (bigger) without impacting the seed. Thus, one may use tissue specific expression and pick the phenotype desired. For example, one may place growth eIF-5A under a xylem specific promoter to achieve an increase in the amount of xylem produced. Thus, any desired promoter may be used to achieve the desired tissue-specific up-regulation.

DHS

[0189] DHS is necessary for the activation of eIF-5A and is expressed in senescing tissues. The present invention thus provides isolated DHS from Arabidopsis thaliana, tomato, carnation, canola, lettuce, alfalfa, banana, cottonwood, and mycosphaerella.

[0190] Thus one embodiment of the present invention is isolated DHS from Arabidopsis thaliana. The amino acid sequence is provided in FIG. 46B and is SEQ ID NO: 6. The polynucleotide encoding the amino acid is provided in FIG. 46A and is SEQ ID NO: 5. The nucleotide sequence in FIG. 46C is shown in SEQ ID NO: 26, while the amino acid sequence in FIG. 46D is shown in SEQ ID NO: 92.

[0191] Another embodiment of the present invention is isolated DHS from tomato. The amino acid sequence is provided in FIGS. 45 A and B and is SEQ ID NO: 2. The polynucleotide encoding the amino acid is provided in FIGS. 45 A and B and is SEQ ID NO: 1.

[0192] Another embodiment of the present invention is isolated DHS from carnation. The amino acid sequence is provided in FIG. 54 and is SEQ ID NO: 10. The polynucleotide encoding the amino acid is provided in FIG. 54 and is SEQ ID NO: 9.

[0193] Another embodiment of the present invention is isolated DHS from canola. The amino acid sequence is provided in FIG. 97 and is SEQ ID NO: 71. The polynucleotide encoding the amino acid is provided in FIG. 97 and is SEQ ID NO: 70.

[0194] Another embodiment of the present invention is isolated DHS from lettuce. FIG. 105 provides a portion of lettuce DHS polynucleotide sequence.

[0195] Another embodiment of the present invention is isolated DHS from alfalfa. The amino acid sequence is provided in FIGS. 107 A and B and is SEQ ID NO: 73. The polynucleotide encoding the amino acid is provided in FIGS. 107 A and B and is SEQ ID NO: 72.

[0196] Another embodiment of the present invention is isolated DHS from banana. The amino acid sequence is provided in FIGS. 108 A and B and is SEQ ID NO: 75. The polynucleotide encoding the amino acid is provided in FIGS. 108 A and B and is SEQ ID NO: 74.

[0197] Another embodiment of the present invention is isolated DHS from cottonwood. The amino acid sequence is provided in FIGS. 109 A and B and is SEQ ID NO: 77. The polynucleotide encoding the amino acid is provided in FIGS. 109 A and B and is SEQ ID NO: 76.

[0198] Another embodiment of the present invention is isolated DHS from mycosphaerella. FIG. 110 provides a portion of lettuce DHS polynucleotide sequence.

[0199] The present invention also provides isolated polynucleotides of DHS that have 90% sequence homology to the above enumerated SEQ ID NOs, and hybridize under high stringency conditions to the complement of the enumerated SEQ ID NOs and which encode DHS.

[0200] The present invention also provides antisense polynucleotides of DHS. The antisense polynucleotides may be of any length as long as they are able to inhibit expression. In some embodiments the antisense polynucleotides comprise the full length coding sequence, directed at the 3'UTR, or directed at the 5'-non-coding sequence Antisense polynucleotides primarily complementary to 5'-non-coding sequences are known to be effective inhibitors of expression of genes encoding transcription factors. Branch, M. A., Molec. Cell Biol., 13:4284-4290 (1993).

[0201] The term "antisense polynucleotide of DHS" as used herein and in the claims encompasses not only those antisense polynucleotides that share 100% homology of the complement of an enumerated SEQ ID NO but also includes those antisense polynucleotides that are a functional variants. Functional variants are as described above. The variant functions as intended by the present invention, that is it is capable of modulating expression of endogenous DHS when introduced into an expression vector and wherein such vector is incorporated into the genome of at least one plant cell.

[0202] One embodiment of the present invention provides expression vectors comprising either DHS polynucleotides (of the present invention as described above) or antisense polynucleotides of DHS (of the present invention as described above). Vectors are as described above.

[0203] The invention also provides a transgenic plant cell transformed with a vector or combination of vectors of the present invention comprising a polynucleotide of DHS either in the sense or antisense orientation, a transgenic plantlet or mature transgenic plant generated from such a cell, or a plant part, such as a flower, fruit, leaves, seeds, etc. of the transgenic plant.

[0204] The present invention also provides methods of inhibiting expression of endogenous DHS. These methods comprise integrating into the genome of at least one cell of a plant, expression vectors of the present invention comprising antisense polynucleotides of DHS. The antisense polynucleotides of DHS are transcribed and inhibit expression of endogenous DHS.

[0205] In another method of inhibiting expression of endogenous DHS, an expression vector containing a DHS polynucleotide of the present invention in a sense orientation is integrated into the genome of at least one cell of a plant. The polynucleotide of DHS is transcribed and the resulting co-expression of exogenous DHS causes a down-regulation or inhibition of expression of endogenous DHS.

[0206] By inhibiting expression of endogenous DHS, resulting transgenic plants have no or substantially less DHS protein to activate eIF-5A. As discussed earlier, eIF-5A must be activated to render it biologically useful. Thus, by inhibiting or reducing the expression of DHS either by antisense polynucleotides or by co-suppression with sense polynucleotides, the resulting transgenic plants will either have no active eIF-5A or reduced active eIF-5A. These transgenic plants will exhibit an increase in biomass of the plant, increased seed yield and/or increased seed size. Transgenic plants having antisense polynucleotides of DHS show an increase in photosynthesis and also have an increased starch content. See Examples 24 and 25.

[0207] Further evidence to support the contention that DHS and eIF-5A play regulatory roles in senescence was provided by treating carnation flowers with inhibitors that are specific for DHS. Spermidine and eIF-5A are the substrates of DHS reaction (Park et al., 1993; Park et al., 1997). Several mono-, di-, and polyamines that have structural features similar to spermidine inhibit DHS activity in vitro (Jakus et al., 1993). Some polyamines, such as spermidine, putrescine, and spermine, have been generally used to extend carnation vase life (Wang and Baker, 1980). Through treatment with different polyamines at different concentrations Wang et al (unpublished b) were able to extend the vase life of carnation flowers by 2 fold. Further studies employing a transient infection system to down-regulate DHS is in progress. Preliminary data indicates that the percent survival rate is almost 4 fold higher at day 8 in cut carnations that were vacuum infiltrated with a transient infection system expressing antisense DHS than untreated flowers (Wang et al., unpublished b).

[0208] A further major loss in agriculture besides the loss of growth due to stress is post harvest stress-induced senescence (McCabe et al., 2001). This is especially true for plants that are partially processed such as cut lettuce. A symptom of cutting lettuce is browning which is a result of phenolics production (Matile et al., 1999). A field trial of lettuce with anti sense polynucleotides of lettuce eIF-5A (LeIF-5A) or antisense full length DHS demonstrated that the transgenic lettuce was significantly more resistant to browning after cutting than the control lettuce. It appears that even though stress induced senescence due to harvesting has distinct circuitry (Page et al., 2001), the translational control upstream of browning and likely other senescence symptoms is regulated at least in part by DHS and eIF-5A. Downstream of the regulation of senescence are the execution genes. These are the effectors of senescence and cause the metabolic changes that bring on the senescence syndrome. It appears that eIF-5A and DHS when down-regulated are capable of dampening down a whole range of symptoms caused by senescence.

[0209] The present invention also relates to antibodies that recognize the three isoforms of eIF-5A(senescence-induced factor eIF-5A); (wounding factor eiF-5A) and (growth factor eIF-5A).

[0210] The present invention also provides a method of identifying senescence-induced eIF-5A, wounding-induced eIF-5A, growth eIF-5A and DHS in other plants and fungi. By using the methods described herein and the sequences provided, probes are designed to isolate/identify the desired isoforms or DHS. Since the isoforms of eIF-5A (senescence-induced eIF-5A, wounding-induced eIF-5A, and growth eIF-5A) are often highly homologous in the coding region (see FIG. 2), to ensure identification and even alter amplification of the desired isoform, probes or primers are preferably designed from the beginning of the 5'UTR and at the end of the 3''UTR. (See FIGS. 3, 4 and 5). A preferred set of primers for amplification of wounding-induced eIF-5A or probes for identification of wounding-induced eIF-5A are as follows. The downstream primer is 5' GAG CTC AAG AAT AAC ATC TCA TAA GAAAC3' (SEQ ID NO: 33) The upstream primer is 5' CTC GAG TGC TCA CTT CTC TCT CTT AGG 3' (SEQ ID NO: 34).

[0211] Before isolating wounding-induced eIF5A from a plant or plant part, it is best to introduce a wounding event to allow the plant to begin expressing wounding-induced eIF-5A. Any wounding event is acceptable and one such exemplary wound events included crushing the leaves at the central vein. Similarly, before isolating senescence-induced eIF-5A, it best to stress the plant tissue to induce senescence.

[0212] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting to the present invention.

EXAMPLES

Example 1

Messenger RNA (mRNA) Isolation

[0213] Total RNA was isolated from tomato flowers and tomato fruit at various developmental stages and from leaves (untreated or after chilling or sorbitol treatment). The tissue (5 g) was briefly ground in liquid nitrogen. The ground powder was mixed with 30 ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8%.-mercaptoethanol). The mixture was filtered through four layers of cheesecloth and centrifuged at 10,000×g at 4° C. for 30 minutes. The supernatant was then subjected to cesium chloride density gradient centrifugation at 26,000×g for 20 hours. The pelleted RNA was rinsed with 75% ethanol, resuspended in 600 μl DEPC-treated water and the RNA precipitated at -70° C. with 0.75 ml 95% ethanol and 30 μl of 3M NaOAc. Ten μg of RNA were fractionated on a 1.2% denaturing formaldehyde agarose gel and transferred to a nylon membrane. Randomly primed ³²P-dCTP-labeled full length DHS cDNA (SEQ ID NO:1) was used to probe the membrane at 42° C. overnight. The membrane was then washed once in 1×SSC containing 0.1% SDS at room temperature for 15 minutes and three times in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. The membrane was exposed to x-ray film overnight at -70° C.

[0214] PolyA.sup.+ mRNA was isolated from total RNA using the PolyA.sup.+ tract mRNA Isolation System available from Promega. PolyA.sup.+ mRNA was used as a template for cDNA synthesis using the ZAP Express® cDNA synthesis system available from Stratagene (La Jolla, Calif.)

Tomato Leaf cDNA Library Screening

[0215] A cDNA library made using mRNA isolated from Match F1 hybrid tomato leaves that had been exposed to 2 M sorbitol for six hours was diluted to approximately 5×10⁶ PFU/ml. The cDNA library was screened using a ³²P-labeled 600 bp RT-PCR fragment. Three positive cDNA clones were excised and recircularized into a pBK-CMV® (Stratagene) phagemid using the method in the manufacturer's instructions. The full length cDNA was inserted into the pBK-CMV vector.

Plasmid DNA Isolation, DNA Sequencing

[0216] The alkaline lysis method described by Sambrook et al., (Supra) was used to isolate plasmid DNA. The full length positive cDNA clone was sequenced using the dideoxy sequencing method. Sanger, et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467. The open reading frame was compiled and analyzed using BLAST search (GenBank, Bethesda, Md.) and alignment of the five most homologous proteins with the derived amino acid sequence of the encoded gene was achieved using a BCM Search Launcher: Multiple Sequence Alignments Pattern-Induced Multiple Alignment Method (See F. Corpet, Nuc. Acids Res., 16:10881-10890, (1987)). Functional motifs present in the derived amino acid sequence were identified by MultiFinder.

Northern Blot Hybridizations of Tomato RNA

[0217] Ten μg of total RNA isolated from tomato flowers at various stages (bud and blossom and senescing petals that are open widely or drying), tomato leaves, and tomato fruit at various stages of ripening (breaker, i.e., green fruit with less than 10% red color, pink, i.e., the entire fruit is orange or pink, and red, either soft or firm) were separated on 1% denatured formaldehyde agarose gels and immobilized on nylon membranes. The full length tomato cDNA labeled with ³²P-dCTP using a random primer kit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). The filters were washed once with 1×SSC, 0.1% SDS at room temperature and three times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried and exposed to X-ray film overnight at -70° C. The results are shown in FIGS. 50-52.

Northern Blot Hybridization of Arabidopsis RNA

[0218] Total RNA from leaves of Arabidopsis plants at five weeks of age (lane 1), six weeks (lane 2) and seven weeks (lane 3) was isolated as above, separated on 1% denatured formaldehyde agarose gels and immobilized on nylon membranes. The full-length Arabidopsis senescence-induced DHS cDNA labeled with ³²P-dCTP using a random primer kit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). The filters were washed once with 1×SSC, 0.1% SDS at room temperature and three times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried and exposed to X-ray film overnight at -70° C. The results are shown in FIG. 55.

Northern Blot Hybridization of Carnation RNA

[0219] Total RNA from petals of carnation plants at various stages of flower development, i.e., tight-bud flowers (lane 1), beginning to open (lane 2), fully open flowers (lane 3), flowers with inrolling petals (lane 4), was isolated as above, separated on 1% denatured formaldehyde agarose gels and immobilized on nylon membranes. The full-length carnation senescence-induced DHS cDNA labeled with ³²P-dCTP using a random primer kit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). The filters were washed once with 1×SSC, 0.1% SDS at room temperature and three times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried and exposed to X-ray film overnight at -70° C. The results are shown in FIG. 56.

Example 2

Sorbitol Induction of Tomato Senescence-Induced DHS Gene

[0220] Tomato leaves were treated with 2 M sorbitol in a sealed chamber for six hours. RNA was extracted from the sorbitol treated leaves as follows.

[0221] Leaves (5 g) were ground in liquid nitrogen. The ground powder was mixed with 30 ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8%-mercaptoethanol). The mixture was filtered through four layers of cheesecloth and centrifuged at 10,000×g at 4° C. for 30 minutes. The supernatant was then subjected to cesium chloride density gradient centrifugation at 26,000×g for 20 hours. The pelleted RNA was rinsed with 75% ethanol, resuspended in 600 μl DEPC-treated water and the RNA precipitated at -70° C. with 0.75 ml 95% ethanol and 30 μl of 3M NaOAc. Ten μg of RNA were fractionated on a 1.2% denaturing formaldehyde agarose gel and transferred to a nylon membrane. Randomly primed ³²P-dCTP-labeled full length DHS cDNA (SEQ ID NO:1) was used to probe the membrane at 42° C. overnight. The membrane was then washed once in 1×SSC containing 0.1% SDS at room temperature for 15 minutes and three times in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. The membrane was exposed to x-ray film overnight at -70° C.

[0222] The results are shown in FIG. 52. As can be seen, transcription of DHS is induced in leaves by sorbitol.

Example 3

Induction of the Tomato DHS Gene in Senescing Flowers

[0223] Tight flower buds and open, senescing flowers of tomato plants were harvested, and RNA was isolated as in Example 2. Ten μg RNA were fractionated on a 1.2% denaturing formaldehyde agarose gel and transferred to a nylon membrane. Randomly primed ³²P-dCTP-labeled full length DHS cDNA (SEQ ID NO.1) was used to probe the membrane at 42° C. overnight. The membrane then was washed once in 1×SSC containing 0.1% SDS at room temperature for 15 minutes and then washed three times in 0.2×SSC containing 0.1% SDS at 65° C. for fifteen minutes each. The membrane was exposed to x-ray film overnight at -70° C.

[0224] The results are shown in FIG. 50. As can be seen, transcription of DHS is induced in senescing flowers.

Example 4

Induction of the Tomato DHS Gene in Ripening Fruit

[0225] RNA was isolated from breaker, pink and ripe fruit as in Example 2. Ten μg RNA were fractionated on a 1.2% denaturing formaldehyde agarose gel and transferred to a nylon membrane. Randomly primed ³²P-dCTP-labeled full length DHS cDNA (SEQ ID NO:1) (FIG. 45) was used to probe the membrane at 42° C. overnight. The membrane then was washed once in 1×SSC containing 0.1% SDS at room temperature for 15 minutes and then washed three times in 0.2×SSC containing 0.1% SDS at 65° C. for fifteen minutes each. The membrane was exposed to x-ray film overnight at -70° C.

[0226] The results are shown in FIG. 51. As can be seen, transcription of DHS is strongest in ripe, red fruit just prior to the onset of senescence leading to spoilage.

Example 5

Induction of Tomato Senescence-Induced DHS Gene by Chilling

[0227] Tomato plants in pots (7-8 weeks old) were exposed to 6° C. for two days, three days or six days in a growth chamber. The light cycle was set for eight hours of dark and sixteen hours of light. Plants were rewarmed by moving them back into a greenhouse. Plants that were not rewarmed were harvested immediately after removal from the growth chamber. RNA was extracted from the leaves as follows.

[0228] Leaves (5 g) were ground in liquid nitrogen. The ground powder was mixed with 30 ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8%-mercaptoethanol). The mixture was filtered through four layers of cheesecloth and centrifuged at 10,000 g at 4° C. for 30 minutes. The supernatant was then subjected to cesium chloride density gradient centrifugation at 26,000 g for 20 hours. The pelleted RNA was rinsed with 75% ethanol, resuspended in 600 WDEPC-treated water and the RNA precipitated at -70° C. with 0.75 ml 95% ethanol and 30 μl of 3M NaOAc. Ten μg of RNA were fractionated on a 1.2% denaturing formaldehyde agarose gel and transferred to a nylon membrane. Randomly primed ³²P-dCTP-labeled full length DHS cDNA (SEQ ID NO:1) was used to probe the membrane at 42° C. overnight. The membrane was then washed once in 1×SSC containing 0.1% SDS at room temperature for 15 minutes and three times in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. The membrane was exposed to x-ray film overnight at -70° C.

[0229] The results are shown in FIG. 53. As can be seen, transcription of DHS is induced in leaves by exposure to chilling temperature and subsequent rewarming, and the enhanced transcription correlates with chilling damage measured as membrane leakiness.

Example 6

Generation of an Arabidopsis PCR Product Using Primers Based on Unidentified Arabidopsis Genomic Sequence

[0230] A partial length senescence-induced DHS sequence from an Arabidopsis cDNA template was generated by PCR using a pair of oligonucleotide primers designed from Arabidopsis genomic sequence. The 5' primer is a 19-mer having the sequence, 5'-GGTGGTGTSTGAGGAAGATC (SEQ ID NO:7); the 3' primer is a 20 mer having the sequence, GGTGCACGCCCTGATGAAGC-3' (SEQ ID NO:8). A polymerase chain reaction using the Expand High Fidelity PCR System (Boehringer Mannheim) and an Arabidopsis senescing leaf cDNA library as template was carried out as follows.

Reaction Components:

TABLE-US-00001

[0231] cDNA 1 μl (5 × 10⁷ pfu) dNTP (10 mM each) 1 μl MgCl₂ (5 mM) + 10x buffer 5 μl Primers 1 and 2 (100 μM each) 2 μl Expand High Fidelity DNA polymerase 1.75 U Reaction volume 50 μl

[0232] Reaction Parameters:

[0233] 94° C. for 3 min

[0234] 94° C./1 min, 58° C./1 min, 72° C./2 min, for 45 cycles

[0235] 72° C. for 15 min.

Example 7

Isolation of Genomic DNA and Southern Analysis

[0236] Genomic DNA was extracted from tomato leaves by grinding 10 grams of tomato leaf tissue to a fine powder in liquid nitrogen. 37.5 ml of a mixture containing 25 ml homogenization buffer [100 mM Tris-HCl, pH 8.0, 100 mm EDTA, 250 mM NaCl, 1% sarkosyl, 1% 2-mercaptoethanol, 10 μg/ml RNase and 12.5 ml phenol] prewarmed to 60° C. was added to the ground tissue. The mixture was shaken for fifteen minutes. An additional 12.5 ml of chloroform/isoamyl alcohol (24:1) was added to the mixture and shaken for another 15 minutes. The mixture was centrifuged and the aqueous phase reextracted with 25 ml phenol/chloroform/isoamylalcohol (25:24:1) and chloroform/isoamylalcohol (24:1). The nucleic acids were recovered by precipitation with 15 ml isopropanol at room temperature. The precipitate was resuspended in 1 ml of water.

[0237] Genomic DNA was subjected to restriction enzyme digestion as follows: 10 μg genomic DNA, 40 μl 10× reaction buffer and 100 U restriction enzyme (XbaI, EcoRI, EcoRV or HinDIII) were reacted for five to six hours in a total reaction volume of 400 μl. The mixture was then phenol-extracted and ethanol-precipitated. The digested DNA was subjected to agarose gel electrophoresis on a 0.8% agarose gel at 15 volts for approximately 15 hours. The gel was submerged in denaturation buffer [87.66 g NaCl and 20 g NaOH/Liter] for 30 minutes with gentle agitation, rinsed in distilled water and submerged in neutralization buffer [87.66 g NaCl and 60.55 g tris-HCl, pH 7.5/Liter] for 30 minutes with gentle agitation. The DNA was transferred to a Hybond-N.sup.+ nylon membrane by capillary blotting.

[0238] Hybridization was performed overnight at 42° C. using 1×10⁶ cpm/ml of ³²P-dCTP-labeled full length DHS cDNA or 3'-non-coding region of the DHS cDNA clone. Prehybridization and hybridization were carried out in buffer containing 50% formamide, 6×SSC, 5×Denhardt's solution, 0.1% SDS and 100 mg/ml denatured salmon sperm DNA. The membrane was prehybridized for two to four hours; hybridization was carried out overnight.

[0239] After hybridization was complete, membranes were rinsed at room temperature in 2×SSC and 0.1% SDS and then washed in 2×SSC and 0.1% SDS for 15 minutes and 0.2×SSC and 0.1% SDS for 15 minutes. The membrane was then exposed to x-ray film at -80° C. overnight. The results are shown in FIG. 49.

Example 8

Isolation of a Senescence-Induced eIF-5a Gene from Arabidopsis

[0240] A full-length cDNA clone of the senescence-induced eIF-5A gene expressed in Arabidopsis leaves was obtained by PCR using an Arabidopsis senescing leaf cDNA library as template. Initially, PCR products corresponding to the 5'- and 3'-ends of the gene were made using a degenerate upstream primer <AAARRYCGMCCYTGCAAGGT> (SEQ ID NO:17) paired with vector T7 primer <AATACGACTCACTATAG> (SEQ ID NO:18), and a degenerate downstream primer <TCYTTNCCYTCMKCTAAHCC> (SEQ ID NO:19) paired with vector T3 primer <ATTAACCCTCACTAAAG> (SEQ ID NO: 20). The PCR products were subcloned into pBluescript for sequencing. The full-length cDNA was then obtained using a 5'-specific primer <CTGTTACCAAAAAATCTGTACC> (SEQ ID NO: 21) paired with a 3'-specific primer <AGAAGAAGTATAAAAACCATC> (SEQ ID NO: 22), and subcloned into pBluescript for sequencing.

Example 9

Isolation of a Senescence-Induced eIF-5a Gene from Tomato Fruit

[0241] A full-length cDNA clone of the senescence-induced eIF-5A gene expressed in tomato fruit was obtained by PCR using a tomato fruit cDNA library as template. Initially, PCR products corresponding to the 5'- and 3'-ends of the gene were made using a degenerate upstream primer (SEQ ID NO:17) paired with vector T7 primer (SEQ ID NO:18), and a degenerate downstream primer (SEQ ID NO:19) paired with vector T3 primer (SEQ ID NO: 20). The PCR products were subcloned into pBluescript for sequencing. The full-length cDNA was then obtained using a 5'-specific primer <AAAGAATCCTAGAGAGAGAAAGG> (SEQ ID NO: 23) paired with vector T7 primer (SEQ ID NO: 18), and subcloned into pBluescript for sequencing.

Example 10

Isolation of a Senescence-Induced eIF-5a Gene from Carnation

[0242] A full-length cDNA clone of the senescence-induced eIF-5A gene expressed in carnation flowers was obtained by PCR using a carnation senescing flower cDNA library as template. Initially, PCR products corresponding to the 5'- and 3'-ends of the gene were made using a degenerate upstream primer (SEQ ID NO:17) paired with vector T7 primer (SEQ ID NO:18), and a degenerate downstream primer (SEQ ID NO:19) paired with vector T3 primer (SEQ ID NO: 20). The PCR products were subdloned into pbluescript for sequencing. The full-length cDNA was then obtained using a 5'-specific primer <TTTTACATCAATCGAAAA> (SEQ ID NO: 24) paired with a 3'-specific primer <ACCAAAACCTGTGTTATAACTCC> (SEQ ID NO: 25), and subcloned into pBluescript for sequencing.

Example 11

Isolation of a Senescence-Induced DHS Gene from Arabidopsis

[0243] A full-length cDNA clone of the senescence-induced DHS gene expressed in Arabidopsis leaves was obtained by screening an Arabidopsis senescing leaf cDNA library. The sequence of the probe (SEQ ID NO: 26) that was used for screening is shown in FIG. 82. The probe was obtained by PCR using the senescence leaf cDNA library as a template and primers designed from the unidentified genomic sequence (AB017060) in GenBank. The PCR product was subcloned into pBluescript for sequencing.

Example 12

Isolation of a Senescence-Induced DHS Gene from Carnation

[0244] A full-length cDNA clone of the senescence-induced DHS gene expressed in carnation petals was obtained by screening a carnation senescing petal cDNA library. The sequence of the probe (SEQ ID NO: 27) that was used for screening is shown in FIG. 83. The probe was obtained by PCR using the senescence petal cDNA library as a template and degenerate primers (upstream: 5' TTG ARG AAG ATY CAT MAA RTG CCT 3') (SEQ ID NO: 28); downstream: 5' CCA TCA AAY TCY TGK GCR GTG TT 3') (SEQ ID NO: 29). The PCR product was subcloned into pBluescript for sequencing.

Example 13

Transformation of Arabidopsis with Full-Length or 3' Region of Arabidopsis DHS in Antisense Orientation

[0245] Agrobacteria were transformed with the binary vector, pKYLX71, containing the full-length senescence-induced Arabidopsis DHS cDNA sequence or the 3' end of the DHS gene (SEQ ID NO:30) (FIG. 80), both expressed in the antisense configuration, under the regulation of double 35S promoter. Arabidopsis plants were transformed with the transformed Agrobacteria by vacuum infiltration, and transformed seeds from resultant To plants were selected on ampicillin.

[0246] FIGS. 65-68 are photographs of the transformed Arabidopsis plants, showing that expression of the DHS gene or 3' end thereof in antisense orientation in the transformed plants results in increased biomass, e.g., larger leaves and increased plant size. FIG. 69 illustrates that the transgenic Arabidopsis plants have increased seed yield.

Example 14

Transformation of Tomato Plants with Full-Length or 3' Region of Tomato DHS in Antisense Orientation

[0247] Agrobacteria were transformed with the binary vector, pKYLX71, containing the full-length senescence-induced tomato DHS cDNA sequence or the 3' end of the DHS gene (SEQ ID NO:31) (FIG. 81), both expressed in the antisense configuration, under the regulation of double 35S promoter. Tomato leaf explants were formed with these Agrobacteria, and transformed callus and plantlets were generated and selected by standard tissue culture methods. Transformed plantlets were grown to mature fruit-producing T₁ plants under greenhouse conditions.

[0248] FIGS. 70-79 are photographs showing that reduced expression of the senescence-induced tomato DHS gene in the transformed plants results in increased biomass, e.g., larger leaf size and larger plants as seen in the transformed Arabidopsis plants, as well as delayed softening and spoilage of tomato fruit.

Example 15

Transformation of Tomato Plants with the 3' Region of Tomato DHS in Antisense Orientation

[0249] Agrobacteria were transformed with the binary vector, pKYLX71, containing the 3' end of the DHS gene (FIG. 81) expressed in the antisense configuration, under the regulation of double 35S promoter. Tomato leaf explants were formed with these Agrobacteria, and transformed callus and plantlets were generated and selected by standard tissue culture methods. Transformed plantlets were grown to mature fruit producing T₁ plants under green house conditions.

[0250] Fruit from these transgenic plants with reduced DHS expression were completely free of blossom end rot under conditions in which about 33% of fruit from control plants developed this disease. Blossom end rot is a physiological disease attributable to nutrient stress that causes the bottom (blossom) end of the fruit to senesce and rot. FIGS. 84A and 84B are photographs showing a control fruit exhibiting blossom end rot and a transgenic fruit that is free of blossom end rot.

[0251] The results indicate that reducing the expression of DHS prevents the onset of tissue and cell death arising from physiological disease.

Example 16

Expression of Arabidopsis thaliana Translation Initiation Factor 5A (AteIF-5A) Isoforms in Wild Type Columbia--Plant Material

[0252] Seeds of Arabidopsis thaliana, ecotype Columbia, were grown in Promix BX soil (Premier Brands, Brampton, ON, Canada) in 6-inch pots. Freshly seeded pots were maintained at 4° C. for 2 days and then transferred to a growth chamber operating at 22° C. with 16-h light/8-h dark cycles. Lighting at 150 mmol radiation m^-2s^-1 was provided by cool-white fluorescent bulbs. Whole rosettes were collected one week intervals at 2 weeks to 7 weeks of age, cauline leaves were collected at 5 weeks, stem, siliques, buds, and flowers were collected at 6 weeks and imbibed seeds (24 hours in water) were also collected, flash frozen in liquid nitrogen and stored at -80° C.

Infection of Arabidopsis thaliana Plants with Pseudomonas syringae

[0253] Seeds of Arabidopsis thaliana ecotype Columbia were sown onto Promix BX soil (Premier Brands, Brampton, ON, Canada) in flats containing 64 growth cells. The seeded flats were maintained at 4° C. for 2 days and transferred to a growth chamber with photoperiod of 9-h light/15-h dark. All plants were treated at 4 weeks of age, though physiologically due to the shortened photoperiod these appear to be slower in development.

[0254] Rosette leaves of 4-week-old plants were infected with avirulent (avr) and virulent (vir) strains Pseudomonas syringae pv. Tomato DC 3000 obtained from Dr. Robin Cameron (university of Toronto, Toronto, Canada). The abaxial surface of the rosette leaves of each plant was inoculated using 1 ml syringe without a needle. Plants were treated using one of four treatments: no inoculation, mock-inoculation with 10 mM MgCl₂, inoculation with avr P. syringae strain (10⁶ cfu/ml 10 mM MgCl₂) or inoculation with vir P. syringae strain (10⁶ cfu/ml 10 mM MgCl₂). Two bacterial counts were made, one immediately after inoculation and the second 3 days later, to ensure that a sufficient amount of bacteria was infiltrated to induce systemic acquired resistance in the avr treatment. The inoculated leaves were harvested at predetermined time points for subsequent analysis.

[0255] Plants with reduced DHS or wounding-induced eIF-5A expression were developed using antisense T-DNA insertions for either gene. These plant lines have shown marked resistance to Pseudomonas syringae pv Tomato DC 300, with transgenic lines exhibiting up to a 99% decrease in bacterial load, relative to the wild type plants. See FIGS. 43 and 44. Data using crop plants have also indicated enhanced pathogen resistance.

Wounding of Arabidopsis thaliana Plants with Hemostat

[0256] 4-week-old plants grown under normal lighting conditions were wounded by crushing with hemostat along the midvein (approximately 10% of the leaf surface) according to Stotz et al (2000). Tissue was harvested at 0 minutes, 1 hour and 9 hours and immediately frozen in liquid nitrogen and stored at -80° C. for further analysis.

RNA Isolation and Northern Blotting

[0257] Total RNA for Northern blot analysis was isolated from Arabidopsis thaliana rosette leaves according to Davis et al. (1986). The RNA was fractionated on a 1% agarose gel and transferred to nylon membranes. (Davis et. al., 1986) Immobilized RNA was hybridized overnight at 42° C. with radiolabeled 3'UTR portions of senescence-induced AteIF-5A, wounding-induced AteIF-5A or growth AteIF-5A. The 3'UTRs were labeled with [α-³²P]-dCTP using a random primer kit (Boehringer Mannheim). The hybridized membranes were washed twice in 2×SSC containing 0.1% SDS at 42° C. for 15 minutes and twice in 1×SSC containing 0.1% SDS at 42° C. for 30 minutes. Hybridization was visualized by autoradiography after an overnight exposure at -80° C.

Antibody Production and Purification

[0258] Eukaryotic translation initiation factor 5A (eIF-5A) isoforms of Arabidopsis thaliana (At) are highly homologous at the amino acid level, especially at the N-terminal region and the central region of the proteins (FIG. 1). In order to obtain antibodies that will be isoform specific, peptides were designed against regions in the isoforms of AteIF-5A that appeared to be unique to each other. An additional cysteine residue was added to each peptide at the N-terminus for conjugation with KLH. The sequences used were: CNDDTLLQQIKS (SEQ ID NO: 35) for senescence-induced AteIF-5A, CTDDGLTAQMRL (SEQ ID NO: 36) for wounding-induced AteIF5A, and CTDEALLTQLKN (SEQ ID NO: 37) for growth AteIF-5A. When these sequences were submitted to protein BLAST (short nearly exact sequences; limited by Arabidopsis thaliana; expected number 20000; word size 2; Matrix PAM90; Gap cost 91) the significant sequences that found in the database were only the matched AteIF-5A and no other. The peptides were synthesized at the University of Western Ontario Peptide Synthesis facility. The carrier protein, Keyhole Limpet Hemocyanin (Sigma), was conjugated to the N-terminal cysteine of the peptide using m-maleimidobenzoyl-N-hydroxysuccinimide ester according to Drenckhahn et al. (1993) and Collawn and Patterson (1999). The rabbits were injected four times at two-week intervals with the linked peptide. Two weeks after the final injection blood is collected by exsanguination of the rabbits and clotting of the collected blood in order to amass the antisera.

Protein Fractionation and Western Blotting

[0259] Tissues list above were homogenized (˜0.5 g/ml) in buffer (50 mM EPPS, pH 7.4, 0.25M sorbitol, 10 mM EDTA, 2 mM EGTA, 1 mM DTT, 10 mM amino-n-caproic acid, Protease Inhibitor Cocktail for Plant tissues (Sigma)) in an eppendorf tube with a small pestle, or in a large mortar and pestle. The homogenates were centrifuged briefly in the microcentrifuge at maximum speed and the pellet was discarded. The total protein was quantified according to Ghosh et al. (1988). SDS-PAGE was performed on Mini protein Dual Slab cells (BioRad, Mississauga, Ontario), and the gels (12% polyacrlyamide) were stained with Coomassie brilliant blue 8250 (Fairbanks et. al. 1971) or transferred to polyvinyldiene difluoride (PVDF) membranes using the semi-dry transfer method (semi-dry transfer cell, Bio-Rad, Hercules, Calif.). The blots were blocked for 30 s in 1 mg/ml polyvinyl alcohol (Miranda et. al., 1993) and for 1 hour in phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 5% (w/v) powdered milk. Primary antibody (from bleeds after second injection) was diluted 1:50 in PBS containing 0.1% (v/v) Tween 20 and 1% (w/v) powdered milk. Antigen was visualized using secondary antibody made in goat against rabbit antibody coupled to alkaline phosphatase (Bioshop, Burlington, Ontario) and the phosphatase substrates, NBT and BCIP (BioRad, Mississauga, ON).

Example 17

Production of Transformed Arabidopsis thaliana Plants Over Expressing the Three eIF-5A Isoforms

Primer Design

[0260] Eukaryotic translation initiation factor 5A (eIF-5A) isoforms of Arabidopsis thaliana (At) are highly homologous in the coding region (FIG. 2). To avoid problems with amplification of the correct genes, primers for senescence-induced AteIF-5A, wounding-induced eIF-5A and growth eIF-5A were designed from the approximate beginning of the 5'UTR and at the end of the 3'UTR as shown in FIGS. 3, 4 and 5 respectively. The 5'UTR and 3'UTR were estimated based on EST information and other sequence information in the GenBank database. The appropriate restriction sites were added to the ends of the primers for ligation in the sense orientation in the pKYLX71 binary vector (FIG. 6). For senescence-induced AteIF-5A the upstream primer is 5' AAGCTT GATCGTGGTCAACTTCCTCTGTTACC 3' (SEQ ID NO: 38) and the downstream primer is 5' GAGCT CAGAAGAAGTATAAAAACCATC 3' (SEQ ID NO: 39). For wounding-induced AteIF-5A the upstream primer is 5' CTC GAGTGCTCACTTCTCTCTCTTAGG 3' (SEQ ID NO: 40) and the downstream primer is 5' GAGCTCA AGAATAACATCTCATAAGAAAC 3' (SEQ ID NO: 41). The upstream primer for growth AteIF-5A is 5' CTC GAGCTAAACTCCATTCGCTGACTTCGC 3' (SEQ ID NO: 42) and the downstream primer is 5' GAGC TCTAGTAAATATAAGAGTGTCTTGC 3' (SEQ ID NO: 43). The restriction sites that were added into the primers were HindIII and SacI for senescence-induced AteIF-5A, XhoI and SacI for wounding-induced AteIF-5A, and XhoI and SacI for growthAteIF-5A as indicated by underlining in the primers listed above.

Isolation of Genomic DNA from Arabidopsis thaliana

[0261] Genomic DNA was isolated from 3-week-old rosette leaf. The tissue was homogenized in extraction buffer (200 mM Tris pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) and the resulting homogenate was vortexed for 15 seconds. The remaining debris was removed by centrifugation in a microcentrifuge at maximum speed for 1 minute. The supernatant was collected and mixed in a 1:1 ratio with isopropanol, vortexed and left at room temperature for 2 minutes. A pellet was collected by centrifugation in a microcentrifuge at maximum speed for 5 minutes, washed with 70% ethanol and vacuum dried for 2 minutes. The dried pellet was resuspended in water and treated with 1:1 volume of chloroform and vortexed. After centrifugation in a microcentrifuge at maximum speed for 2 minutes the top layer was collected and treated with 20 μl salt (3M sodium acetate) and 2 volumes of ethanol for precipitation at -20° C. for 30 minutes. The purified genomic DNA was then centrifuged at maximum speed for 30 minutes in a microcentrifuge, dried and resuspended in water for PCR.

PCR from Genomic DNA

[0262] PCR was performed with the primers described above. The PCR reaction mixture contained 1×Tsg or Taq polymerase reaction buffer, 1 U of Tsg or Taq polymerase, 0.2 mM dNTP, 2 mM MgCl₂, and 15 pmols of each specific primer accordingly. The reaction began with a hot start at 95° C. for 10 minutes and first cycle consisted of 1 minute denaturing temperature of 95° C., 2 minutes annealing temperature of 55° C., and a 2 minute extension temperature of 72° C. The following 29 cycles proceeded a touchdown program where the annealing temperature was decreased by 0.5° C. per cycle, and the final cycle had an annealing temperature of 40° C. The final extension of 72° C. was held for 10 minutes. The PCR products were separated by 1% agarose gel electrophoresis, cut out and retrieved by Millipore Ultrafree-DA for DNA Extraction from Agarose spin columns (Millipore Corporation, Bedford, Mass.) according to directions.

Ligation into pGEM®-TEasy

[0263] Purified PCR products were ligated into pGEM®-T Easy Vector (FIG. 7) according to directions provided by Promega. Briefly, PCR products were mixed in a 3:1 ratio with pGEM T-Easy Vector, 3 Weiss Units T4 DNA ligase in Rapid Ligation Buffer (30 mM Tris-HCl, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, and 5% polyethylene glycol (MW8000, ACS Grade) pH 7.8) provided in the Promega pGEM®-T Easy Vector System (Promega Corporation, Madison Wis.). The ligation reaction was incubated overnight at 15° C. and transformed into competent E. coli DH5-α cell suspension (made competent using RbCl/CaCl; Kushner, 1978). The transformation mixture was first incubated on ice for 30 minutes, heat shocked for 90 seconds at 42° C., and allowed to recover at 37° C. for 1 hour after the addition of 1 ml 2×YT broth. The transformed cells were pelleted, resuspended in a small volume of 2×YT broth and plated on agar plates containing 50 μg/ml ampicillin for selection. Only transformants are able to grow on the ampicillin-containing plates as the pGEM®-T Easy Vector provides ampicillin resistance to the cells. Transformants were selected and screened for the PCR product insert ligated into the pGEM®-T Easy Vector.

Screening for PCR Product Inserts in pGEM®-TEasy Vector through Restriction Enzyme Digestions

[0264] Colonies that grew on selection media were grown in 5 ml 2×YT broth containing 50 μg/ml ampicillin overnight at 37° C. The recombinant plasmids from the selected colonies were purified using Wizard Prep DNA Purification Kit (Promega). The plasmid DNA was digested with EcoRI for 1 hour at 37° C. and visualized on a 1% agarose gel for verification that the AteIF-5As insert sizes were present. The positive plasmids were then sequenced by the Core Molecular Biology Facility (University of Waterloo, Waterloo, ON) for confirmation that the sequence is suitable for over expression in planta.

Ligation into pKYLX71

[0265] The constructs of pGEM:wounding-induced AteIF-5A, and pGEM:growth AteIF-5A were double digested with XhoI and SacI and sub-cloned into the binary vector, pKYLX71 that had also been digested with XhoI and SacI. These enzyme digestions ensured that wounding-induced AteIF-5A and growth AteIF-5A would be inserted in the sense orientation in the binary vector pKYLX71 under the control of the cauliflower mosaic virus double 35S promoter. The ligation reactions used 1 μg of binary vector and 3 μg of either wounding-induced AteIF-5A or growth AteIF-5A. Ligation took place in ligation buffer (30 mM Tris-HCl, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, and 5% polyethylene glycol (MW8000, ACS Grade) pH 7.8) with 3 Weiss units of T4 DNA Ligase (Fermentas). The ligation reaction was incubated overnight at 15° C. and transformed into competent E. coli DH5-α cell suspension (made competent using RbCl/CaCl; Kushner, 1978). The transformation mixture was first incubated on ice for 30 minutes; heat shocked for 90 seconds at 42° C. and allowed to recover at 37° C. for 1 hour after the addition of 1 ml 2×YT broth. The transformed cells were pelleted, resuspended in a small volume of 2×YT broth and plated on agar plates containing 50 μg/ml tetracycline for selection. Only transformants are able to grow on the tetracycline-containing plates as the binary vector pKYLX71 provides tetracycline resistance to bacterial cells. Transformants were selected and screened for wounding-induced AteIF-5A or growth AteIF5A insert by PCR and double digestion with XhoI and SacI. Following PCR amplification (same as was done with genomic DNA explained above) and digestion, the products were separated using 1% agarose electrophoresis for conformation of the correct sized insert.

Agrobacterium Electroporation and Selection

[0266] The constructs pKYLX71:wounding-induced AteIF-5A and pKYLX71:growth AteIF-5A was electroporated into competent Agrobacterium tumefaciens GV3010. The preparation of competent Agrobacterium cells a single colony was inoculated in 5 ml of 2×YT broth containing 50 μg/ml of rifampicin, and 50 μg/ml gentamycin. This grew overnight at 28° C. in a Form a Scientific Orbital Shaker (Fisher Scientific) at 280 rpm and was used to inoculate 30 ml cultures of 2×YT also with 50 μg/ml of rifampicin, and 50 μg/ml gentamycin at various dilutions (1:500, 1:1000, 1:2000). The newly inoculated cultures grew until OD₆₀₀ was between 0.5 and 0.8 before being cooled and centrifuged down in an SS-34 rotor (Sorvall) at 2000 g for 15 minutes. The pellets were resuspended in 50 ml of ice-cold water and centrifuged at 2000 g for 15 minutes. This washing procedure was repeated for a total of four times to remove the salts and the dead cells from the culture. The final pellet was resuspended in 40 ml ice cold 10% (v/v) glycerol and centrifuged at 2000 g for 15 minutes and repeated once. The pellet was then resuspended in 100 μl ice-cold 10% glycerol and mixed well. Cells were split up into aliquots of 100 μl and stored on ice.

[0267] For electroporation of the DNA constructs into the competent Agrobacterium cells the 100 μl aliquots were each mixed well with 500 ng of DNA construct. The bacteria:vector mixture was then transferred to a pre-cooled electroporation cuvette and placed in the Gene Pulser (Biorad) adjusted to the following settings: 2.5 kV, 25 μF, and 200Ω. After electroporation 1 ml 2×YT broth was added and the whole suspension was transferred to a culture tube. The electroporated cultures were incubated at 28° C., 280 rpm, for 3 hours to allow them to recover and then 2 ml 2×YT both was added as well as 50 μg/ml of rifampicin, and 50 μg/ml gentamycin. After 2 days of growing in culture the electroporated cells were plated on tetracycline, gentamycin and rifampicin (all at 50 μg/ml) and colonies grew after an addition 2 days. The resulting colonies were screened for pKYLX71:wounding-induced AteIF-5A or pKYLX71:growth AteIF-5A by PCR and double digestion with SacI and XhoI, and visualized by separation on a 1% agarose gel.

Plant Transformation

[0268] A positive colony of Agrobacterium tumefaciens GV3010 containing either pKYLX71:wounding-induced AteIF-5A or pKYLX71:growth AteIF-5A were used for the transformation of wild type Arabidopsis thaliana ecotype Columbia. In preparation of the bacterial slurry used for plant transformation a single colony positive for pKYLX71:wounding-induced AteIF-5A or pKYLX71:growth AteIF-5A construct was inoculated in 5 ml of 2×YT broth containing 50 μg/ml of tetracycline, 50 μg/ml of rifampicin, and 50 μg/ml gentamycin. This grew for 2 days at 28° C. in a Form a Scientific Orbital Shaker (Fisher Scientific) at 280 rpm and was used to inoculate 35 ml (total) 2×YT also with 50 μg/ml of rifampicin, and 50 μg/ml gentamycin. The 35 ml culture was grown overnight at 28° C., 280 rpm, and used to inoculate 535 ml (total) 2×YT with 50 μg/ml of rifampicin, and 50 μg/ml gentamycin. Again the culture was grown overnight at 28° C., 280 rpm, to an OD₆₀₀ of about 2.0.

[0269] The cultures were transferred to two 250 ml tubes before centrifugation for 15 minutes at 1945 g at 4° C. in a GSA rotor (Sorvall). The pellets were resuspended in 500 ml of infiltration media (1.1 g MS salts, 25 g sucrose, 0.25 g MES, pH5.7 with KOH, 100 ng/ml benzylaminopurine and 50 μl Vac-In-Stuff (Silwet L-77; Lehle Seeds)) and placed in a large plastic dish in a vacuum desiccator with 4 large rubber stoppers. Five pots containing 8 plants each at the right stage of development were used sequentially for infiltration. Each pot was first inverted over a trash can to remove any loose soil, then was placed (still inverted) into plastic container in the glass desiccator so that the 4 large rubber stoppers acted as stand for the inverted pot thus allowing the bolts to be dipped into the Agrobacterium slurry, but not the rosettes. The plants were then subjected to a vacuum (400 mm Hg) in this inverted state for 10 minutes. The vacuum infiltrated plants were then allowed to recover and grown as usual in the growth chamber conditions explained in the plant material section. After several weeks when the siliques were dry and seed matured, the seeds were collected with each pot pooled together.

Selecting Plant Transformants and Segregation Analysis

[0270] To identify primary transformants, seeds from the vacuum-infiltrated plants were surface sterilized in a solution of 1% (v/v) sodium hypochlorite and 0.1% (v/v) Tween 80 for 20 minutes on a rotator (Barnstead/Thermolyne), rinsed four times with sterile water, and resuspended in a sterile 0.8% agar. The resuspended seeds were then planted onto sterile, half-strength Murashige and Skoog (MS) medium (2.2 g/L) supplemented with 1% (w/v) sucrose, 0.5 g/L 2-[N-Morpholino]ethanesulfonic acid (MES), 0.7% (w/v) bacteriological agar and 40 to 50 μg/mlkanamycin (Murashige and Shoog, 1962). Only transformants are able to grow on the kanamycin-containing plates since the binary vector provides the kanamycin resistance gene to the transformant seedlings (FIG. 6). Seedlings that do not harbour the binary vector become yellow and die, as there is no kanamycin resistance gene. Wild-type seedlings were used as controls and plated onto MS medium without kanamycin added to the medium, as well seeds from a homozygous line containing empty pKYLX71 vectors were seeded as controls on kanamycin containing plates. The empty vector control is useful in demonstrating the effect kanamycin has on growth of the seedlings as well as the effect of random integration of the binary vector into the genome of Arabidopsis thaliana. A small amount of wild type seed was plated onto a small area of each plate containing MS medium and 40 to 50 μg/ml kanamycin. This was done in order to make sure the medium was selective enough for the transformants and to test the strength of the kanamycin.

[0271] The seeded plates were kept at 4° C. for 3 days to synchronize the germination. After 3 days the plates were transferred to growth chambers where they grew for an additional 7 days under 16-h light/8-h dark cycles at 20±2° C. Lighting was maintained at 150 μmol radiation m^-2s^-1 and was provided by cool-white fluorescent bulbs. The efficiency for transformation of Arabidopsis thaliana plants with the pKYLX71:wounding-induced AteIF-5A and pKYLX71:growth AteIF-5A vectors was determined.

[0272] After a total of 10 days since seeding, the 14 transformants or the 16 transformants for Sense wounding-induced AteIF-5A and Sense growth AteIF-5A respectively were transplanted to Promix BX soil (Premier Brands, Brampton, ON, Canada) in flats containing 32 cells. These transplanted Ti generation plants were then transferred into another growth chamber operating at 22° C. with 16-h light/8-h dark cycles. Lighting at 150 μmol radiation m^-2s^-1 was provided by cool-white fluorescent bulbs. The T1 generation plants grew to maturity and produced T2 generation seeds. These were harvested and stored at -20° C. until further screening was done. The T1 generation was named 1, 2, 3, etc. All 16 lines of Sense growth AteIF-5A plants survived and produced seeds, but only 9 out of 14 transformants of the Sense wounding-induced AteIF-5A plants survived and produced seeds.

[0273] The selection of T2 generation transformants was conducted in the same way as the T1 generation transformants Line 12 of the Sense growth AteIF-5A plants produced no transformants on the selectable media and was not included in any further work. Lines 1 through to 16 (minus line 12) of the Sense growth AteIF-5A plants each had 8 sublines carried through. These were named A through H so that for example in the T1 line 1, the T2 generation plants were named 1A, 1B, 1C, etc. Lines 1, 2, 3, 4, 5, 7, 9, and 11 of the Sense wounding-induced AteIF-5A plants each had 8 sublines (A-H) carried through. Line 12 T1 plants had only produced about 30 T2 seeds and only 1 subline in the T2 generation will be carried through. T2 plants of Sense wounding-induced AteIF-5A are still growing and being characterized. The T2 plants for the Sense growth AteIF-5A have matured and produced seeds, which were harvested and stored at -20° C. until further analysis.

[0274] The selection of the T3 generation transformants of Sense growth AteIF-5A was conducted in the same manner as the T2. Eight lines were chosen based on phenotype analysis as well as the degree of over expression of Sense growth AteIF-5A. The levels of expression were broken down into four categories: high-level expression, medium-level expression, low-level expression, and no expression (due to co-suppression). Two lines were chosen for each of the levels of expression and 12 plants from each line were transplanted. The corresponding lines for these four levels of expression are: 1A, 2D, 4D, 15A, 8D, 9H, 11C and 16C. The T3 generation for Sense growth AteIF-5A plants are still growing and being characterized.

Example 18

Phenotype Analysis of Sense Wounding-Induced AteIF5A and Sense Growth AteIF5A

Photographic Record

[0275] Morphological phenotypes of the Sense wounding-induced AteIF-5A and Sense growth AteIF-5A lines were recorded photographically during segregation, as were the phenotypes of the corresponding control wild type plants (Arabidopsis thaliana ecotype Columbia) and plants transformed with an empty binary vector pKYLX71.

Seed Measurements

[0276] T3 seeds collected from T2 plants of Sense growth AteIF-5A were measured for total seed yield (both weight and volume), seed size (length and width), and calculated individual weight and volume of produced seed. Total seed yield by weight was measured on a Sartorius analytical digitized scale, and the volume was determined by pouring and packing down the total seed yielded by each plant into a glass 1 ml syringe that was graduated every 100 μl. To determine the seed size by length, width and calculated volume, the seeds were placed on a slide containing a micrometer and viewed on an Olympus BX51 Microscope. Photographs of the seeds on the micrometer were taken with a Spot Insight Color Camera (Diagnostic Instruments Inc.) attached to a Compaq Evo D500 (Compaq Company Corporation; Intel® Pentium 4 CPU 1.7 GHz, 262 MG RAM, running Windows 2000). Using Image-Pro Express Version 4.0 for Windows. Measurements of 10 seeds in each subline were made using the micrometer in the image for size calibration. The measurements were imported into Microsoft Excel, and calculations such as standard error and volume were performed.

Example 19

Biochemical Analysis of Sense Wounding-Induced AteIF5A and Sense Growth AteIF5A-Protein Fractionation and Western Blotting

[0277] The first cauline leaf from each subline of Sense growth AteIF-5A T2 plants were collected and proteins extracted as described above. Total protein from lines 1A, 2A, up to 16A were fractionated by 12% SDS-PAGE and transferred to a PVDF membrane. The blot was probed with growth aAteIF-5A at a 1:50 dilution. Control total protein was extracted from the first cauline leaf from wild type and empty binary vector control plants.

Example 20

Expression of Arabidopsis thaliana Translation Initiation Factor 5A (AteIF-5A) Isoforms in Wild Type Columbia

[0278] Several tissues were collected at different developmental stages and the extracted proteins from these tissues were used for Western blotting. The Western blot in FIG. 8 demonstrates that senescence-induced AteIF-5A is not present in the 2 week old rosette leaves, but is upregulated in the 3 week old rosette leaves and increases in abundance until 5 weeks and declines in abundance, but is still present at 7 weeks. No senescence AteIF-5A was detected in the PEG treated plants or control, but was present in the flower lane (which included senescent flowers) and in the imbibed seed lane reflecting senescence of cotyledonary tissues. When the blot was probed with the wounding-induced aATeIF-5A antibody, faint bands appeared in the siliques, imbibed seed and stem lanes. The band seen in the siliques and stem lanes may be due to the wounding that occurred with collection of the tissue. Since it is difficult to collect the siliques and stem, they were not flash frozen immediately allowing for some up-regulation of the wounding-induced isoform of AteIF-5A. The only band that appeared when the blot was probed with growth aAT-eIF5A was imbibed seeds, keeping with the notion that this is the isoform involved in cell division.

[0279] Plants that were treated with either no treatment, mock inoculation with MgCl₂, avr P. syringae or with vir P. syringae were collected at several time points to analyze the expression of the AteIF-5As during pathogen ingress. The avr strain is recognizable by the plant and induces the hypersensitive response that leads to cell death or necrosis in the region of infection, thus disallowing the pathogen to cause disease. Furthermore the localized response eventually becomes a systemic response in order to protect the plant from further ingress. This is known as Systemic Acquired Resistance (SAR), which involves the expression of a suite of genes known as the Pathogenesis Response (PR) genes. On the other hand the vir strain will not be recognized by the plant, and will not induce a hypersensitive response and will lead to disease. The diseased state of Arabidopsis thaliana includes yellowing leaves and cell death after a few days post infection. After 72 hours post treatment control plants, mock treated plants, avr treated plants and vir treated plants were collected for western blotting with the three aAteIF-5A antibodies (FIG. 9). At this point both SAR and disease were visible in the avr treated and the vir treated plants respectively. When probed with the senescence-induced aAteIF-5A antibody, a band that was relatively the same in all the samples was observed. Since all of the plants were 4 weeks old this came with no surprise, since the senescence isoform was seen starting at 3 weeks in FIG. 8. When the blot was then probed with the wounding-induced aAteIF-5A antibody, a faint band was detectable in the untreated, mock treated and avr treated plants where there was a strong band detected in the vir treated plants. This upregulation of the wounding isoform may be due to cell death caused by disease (also a type of cellular wounding). The blot probed with growth aAteIF-5A did not show any bands and thus was not included in the figure. As the senescence-induced AteIF-5A did not change in expression during these treatments demonstrates its specificity for natural senescence. The increase in wounding-induced AteIF-5A expression also demonstrates its specificity for death due to wounding. To further investigate this possibility, an experiment was performed with wounding leaves of Arabidopsis thaliana.

[0280] The wounding experiment showed similar results as the pathogenesis experiment (FIG. 10). Northern blots were used to show the transcriptional change in of senescence-induced AteIF-5A, wounding-induced AteIF-5A and growth AteIF-5A. The probes were specific to each of the AteIF-5As and consisted of the 3'UTR of each. It was observed that like the pathogenesis experiment senescence-induced AteIF-5A expression did not change, as these were 4-week-old plants and samples were only taken over a 9-hour interval. This again is consistent with the fact that senescence-induced AteIF-5A is natural senescence specific isoform. The expression of wounding-induced AteIF-5A however did increase after 9 hours. There is probably some translational control occurring, as the transcript appears fairly constitutive (FIG. 10), but the protein does not appear as highly expressed when not induced (FIG. 9). The transcript for growth AteIF-5A was barely detectable in all the samples, and shows a decline in expression post wounding.

Example 21

Production of Transformed Arabidopsis thaliana Plants Over Expressing the Three eIF-5A Isoforms

[0281] The AteIF-5As were isolated from genomic DNA by PCR (FIG. 11). The products were ligated in pGEM (FIG. 12) and the sequence was verified for suitability for over-expression in planta. Wounding-induced AteIF-5A and growth AteIF-5A were double digested out of pGEM with XhoI and SacI and ligated in the sense orientation behind the cauliflower mosaic virus 35S² promoter in pKYLX71. Positive ligation was confirmed by digestion and PCR (FIG. 13). The pKYLX71:senescence-induced AteIF-5A and the pKYLX71:growth AteIF-5A were then electroporated into Agrobacterium tumefaciens GV3010 for transformation via vacuum infiltration of Arabidopsis thaliana wild type of the ecotype Columbia. After plant transformation the seeds were collected and transformants selected for on Kanamycin containing MS plates.

Arabidopsis thaliana Plants Over Expressing Wounding-Induced AteIF-5A (Sense Wounding-Induced AteIF-5A)

[0282] T1 generation plants were seeded on MS plates containing 50 μg/ml Kanamycin and were stored at 4° C. for 3 days and in the growth chamber for 7 days (FIG. 14). There were 14 transformants that were transplanted to soil. A common phenotype in these 14 T1 generation plants was stunted growth. Lines 1, 4, 6, 8, 10, 11, 12, 13, and 14 were severely stunted in their growth and 6, 8, 10, 13 and 14 did not produce any seed. Lines 2 and 3 were moderately stunted whereas lines 5, 7 and 9 grew similarly to wild type plants (FIG. 15 and FIG. 16). Some other phenotypes observed in the T1 generation of Sense wounding-induced AteIF-5A plants included yellow leaves, purple cotyledons, curled up leaves and differences in flower shape. It is interesting to note that the appearance in the stunted growth was not observed until the plants were transplanted to soil. A possible explanation of this would be that during transplant the roots are damaged slightly (a consequence of transplanting that is unavoidable) and were unable to recover. In fact a preliminary experiment where seeds were soaked in a Kanamycin solution and seeded to soil directly no stunted plants were observed (whereas previously 70% of the plants had some degree of stunting), as no root damage would be invoked without transplantation.

[0283] Lines 1, 2, 3, 4, 5, 7, 8, 11 and 12 produced T2 seeds and were carried through (FIG. 17). Each T2 line has sublines A-H, except for 12, which only grew one transformant, and are currently being analyzed.

Arabidopsis thaliana Plants Over Expressing Growth AteIF-5A (Sense Growth AteIF-5A)

[0284] The T1 generation seeds of Sense growth AteIF-5A were grown on selective media and 16 transformants grew (FIG. 18). The transformants were photographed over their lifetime. The phenotypes varied from similar to wild type (Lines 1, 2, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, and 16) to moderately stunted and yellow (Lines 2, 4 and 9; FIG. 19). All the lines were carried through to T2 and each line had 8 sublines labeled A-H. Line 12 did not produce any transformants in T2 and was deemed to be wild type. The T2 generation plants had much more exaggerated phenotypes than that of T1 generation plants. The lines that were carried to T3 will be discussed in detail.

[0285] The Sense growth AteIF-5A T2 generation lines were characterized in groups according to the level of expression of the growth AteIF-5A transgene. A Western blot was performed on protein extracted from cauline leaves from each line (FIG. 20). Since most of the sublines A-H demonstrated similar phenotypes within a line, the Western blot was only done with subline A of each line to get a general overview of level of expression of growth AteIF-5A. Protein from the cauline leaves of wild type plants and plants containing the empty binary vector were used as controls on the gels. The level of expression observed in these sublines can be categorized as high (Lines 1, 2, 3, 10, 13), medium (Lines 4, 5, 6, 15), low (Lines 7,8,9,14) or none (Lines 11, 16, wild type and binary control). The blots were also probed with antibodies against senescence-induced AteIF-5A and wounding-induced AteIF-5A. These westerns indicated that the increase in expression in the Sense growth AteIF-5A lines is due to growth AteIF-5A and not a general upregulation of other AteIF-5A isoforms, as no significant amount of either isoform was detected. This also demonstrated that the specificity of the isoform specific antibodies is acceptable.

[0286] The Sense growth AteIF-5A lines be carried through to the T3 generation were chosen based on phenotype as well as the level of expression of growth AteIF-5A (See Table 1 for a summary of phenotypes within each line). Two lines from each category of level of expression were chosen. The lines that will be carried through are 1A, 2D, 4D, 15A, 8D, 9H, 11C, and 16C.

[0287] Line 1 according to the western blot in FIG. 20, has a high level of growth AteIF-5A expression. These plants had large, dark green rosettes with leaves that were quite round in comparison to wild type plants (FIG. 21). The rosettes of line 1 also had a whorled phenotype, where the leaves all curl in the same direction. These Sense growth AteIF-5A plants bolted slightly later than wild type. Line 2 also demonstrated high level of growth AteIF-5A expression, but differed from line 1 in that these plants were small and yellowed (FIG. 22). Line 2 plants also bolted later than the wild type and binary control plants, as well produced smaller bolts (about half the size) and fewer siliques.

[0288] Of the medium level of expression lines, line 4 appeared similar to wild type in leaf/rosette size and in bolt size, though appeared to bolt just a few days before the wild type and binary control plants. The second line with a medium level of expression of growth AteIF-5A is line 15. These plants are, like line 4, very similar to wild type, but the area that the rosette occupied was larger than the controls (FIGS. 23 and 24). The leaves of the rosette also appeared to be rounder at the tips than the controls. The bolts however did not appear to have any distinctive phenotype.

[0289] The low expressing Sense growth AteIF-5A lines that will be carried through to T3 are from lines 8 and 9. Line 8 had very large leaves and large rosettes compared to the control plants (FIG. 25). The leaves also appeared to be wider and rounder than the control plants. The time of bolting, bolt size and number seemed to be consistent with the controls. The Sense growth AteIF-5A line 9 had similar leaf shape as in line 8, but was far more yellow and smaller (FIG. 26). As in line 2 (one of the high expressing lines), these plants show stunted growth, shorter bolts, but unlike line 2, line 9 bolted about the same time as the control plants.

[0290] The two lines 11 and 16 of the Sense growth AteIF-5A plants according to the western blot (FIG. 20) have no upregulated expression of growth AteIF-5A. This may be due to cosuppression of the transgene as well as the endogenous gene. Though these plants do look similar to the controls (FIG. 27 and FIG. 28), it is believed that the transgene is incorporated into the genome of lines 11 and 16 for several reasons. Firstly, they do have Kanamycin resistance as demonstrated by the selectivity on the Kanamycin containing MS plates. Secondly, the rosette size, leaf size, and bolt size of line 16 (FIG. 28) are at least 50% larger than the controls. But the strongest evidence is in the size and composition of the T3 seeds that they produced.

[0291] The T3 seeds were measured from all lines of T2 Sense growth AteIf-5A plants. Photographs were taken of each line (the largest and the smallest highlighted in FIG. 29), and measurements were made in silico with a micrometer in the photographs used for calibration. For each line and for the controls, ten of the largest seeds in the field of view were measured and used for calculations. It was found that the high expression line 2 had seeds that were up to 3 times as large as the wild type and binary controls. Whereas the lines that demonstrated the lowest expression (Lines 11 and 16) had some of the smallest seeds that were only about 88% the size of wild type or binary control seeds. The average seed size for each line was expressed as nm³ (FIG. 30) and was calculated using an equation for the volume of an ellipsoid as seeds from Arabidopsis thaliana are approximately ellipsoid. The measured size of the control seeds fell into published guidelines as determined by Boyes et al (2001). From the measured size of individual seeds and the total seed yield (both weight and volume), the average individual seed weight was calculated and plotted (FIG. 31). It appeared that most of the lines that demonstrated a different size than that of the control seeds also had the same trend in individual seed weight. In fact when the seed weight was plotted against the seed size (volume) the relationship was mostly linear with an R²=0.7412. There were 5 lines that were outliers that had either an increased density (3 of them) or a decreased density (2 of them). One of the lines with the increased density is 8D and will be carried through T3 generation. The total seed yield from all the T2 generation plants were quite variable, with few trends. One notable line however is the medium expressing Sense growth AteIF-5A line 4D, which produced the most seeds (both weight and volume). In fact 4D produced 2.5 fold more than the control plants and will be carried through T3.

[0292] T3 seeds were plated on selection media as described previously. Lines 1A, 2D, 4D, 1 5A, 8D, 9H, 11C and 16C were transplanted to soil. Several other sublines of Sense growth AteIF-5A line 1 did not germinate, as well as line 2H, which had the largest seeds of all the sublines did not germinate. Plants from line 11 (one of the cosuppression lines) were not as healthy as typically found at this age. These seeds were also one of the smallest measured. It appears that these lines are still segregating, as there were still non-Kanamycin resistant plants as well as seeds that did not germinate from all the lines. This is probably a side effect of the transgene and not technique as the control seeds that were treated in the same manner, all germinated.

Example 22

Characterization of arabidopsis Senescence-Induced eIF-5A

[0293] Methodology for Obtaining Full-Length Arabidopsis Senescence-Induced eIF-5A

[0294] Degenerate primers based on several plant eIF-5A genes, in combination with vector primers T3 & T7 were used in order to PCR an eIF-5A gene from an Arabidopsis cDNA library. Specifically, the 5' region of the eIF-5A gene was obtained from a PCR reaction utilizing both the T3 primer (located upstream of the F5A gene in the library vector) and one of the downstream (reverse-orientation) degenerate primers. Likewise, the 3' region of the gene was obtained from a PCR reaction utilizing both the T7 primer (located downstream of the eIF-5A gene in the library vector) and one of the upstream (forward-orientation) degenerate primers. The full-length eIF-5A gene was derived from alignment analysis of the 5' region and 3' region of the gene.

[0295] There are 2-3 major products for each PCR reaction. These fragments were cloned to pbluescript plasmid and sequenced. The eIF-5A positive PCR fragments were identified based on the mapping analysis against the gene bank. There is only one upstream and downstream positive eIF-5A PCR fragments for Arabidopsis.

[0296] The specific 5'- and 3'-end primers for the Arabidopsis eIF-5A gene were designed according to the 5' and 3' PCR fragment sequencing results. The full-length Arabidopsis eIF-5A gene was obtained from a PCR reaction utilizing their specific 5'- and 3'-end primers and the corresponding cDNA library as a template. The full-length gene was further confirmed by sequencing. In the end, we cloned one Arabidopsis eIF-5A isoform gene, which was termed senescence-induced eIF-5A.

T3 and T7 Primers:

TABLE-US-00002

[0297] (SEQ ID NO: 20) T3: 5'- ATT AAC CCT CAC TAA AG -3' (SEQ ID NO: 18) T7: 5'- AAT ACG ACT CAC TAT AG -3'

Degenerate Primers for Arabidopsis eIF5A:

TABLE-US-00003 Forward (upstream) primer: (SEQ ID NO: 17) 5'- AAA RRY CGM CCY TGC AAG GT -3' Reverse (downstream) primer: (SEQ ID NO: 19) 5'- TCY TTN CCY TCM KCT AAH CC -3'

Subcloning Arabidopsis Antisensefull-Length Senescence-Induced eIF-5A into pKYLX71 Vector (Containing the SAG12 Promoter)

[0298] Specific (Homologous) Primers for Arabidopsis senescence-induced eIF-5A, antisense full-length construct: Forward Full-length senescence-induced eIF-5A primer (30-mer): 5'-CCGAGCTCCTGTTACCAAAAAATCTGTACC-3' (SEQ ID NO: 48) (note: underlined portion is the SacI recognition sequence, used for ligating the 5'-end of the PCR fragment into the SacI site in the Multiple Cloning Site (MCS) of pBluescript). Reverse full-length senescence-induced eIF-5A primer (36-mer): 5'-ACCTCGAGCGGCCGCAGAAGAAGTATAAAAACCATC-3' (SEQ ID NO: 49) (note: underlined portion is the NotI recognition sequence, used for ligation into the MCS of pBluescript).

[0299] The orientation of the SacI and NotI sites within the MCS of the pBluescript vector was such that the gene was subcloned in its antisense orientation (i.e. the NotI site is upstream of the SacI site).

Example 23

SAG 12 Promoter was Used to Express the Antisense Senescence-Induced Arabidopsis Full-Length eIF-5A)

[0300] Experimental evidence shows that transcription of a set of "senescence-associated genes" or SAGs increases during the onset of senescence (Lohman et al., 1994; Weaver et al., 1998). In fact, senescence appears to begin with the synthesis of new mRNAs and probably down-regulation of other mRNAs, indicating that selective synthesis of proteins is necessary for senescence (Nooden, 1988). That the leaf senescence program is accompanied by changes in gene expression was first demonstrated by Watanabe and Imaseki (1982) using in vitro translation followed by gel electrophoresis to detect changes occurring in translatable mRNA populations. This initial work and subsequent analysis of the in vitro translated proteins revealed the abundance of most mRNAs diminished significantly during the progression of senescence while other translatable mRNAs increased (Watanabe and Imaseki, 1982; Davies and Grierson, 1989; Becker and Apel, 1993; Buchanan-Wollaston, 1994; Smart et al., 1995). Differential screening of cDNA libraries made from mRNAs of senescent leaf tissues also demonstrated that the expression of many genes is down-regulated, whereas the expression of other genes is up-regulated during senescence. SAGs have been identified from a variety of plant species, including Arabidopsis (Hensel et al., 1993; Taylor et al., 1993; Lohman et al., 1994; Oh et al., 1996), asparagus (King et al., 1995), barley (Becker and Apel, 1993), Brassica napus (Buchanan-Wollaston, 1994), maize (Smart et al., 1995), radish (Azumi and Watanabe, 1991) and tomato (Davies and Grierson, 1989; Drake et al., 1996). Senescence can be morphologically identified as a characteristically patterned leaf yellowing that begins at the edges of a leaf and reaches the veins last (Weaver et al., 1998). Visible senescence in Arabidopsis thaliana rosette leaves appears approximately 21 days after germination with dramatic upregulation of SAG 12 at the time (Noh an Amasino, 1999). SAG 12 is a gene with the closest specificity for natural senescence and is thus termed a senescence marker. With no detectable expression in young leaves, SAG 12 is induced in older leaves after they are ˜20% yellow but cannot be induced by treatment that does not induce yellowing of leaves (Weaver et al., 1998). Its high degree of specificity for natural senescence can be explained by the fact that the gene product of SAG 12 shows similarity to cysteine proteases and may be involved in protein turnover during senescence (Lohman et al., 1994; Weaver et al., 1998).

Description of Transgenic Plants

[0301] Transgenic Arabidopsis plants were generated expressing the full-length antisense senescence-induced eIF-5A transgene under the control of the SAG 12 (leaf senescence-specific) promoter, which is activated at the onset of natural leaf senescence, approximately 21 days after germination (Noh and Amasino, 1994), but not in the event of stress-induced senescence. At this point, the transgenic plants express phenotypes characteristic of suppressed full-length senescence-induced eIF-5A expression. Rosette leaves were harvested from 3 to 8-week-old transgenic Arabidopsis antisense full-length senescence-induced eIF-5A plants.

Methodology for the Production of Homozygous Transgenic Antisense Senescence-Induced eIF-5A Arabidopsis thaliana Plants Under Control of the SAG 12 Promoter

[0302] Inserting the SAG 12-Antisense-Full-Length Senescence-Induced eIF-5A Construct in pKYLX71

[0303] First, the plasmid pKYLX71 was cut with EcoRI and HindIII to remove its double 35S promoter, and resultant sticky ends were filled in with Klenow enzyme to create blunt ends. pKYLX71 without the promoter was then ligated to re-circularize the plasmid.

[0304] Secondly, the Arabidopsis SAG 12 promoter was amplified from genomic DNA by PCR using primers containing SalI and XbaI, as described below. This promoter sequence was then inserted into the Multiple Cloning Site (MCS) of pBlueScript using the restriction enzymes SalI and XbaI followed by ligation with T4 DNA ligase.

[0305] The forward SAG 12 Primer was 5'-GGC CGTCGACGATATCTCTTTTTATATTCAAAC-3' (SEQ ID NO: 50) (underlined portion is Sail recognition site, used for ligating the 5'-end of the PCR fragment into the Sail site in the Multiple Cloning Site (MCS) of pBluescript). The Reverse SAG 12 Primer was 5'-CGTCTAGACATTGTTTTAGGAAAGTTAAATGA-3' (SEQ ID NO: 51) (underlined portion is the XbaI recognition site, used for ligating the 5'-end of the PCR fragment into the SacI site in the Multiple Cloning Site (MCS) of pBluescript).

[0306] Thirdly, to create the pBlueScript-SAG 12:antisense-full length-senescence-induced eIF-5A construct, full length senescence-induced eIF-5A was amplified by PCR from the Arabidopsis cDNA library using primers with SacI and NotI restriction sites, as outlined below, and subcloned into the pBluescript-SAG 12 described in the previous paragraph. Note that the orientation of the SacI and NotI sites within the MCS of the pBluescript-SAG 12 vector was such that the gene was subcloned in its antisense orientation (i.e. the NotI site is upstream of the SacI site).

[0307] The forward full-length senescence-induced eIF-5A Primer was 5'-CCGAGCTCCTGTTACCAAAAAATCTGTACC-3' (SEQ ID NO: 48) (note: underlined portion is the SacI recognition sequence, used for ligating the 5'-end of the PCR fragment into the SacI site in the Multiple Cloning Site (MCS) of pBluescript-SAG 12 vector). The reverse Full-length senescence-induced eIF-5A Primer was 5'-ACCTCGAGCGGCCGCAGAAGAAGTATAAAAACCATC-3' (SEQ ID NO: 49) (note: underlined portion is the NotI recognition sequence, used for ligation into the Multiple Cloning Site (MCS) of pBluescript-SAG 12 vector).

[0308] Finally, the desired construct was created in the binary vector, pKYLX71, by digesting pKYLX71 was digested with SacI and XhoI, and also cutting out the SAG 12:full-length senescence-induced eIF-5A cassette from pBluescript with Sail and SacI.

[0309] The XhoI and Sail sticky ends are partially complementary. Hence, these two sets of digested overhangs (specifically, SacI with SacI, and XhoI with SalI) were able to be ligated together with T4 DNA ligase, creating the final construct (SAG 12:antisense-senescence-induced eIF-5A in pKYLX71).

Transformation and T1 Seed Harvest

[0310] The pKYLX71-SAG 12:antisense-eIF-5A construct was proliferated in E. coli DHα cells, isolated and electroporated into a competent Agrobacterium strain. The bacteria were then used to infiltrate 4.5 week old wildtype Arabidopsis plants and the resulting infiltrated plants were designated as "T₀" plants, which were then grown to the end of their life-cycle. Seeds were harvested, collected and designated as T₁, seeds. 10 plates of T₁, seeds were plated and screened for kanamycin resistance (1/2MS salt and 50 μg kanamycin/mL) with wildtype as a control; only those seeds containing pKYLX71-SAG 12-antisense-eIF-5A construct survive and grow on kanamycin (K50) media. 24 T₁, seedlings were chosen from these plates and placed in soil. The seeds harvested from T₁. transgenic plants were labeled as T₂ seeds. Each seedling yielded one plant line (#1=1 line containing 1 plant, #2=1 line containing 1 plant, etc.).

Screening and Identification of Phenotypes

[0311] Once kanamycin resistant T₁, seeds were identified, successive generations of T₂, T₃ and T₄ plants were grown. By screening seeds on K50 media, it was possible to distinguish between those plants which inherited the genetic construct and were homozygous for the construct. A phenotypic expression of stunted growth was observed in one T₃ plant line when grown in a pot. However, when the same set of seeds was re-grown in identical conditions, the phenotype was not observed.

[0312] From the 24 T₁ plants, 4 lines were chosen on the basis of high seed yield (lines T2.14, T2.18, T2.19 and T2.23) and plated on K50 media with wildtype seeds as a control. Approximately 75% of the seeds from each line survived on K50 media and fell into size categories of Small, Medium and Large. From each line, small, medium and large seedlings were removed from plates and planted in soil. Under greenhouse conditions, the Small seedlings did not recover as quickly as their Medium and Large counterparts. At week 6, the Small plants were just beginning to show signs of bolting while the other plants had bolted and flowered. In total, six transgenic T₂ plants (from a total of 3 lines×8 plants=96 transgenic plants) demonstrated dramatic delay in bolting and were deemed "Late Bolt" plants. The seed yields of these plants were also dramatically lower than other transgenics.

[0313] From the 96 T₂ plants, 3 lines were selected to produce T₃ plants (T3.19.S8 and T3.14.L7 which were Late Bolts; and, T3.23.S3 which was not a Late Bolt). When planted on K50 media plates, these lines showed homozygous survival. 13 seedlings were transplanted into pots (10 seedlings per pot). From this set of plants, a dramatic dwarf phenotype was observed in T3.14.L7 plant line. T₄ seeds were collected, and lower seed yield was observed in that line. A dense growth (dense silique growth, more branches) phenotype was observed in line T3.19.S8, while a phenotype similar to wildtype was observed in line T3.23.S3. Seed sizes from the 3 transgenic lines were compared but no statistically significant differences were determined Chlorophyll levels were also analyzed but no statistically significant differences from wildtype control were determined.

[0314] T₄ seeds of lines T3.19.S8, T3.14.L7 and T3.23.S3 were screened on K50 to obtain the next generation of plants and showed evidence of inherited gene construct (uniform green growth on plates) compared with wild-type seed that died. However, when planted in individual flats, the dwarf phenotype was not expressed suggesting that the eIF-F5A antisense transgene had been lost. Finally, seeds collected from all T₅ plants were screened on K50 plates and showed evidence of kanamycin resistance. Work is now underway to confirm that the antisense transgene has been lost, and these T4 plants are azygous.

[0315] Eight daughter lines were chosen from mother lines T2.14, T2.19 and T2.23 and screened on K50 media with wild-type seeds as a control. Three lines were chosen based on low seed yield: T3.14.L8, T3.14.S8, and T3.23.S1. The other five lines chosen are: T3.18.S7, T3.18.S2, T3.19.S1, T3.19.S5, and T3.23.S6. All the lines screened on K50 media showed homozygous survival, while T3.14.L8, T3.14.S8 and T3.23.S6 showed heterozygous survival. Seedlings from lines T3.14.L8 and T3.14.S8 that survived were white in color with green vascular tissue, while seedlings from T3.23.S6 that survived were entirely dark green in color. These seedlings were selected for transplantation. In total, 28 seedlings from each line were transplanted into cells and grown in greenhouse conditions.

[0316] At week 3, all lines started bolting except for lines T3.14.L8 and T3.23.S1 and several plants within lines T3.18.S7, T3.18.S2, T3.19.S1, T3.19.S5, T3.23.S1 and T3.23.S6. An irregular rosette leaf morphology (elongation of 2^nd pair leaves phenotype) was observed in T3.14.L8 and T3.14.S8 lines. At week 5, additional irregular leaf morphologies of increased number of rosette leaves and crinkle-edged rosette leaves phenotypes were also observed in lines T3.18.S7 and T3.23.S6. Rosettes smaller than wild-type were observed in lines T3.23.S1, T3.19.S1, and T3.19.S5. At week 7, spindly stem and no stem elongation phenotypes were observed in lines T3.18.S7, T3.18.S2, T3.19.S1, T3.19.S5, T3.23.S1 and T3.23.S6. The first and second cauline leaf of each plant was collected at week 5 and 6, respectively, for investigation of senescence eIF-5A protein expression.

Example 24

Determination of Oxygen Output The leaves were harvested and the areas were measured before they were weighed. The leaves were ground to a fine powder using 1 mL of cold degassed grinding buffer with a mortar and pestle. Then the homogenate was transferred into an eppendorf tube and placed immediately on ice. For tomato leaves, the homogenate isolated required to be filtered through a piece of Miracloth.

[0317] 50 μl of homogenate from all samples were added into 10 ml test tubes containing 5 ml grinding buffer and 25 μl DCPIP (2,6-dichlorophenol indophenol). The samples were shaken well and then one set of samples were placed for 15 mins under illumination by a pair of lamps and the second set of samples were placed in the dark for 15 mins. After the minute incubation, 50 μL of DCMU (3-(3,4-dichlorophenyl)-1,1 dimethylurea) was added to both set of samples in order to stop the reaction and then centrifuged in a microcentrifuge for 2 mins at 14,000 g. The absorbencies of the supernatant collected were read at 590 nm using grinding buffer as a blank.

[0318] The molar extinction coefficient for this assay is 16×10³, that is, a change in concentration of 1 mole per liter changes the absorbance of the solution by 16×10³ μmole of DCPIP reduced/h/ml=(difference in absorbance)× 1/16×10³ (moles/l)]×[reaction volume (ml)/10³ (ml/l)]×[10⁶ (μmole/mole)]×[60 (min/hr)/reaction time (min)]×[1/sample volume (ml)].

[0319] For every 2 moles of DCPIP that are reduced, 1 mole of O₂ is generated.

Reference: Allen J. F. and Holmes N. G., 1986 Electron Transport and Redox Titration s in Photosynthesis: Energy Transduction. Edited by M. F. Hipkins & N. R. Baker., IRL Press, Oxford Pp 107-108.

Example 25

Quantitative Determination of Starch

[0320] Starch content in tomato stems was determined using a method adapted from Lustinec et al. Quantitative determination of starch, amylose, and amylopectin in plant tissues using glass fiber paper. Anal. Biochem. 132:265-271 (1983). Tomato stem tissue was homogenized in three volumes of water using an Omnimixer (12 reps of 5 sec each), followed by a Polytron homogenizer (30 sec). Homogenate was stored in 10 ml aliquots at -20° C. prior to analysis. For analysis, 10 ml homogenate was thawed and mixed with an equal volume of concentrated perchloric acid (HClO₄, 70% w/w) and incubated for 20 min at room temperature to dissolve the starch. Simultaneously, several solutions of potato starch (in the range of 0.1-1.0 mg/ml) were processed alongside the tomato stem sample to generate a standard curve. The homogenate (or potato starch standard solution) was stirred and filtered through Whatman GF/A glass microfiber paper (9.0 cm diameter) using a vacuum flask attached to an aspirator. One ml of filtrate was mixed with 3 ml of iodine solution A (8 mM I₂, 17 mM KI, 514 mM NaCl) and incubated for 30 min at 4° C. to form a starch-iodine precipitate. The precipitate was collected on Whatman GF/A glass microfiber paper (9.0 cm diameter) using a vacuum flask attached to an aspirator, and then wash the filtrate with the following solutions: once with 10 mL iodine solution B (83 mM I₂, 180 mM KI, 8% perchloric [HClO₄] acid); once with 5 mL ethanol-NaCl solution (67% ethanol, 342 mM NaCl); twice with 3 ml ethanol-NaOH solution (67% ethanol, 250 mM NaOH). Once ethanol had evaporated, the microfiber paper was removed from aspirator and inserted into screw-capped glass tube. Sulfuric [H₂SO₄] acid (9 mL of 0.75 M solution) was added to the tube and the tube was incubated in a boiling water bath for 30 min. Three 1 mL-aliquots of eluate were pipetted into glass test tubes and mixed with 1 mL of 5% phenol, quickly followed by 5 mL of concentrated H₂SO₄. The tubes were vortexed and incubated at room temperature for 30 min to allow the color to develop. Simultaneously, a blank for the spectrophotometer measurements was prepared by mixing 1 mL of 0.75 M H₂SO₄ with 1 mL of 5% phenol, and quickly adding 5 mL concentrated H₂SO₄; the blank was also incubated at room temperature for 30 min. A spectrophotometer was calibrated at 480 nm using the blank, and the O.D. of all samples and potato starch standards were measured and recorded. A standard curve was prepared using the potato starch solutions, and used to interpolate the quantity of starch in each sample.

Example 26

[0321] Arabidopsis thaliana (Columbia ecotype) was transformed by the Arabidopsis thaliana sense Senescence-induced eIF-5A (At-eIF) and Tomato sense senescence-induced eIF-5A genes independently. These genes were constitutively expressed in the whole life cycle of the transgenic plants. The inflorescence stems of these plants exhibited a significant increase of xylem development. See FIGS. 89-94.

[0322] The seeds of transgenic and control plants were sown on 1/2MS medium agar plates, and kept in a growth chamber at 22° C., 80% rh, and 16 h light/day, for 9 days. Then, the seedlings were transferred to 32-well-flats with a commercial soil, and were maintained under the same conditions as above, for 48 days. The main inflorescence stems were selected for microscopic observation. Cross sections were hand-cut from the base of the stems within 2 mm above the rosette. The sections were stained with the phloroglucinol-HCl method. We found that the stem xylem at this age has achieved its maximum development. A comparison was made between transgenic and control plants in the sizes (sectional areas) of xylem. In addition, measurements were done for phloem and pith in both transgenic and control plants.

[0323] Measurement of tissue areas was as follows. Cross sections were photographed with a Zeiss microscope, and the micrographs were digitalized using Photoshop®. These images were printed out on paper and different tissues were cut out, and their areas were measured by an area-measuring meter. To calculate the actual area of each tissue, the following formula was used: The actual area=(The area of an individual tissue on paper)/(Magnification)²

[0324] It thus appears that senescence-induced eIF-5A is also involved in programmed cell death associated with xylogenesis. Constitutive antisense suppression of senescence-induced AteIF-5A in Arabidopsis reduced the thickness of the inflorescence stem as well as the number of xylem cell layers. By contrast, the inflorescence stems of plants in which Arabidiposis or tomato senescence-induced eIF-5A was constitutively over-expressed were, on average, 1.7-fold thicker than those of corresponding wild-type plants, and the total xylem area per cross-section of inflorescence stem was 2 fold higher. The over-expressing transgenic plants also had greatly increased rosette leaf biomass and grew faster than wild-type plants, which may reflect enhanced nutrient uptake. The same phenotype was observed when the senescence-induced isoform of eIF-5A from tomato was over-expressed in Arabidopsis plants. These results collectively indicate that the senescence-induced isoform of eIF-5A not only regulates leaf and flower senescence, but is also involved in xylogenesis.

Example 27

Suppression of Deoxyhypusine Synthase Delays Browning of Pre-Packaged Cut Lettuce in Ambient Atmosphere

[0325] Commercially-available pre-packaged salad is commonly stored under conditions of controlled atmosphere, whereby the level of oxygen is greatly reduced below its atmospheric concentration in order to extend the shelf life of the product. The most common symptom of spoiled pre-packaged salad is browning on the cut surfaces of lettuce. Although controlled atmosphere packaging does achieve a delay in browning, it can also result in off-odour and off-flavour. In this study, down-regulation of deoxyhypusine synthase (DHS) was shown to have potential as an alternative strategy for delaying browning on the cut surfaces of lettuce. DHS catalyzes the activation of eukaryotic translation initiation factor 5A (eIF5A), which acts as a nucleocytoplasmic shuttle protein for select populations of mRNAs. DHS appears to play a role in browning of cut lettuce inasmuch as suppression of DHS expression (by antisense technology) resulted in a significant delay in the onset of browning under atmospheric conditions. Specifically, 80% of the cut segments of wildtype lettuce plants showed browning at 6 days after cutting, whereas only 27%, on average, of the cut segments of transgenic plants from 5 segregating lines turned brown over the same period, with some individual plants showing 0% browning. See FIGS. 51 and 53.

Example 28

Suppression of Deoxyhypusine Synthase Expression in Canola Increases Seed Yield

[0326] Deoxyhypusine synthase (DHS) mediates the first of two enzymatic reactions that convert inactive eukaryotic translation initiation factor-5A (eIF-5A) to an activated form able to facilitate translation. A full-length cDNA clone encoding canola (Brassica napus cv Westar) DHS was isolated from a cDNA expression library prepared from senescing leaves. DHS was suppressed in transgenic canola plants by expressing the antisense 3'-UTR of canola DHS cDNA under the regulation of the constitutive cauliflower mosaic virus (CaMV-35S) promoter. Plants expressing this antisense transgene had reduced levels of leaf DHS protein and exhibited delayed natural leaf senescence. Suppression of DHS expression also increased rosette leaf size by 1.5 to 2 fold, and enhanced seed yield by up to 90%. These pleiotropic effects of DHS suppression in canola are in agreement with results obtained previously for Arabidopsis (Wang et al., 2003, Plant Mol. Biol. 52: 1223-1235), and indicate that this protein plays a central role in plant development and senescence.

Example 29

Extending the Vase Life of Carnation Flowers by Administering Inhibitors of Deoxyhypusine Synthase and by Antisense Suppression of Deoxyhypusine Synthase

[0327] A full-length cDNA clone (AF296079) encoding deoxyhypusine synthase (DHS) was isolated from carnation petals. DHS mediates the first of two enzymatic reactions that convert inactive eukaryotic translation initiation factor-SA (eIF-5A) to an activated form able to facilitate translation. Northern analysis revealed that DHS expression is correlated with senescence of carnation flower petals. Treatment of cut carnation flowers with inhibitors of the DHS reaction, including diaminobutane (putrescine), diaminopropane, diaminohexane, diaminooctane and spermidine, extended the vase life of the flowers by up to 83%. In order to evaluate the role of DHS in carnation flower senescence more definitively, expression of the protein was suppressed in transgenic plants by introducing the antisense 3'-UTR of carnation DHS cDNA under regulation of the constitutive cauliflower mosaic virus promoter through Agrobacterium transformation. Three lines of transgenic flowers with reduced DHS expression were analyzed and found to have longer vase-life relative to wild-type flowers. Indeed, one of the lines exhibited an increase in vase life of >100%. These findings indicate that DHS plays a central role in flower senescence.

Example 30

The Delayed Bolting Phenotype Induced by Suppression of Deoxyhypusine Synthase in arabidopsis can be Rescued by Treatment with GA3

[0328] Deoxyhypusine synthase (DHS) is a ubiquitous enzyme required for post-translational activation of eukaryotic translation initiation factor 5A (eIF-5A) and appears to be essential for normal plant growth and development. DHS was suppressed in Arabidopsis by expressing full-length antisense Arabidopsis DHS cDNA in transgenic plants under the regulation of the senescence-specific SAG12 promoter. Plants expressing the transgene had reduced levels of leaf DHS protein, and exhibited delayed bolting and a pronounced delay (2 to 5 weeks) in the onset of leaf senescence. The bolts were also shorter, although this did not result in a reduction in biomass or seed yield. Treatment of the transgenic plants with GA3 reversed the delayed bolting phenotype. A similar phenotype was obtained by antisense suppression of DHS under the regulation of GCI, a glucacorticoid-inducible promoter that can be activated by administering dexamethasone (DEX). Again, administering GA3 rescued this phenotype; that is, the GA3-treated transgenic plants bolted normally, the bolts were of normal size and there was no delay in the onset of leaf senescence. These results collectively indicate that DHS, through activation of one or more of the three isoforms of eIF-5A in Arabidopsis, influences GA metabolism.

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[0393] Jakus, J., E. C. Wolff, M. H. Park, and J. E. Folk. 1993. Features of the spermidine-binding site of deoxyhypusine synthase as derived from inhibition studies: effective inhibition by bis- and mono-guanylated diamines and polyamines. J. Biol. Chem. 268, 13151-13159.

[0394] Kushner, S. R. 1978. An improved method for transformation of Escherichia coli with ColE1 derived plasmids. In: Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (H. W. Boyer and S, Nicosia, eds.), Elsevier/North-Holland Press, New York. Pp. 17-23.

[0395] Liu, Y. P., M. Nemeroff, Y. P. Yan, and K. Y. Chen. 1997. Interaction of eukaryotic initiation factor 5A with the human immunodeficiency virus type 1 Rev response element RNA and U6 snRNA requires deoxyhypusine or hypusine modification. Biol. Signals 6(3), 166-74.

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[0397] McCabe, M. S, L. C. Garratt, F. Schepers, W. J. R. M. Jordi, G. M. Stoopen, E. Davelaar, J. H. A. van Rhijn, J. B. Power, and M. R. Davey. 2001. Effects of P.sub.SAG12-IPT gene expression on development and senescence in transgenic lettuce. Plant Physiol. 127: 505-516.

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[0405] Park, M. H., E. C. Wolff, and J. E. Folk. 1993. Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. BioFactors 4(2), 95-104.

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[0407] Rosorius, 0., B. Reicher, F. Kraetzer, P. Heger, M.-C. Dabauvalle, and J. Hauber. 1999. Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1. J. Cell Sci. 112: 2369-2380. Stotz 2000.

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Sequence CWU 1

1

16511584DNALycopersicon esculentumCDS(54)..(1196) 1cgcagaaact cgcggcggca gtcttgttcc gtacataatc ttggtctgca ata atg 56 Met 1gga gaa gct ctg aag tac agt atc atg gac tca gta aga tcg gta gtt 104Gly Glu Ala Leu Lys Tyr Ser Ile Met Asp Ser Val Arg Ser Val Val 5 10 15ttc aaa gaa tcc gaa aat cta gaa ggt tct tgc act aaa atc gag ggc 152Phe Lys Glu Ser Glu Asn Leu Glu Gly Ser Cys Thr Lys Ile Glu Gly 20 25 30tac gac ttc aat aaa ggc gtt aac tat gct gag ctg atc aag tcc atg 200Tyr Asp Phe Asn Lys Gly Val Asn Tyr Ala Glu Leu Ile Lys Ser Met 35 40 45gtt tcc act ggt ttc caa gca tct aat ctt ggt gac gcc att gca att 248Val Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp Ala Ile Ala Ile50 55 60 65gtt aat caa atg cta gat tgg agg ctt tca cat gag ctg ccc acg gag 296Val Asn Gln Met Leu Asp Trp Arg Leu Ser His Glu Leu Pro Thr Glu 70 75 80gat tgc agt gaa gaa gaa aga gat gtt gca tac aga gag tcg gta acc 344Asp Cys Ser Glu Glu Glu Arg Asp Val Ala Tyr Arg Glu Ser Val Thr 85 90 95tgc aaa atc ttc ttg ggg ttc act tca aac ctt gtt tct tct ggt gtt 392Cys Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Val 100 105 110aga gac act gtc cgc tac ctt gtt cag cac cgg atg gtt gat gtt gtg 440Arg Asp Thr Val Arg Tyr Leu Val Gln His Arg Met Val Asp Val Val 115 120 125gtt act aca gct ggt ggt att gaa gag gat ctc ata aag tgc ctc gca 488Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu Ile Lys Cys Leu Ala130 135 140 145cca acc tac aag ggg gac ttc tct tta cct gga gct tct cta cga tcg 536Pro Thr Tyr Lys Gly Asp Phe Ser Leu Pro Gly Ala Ser Leu Arg Ser 150 155 160aaa gga ttg aac cgt att ggt aac tta ttg gtt cct aat gac aac tac 584Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr 165 170 175tgc aaa ttt gag aat tgg atc atc cca gtt ttt gac caa atg tat gag 632Cys Lys Phe Glu Asn Trp Ile Ile Pro Val Phe Asp Gln Met Tyr Glu 180 185 190gag cag att aat gag aag gtt cta tgg aca cca tct aaa gtc att gct 680Glu Gln Ile Asn Glu Lys Val Leu Trp Thr Pro Ser Lys Val Ile Ala 195 200 205cgt ctg ggt aaa gaa att aat gat gaa acc tca tac ttg tat tgg gct 728Arg Leu Gly Lys Glu Ile Asn Asp Glu Thr Ser Tyr Leu Tyr Trp Ala210 215 220 225tac aag aac cgg att cct gtc ttc tgt cct ggc ttg acg gat gga tca 776Tyr Lys Asn Arg Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly Ser 230 235 240ctt ggt gac atg cta tac ttc cat tct ttc aaa aag ggt gat cca gat 824Leu Gly Asp Met Leu Tyr Phe His Ser Phe Lys Lys Gly Asp Pro Asp 245 250 255aat cca gat ctt aat cct ggt cta gtc ata gac att gta gga gat att 872Asn Pro Asp Leu Asn Pro Gly Leu Val Ile Asp Ile Val Gly Asp Ile 260 265 270agg gcc atg aat ggt gaa gct gtc cat gct ggt ttg agg aag aca gga 920Arg Ala Met Asn Gly Glu Ala Val His Ala Gly Leu Arg Lys Thr Gly 275 280 285atg att ata ctg ggt gga ggg ctg cct aag cac cat gtt tgc aat gcc 968Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Val Cys Asn Ala290 295 300 305aat atg atg cgc aat ggt gca gat ttt gcc gtc ttc att aac acc gca 1016Asn Met Met Arg Asn Gly Ala Asp Phe Ala Val Phe Ile Asn Thr Ala 310 315 320caa gag ttt gat ggt agt gac tct ggt gcc cgt cct gat gaa gct gta 1064Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val 325 330 335tca tgg gga aag ata cgt ggt ggt gcc aag act gtg aag gtg cat tgt 1112Ser Trp Gly Lys Ile Arg Gly Gly Ala Lys Thr Val Lys Val His Cys 340 345 350gat gca acc att gca ttt ccc ata tta gta gct gag aca ttt gca gct 1160Asp Ala Thr Ile Ala Phe Pro Ile Leu Val Ala Glu Thr Phe Ala Ala 355 360 365aag agt aag gaa ttc tcc cag ata agg tgc caa gtt tgaacattga 1206Lys Ser Lys Glu Phe Ser Gln Ile Arg Cys Gln Val370 375 380ggaagctgtc cttccgacca cacatatgaa ttgctagctt ttgaagccaa cttgctagtg 1266tgcagcacca tttattctgc aaaactgact agagagcagg gtatattcct ctaccccgag 1326ttagacgaca tcctgtatgg ttcaaattaa ttatttttct ccccttcaca ccatgttatt 1386tagttctctt cctcttcgaa agtgaagagc ttagatgttc ataggttttg aattatgttg 1446gaggttggtg ataactgact agtcctctta ccatatagat aatgtatcct tgtactatga 1506gattttgggt gtgtttgata ccaaggaaaa atgtttattt ggaaaacaat tggattttta 1566atttattttt tcttgttt 15842381PRTLycopersicon esculentum 2Met Gly Glu Ala Leu Lys Tyr Ser Ile Met Asp Ser Val Arg Ser Val1 5 10 15Val Phe Lys Glu Ser Glu Asn Leu Glu Gly Ser Cys Thr Lys Ile Glu 20 25 30Gly Tyr Asp Phe Asn Lys Gly Val Asn Tyr Ala Glu Leu Ile Lys Ser 35 40 45Met Val Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp Ala Ile Ala 50 55 60Ile Val Asn Gln Met Leu Asp Trp Arg Leu Ser His Glu Leu Pro Thr65 70 75 80Glu Asp Cys Ser Glu Glu Glu Arg Asp Val Ala Tyr Arg Glu Ser Val 85 90 95Thr Cys Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly 100 105 110Val Arg Asp Thr Val Arg Tyr Leu Val Gln His Arg Met Val Asp Val 115 120 125Val Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu Ile Lys Cys Leu 130 135 140Ala Pro Thr Tyr Lys Gly Asp Phe Ser Leu Pro Gly Ala Ser Leu Arg145 150 155 160Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn 165 170 175Tyr Cys Lys Phe Glu Asn Trp Ile Ile Pro Val Phe Asp Gln Met Tyr 180 185 190Glu Glu Gln Ile Asn Glu Lys Val Leu Trp Thr Pro Ser Lys Val Ile 195 200 205Ala Arg Leu Gly Lys Glu Ile Asn Asp Glu Thr Ser Tyr Leu Tyr Trp 210 215 220Ala Tyr Lys Asn Arg Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly225 230 235 240Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Lys Lys Gly Asp Pro 245 250 255Asp Asn Pro Asp Leu Asn Pro Gly Leu Val Ile Asp Ile Val Gly Asp 260 265 270Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Gly Leu Arg Lys Thr 275 280 285Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Val Cys Asn 290 295 300Ala Asn Met Met Arg Asn Gly Ala Asp Phe Ala Val Phe Ile Asn Thr305 310 315 320Ala Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala 325 330 335Val Ser Trp Gly Lys Ile Arg Gly Gly Ala Lys Thr Val Lys Val His 340 345 350Cys Asp Ala Thr Ile Ala Phe Pro Ile Leu Val Ala Glu Thr Phe Ala 355 360 365Ala Lys Ser Lys Glu Phe Ser Gln Ile Arg Cys Gln Val 370 375 3803369PRTHomo sapiens 3Met Glu Gly Ser Leu Glu Arg Glu Ala Pro Ala Gly Ala Leu Ala Ala1 5 10 15Val Leu Lys His Ser Ser Thr Leu Pro Pro Glu Ser Thr Gln Val Arg 20 25 30Gly Tyr Asp Phe Asn Arg Gly Val Asn Tyr Arg Ala Leu Leu Glu Ala 35 40 45Phe Gly Thr Thr Gly Phe Gln Ala Thr Asn Phe Gly Arg Ala Val Gln 50 55 60Gln Val Asn Ala Met Ile Glu Lys Lys Leu Glu Pro Leu Ser Gln Asp65 70 75 80Glu Asp Gln His Ala Asp Leu Thr Gln Ser Arg Arg Pro Leu Thr Ser 85 90 95Cys Thr Ile Phe Leu Gly Tyr Thr Ser Asn Leu Ile Ser Ser Gly Ile 100 105 110Arg Glu Thr Ile Arg Tyr Leu Val Gln His Asn Met Val Asp Val Leu 115 120 125Val Thr Thr Ala Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala 130 135 140Pro Thr Tyr Leu Gly Glu Phe Ser Leu Arg Gly Lys Glu Leu Arg Glu145 150 155 160Asn Gly Ile Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Glu Asn Tyr 165 170 175Cys Lys Phe Glu Asp Trp Leu Met Pro Ile Leu Asp Gln Met Val Met 180 185 190Glu Gln Asn Thr Glu Gly Val Lys Trp Thr Pro Ser Lys Met Ile Ala 195 200 205Arg Leu Gly Lys Glu Ile Asn Asn Pro Glu Ser Val Tyr Tyr Trp Ala 210 215 220Gln Lys Asn His Ile Pro Val Phe Ser Pro Ala Leu Thr Asp Gly Ser225 230 235 240Leu Gly Asp Met Ile Phe Phe His Ser Tyr Lys Asn Pro Gly Leu Val 245 250 255Leu Asp Ile Val Glu Asp Leu Arg Leu Ile Asn Thr Gln Ala Ile Phe 260 265 270Ala Lys Cys Thr Gly Met Ile Ile Leu Gly Gly Gly Val Val Lys His 275 280 285His Ile Ala Asn Ala Asn Leu Met Arg Asn Gly Ala Asp Tyr Ala Val 290 295 300Tyr Ile Asn Thr Ala Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg305 310 315 320Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Val Asp Ala Gln Pro 325 330 335Val Lys Val Tyr Ala Asp Ala Ser Leu Val Phe Pro Leu Leu Val Ala 340 345 350Glu Thr Phe Ala Gln Lys Met Asp Ala Phe Met His Glu Lys Asn Glu 355 360 365Asp 46PRTArtificial SequenceDescription of Artificial Sequence Synthetic conserved peptide fragment 4Thr Gly Lys His Gly His1 552272DNAArabidopsis thalianaCDS(68)..(265)CDS(348)..(536)CDS(624)..(842)CDS(979)..(1065)CDS(1- 154)..(1258)CDS(1575)..(1862) 5gaactcccaa aaccctctac tactacactt tcagatccaa ggaaatcaat tttgtcattc 60gagcaac atg gag gat gat cgt gtt ttc tct tcg gtt cac tca aca gtt 109 Met Glu Asp Asp Arg Val Phe Ser Ser Val His Ser Thr Val 1 5 10ttc aaa gaa tcc gaa tca ttg gaa gga aag tgt gat aaa atc gaa gga 157Phe Lys Glu Ser Glu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly15 20 25 30tac gat ttc aat caa gga gta gat tac cca aag ctt atg cga tcc atg 205Tyr Asp Phe Asn Gln Gly Val Asp Tyr Pro Lys Leu Met Arg Ser Met 35 40 45ctc acc acc gga ttt caa gcc tcg aat ctc ggc gaa gct att gat gtc 253Leu Thr Thr Gly Phe Gln Ala Ser Asn Leu Gly Glu Ala Ile Asp Val 50 55 60gtc aat caa atg gttcgtttct cgaattcatc aaaaataaaa attccttctt 305Val Asn Gln Met 65tttgttttcc tttgttttgg gtgaattagt aatgacaaag ag ttt gaa ttt gta 359 Phe Glu Phe Val 70ttg aag cta gat tgg aga ctg gct gat gaa act aca gta gct gaa gac 407Leu Lys Leu Asp Trp Arg Leu Ala Asp Glu Thr Thr Val Ala Glu Asp 75 80 85tgt agt gaa gag gag aag aat cca tcg ttt aga gag tct gtc aag tgt 455Cys Ser Glu Glu Glu Lys Asn Pro Ser Phe Arg Glu Ser Val Lys Cys 90 95 100aaa atc ttt cta ggt ttc act tca aat ctt gtt tca tct ggt gtt aga 503Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Val Arg 105 110 115gat act att cgt tat ctt gtt cag cat cat atg gtttgtgatt tttgctttat 556Asp Thr Ile Arg Tyr Leu Val Gln His His Met 120 125caccctgctt ttttatagat gttaaaattt tcgagcttta gttttgattt caatggtttt 616tctgcag gtt gat gtt ata gtc acg aca act ggt ggt gtt gag gaa gat 665 Val Asp Val Ile Val Thr Thr Thr Gly Gly Val Glu Glu Asp 130 135 140ctc ata aaa tgc ctt gca cct aca ttt aaa ggt gat ttc tct cta cct 713Leu Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ser Leu Pro 145 150 155gga gct tat tta agg tca aag gga ttg aac cga att ggg aat ttg ctg 761Gly Ala Tyr Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu160 165 170 175gtt cct aat gat aac tac tgc aag ttt gag gat tgg atc att ccc atc 809Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile 180 185 190ttt gac gag atg ttg aag gaa cag aaa gaa gag gtattgcttt atctttcctt 862Phe Asp Glu Met Leu Lys Glu Gln Lys Glu Glu 195 200tttatatgat ttgagatgat tctgtttgtg cgtcactagt ggagatagat tttgattcct 922ctcttgcatc attgacttcg ttggtgaatc cttctttctc tggtttttcc ttgtag aat 981 Asngtg ttg tgg act cct tct aaa ctg tta gca cgg ctg gga aaa gaa atc 1029Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg Leu Gly Lys Glu Ile 205 210 215aac aat gag agt tca tac ctt tat tgg gca tac aag gtatccaaaa 1075Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys220 225 230ttttaacctt tttagttttt taatcatcct gtgaggaact cggggattta aattttccgc 1135ttcttgtggt gtttgtag atg aat att cca gta ttc tgc cca ggg tta aca 1186 Met Asn Ile Pro Val Phe Cys Pro Gly Leu Thr 235 240gat ggc tct ctt ggg gat atg ctg tat ttt cac tct ttt cgt acc tct 1234Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Arg Thr Ser 245 250 255ggc ctc atc atc gat gta gta caa ggtacttctt ttactcaata agtcagtgtg 1288Gly Leu Ile Ile Asp Val Val Gln 260 265ataaatattc ctgctacatc tagtgcagga atattgtaac tagtagtgca ttgtagcttt 1348tccaattcag caacggactt tactgtaagt tgatatctaa aggttcaaac gggagctagg 1408agaatagcat aggggcattc tgatttaggt ttggggcact gggttaagag ttagagaata 1468ataatcttgt tagttgttta tcaaactctt tgatggttag tctcttggta atttgaattt 1528tatcacagtg tttatggtct ttgaaccagt taatgtttta tgaaca gat atc aga 1583 Asp Ile Arggct atg aac ggc gaa gct gtc cat gca aat cct aaa aag aca ggg atg 1631Ala Met Asn Gly Glu Ala Val His Ala Asn Pro Lys Lys Thr Gly Met270 275 280 285ata atc ctt gga ggg ggc ttg cca aag cac cac ata tgt aat gcc aat 1679Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn 290 295 300atg atg cgc aat ggt gca gat tac gct gta ttt ata aac acc ggg caa 1727Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Gly Gln 305 310 315gaa ttt gat ggg agc gac tcg ggt gca cgc cct gat gaa gcc gtg tct 1775Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser 320 325 330tgg ggt aaa att agg ggt tct gct aaa acc gtt aag gtc tgc ttt tta 1823Trp Gly Lys Ile Arg Gly Ser Ala Lys Thr Val Lys Val Cys Phe Leu 335 340 345att tct tca cat cct aat tta tat ctc act cag tgg ttt tgagtacata 1872Ile Ser Ser His Pro Asn Leu Tyr Leu Thr Gln Trp Phe350 355 360tttaatattg gatcattctt gcaggtatac tgtgatgcta ccatagcctt cccattgttg 1932gttgcagaaa catttgccac aaagagagac caaacctgtg agtctaagac ttaagaactg 1992actggtcgtt ttggccatgg attcttaaag atcgttgctt tttgatttta cactggagtg 2052accatataac actccacatt gatgtggctg tgacgcgaat tgtcttcttg cgaattgtac 2112tttagtttct ctcaacctaa aatgatttgc agattgtgtt ttcgtttaaa acacaagagt 2172cttgtagtca ataatccttt gccttataaa attattcagt tccaacaaca cattgtgatt 2232ctgtgacaag tctcccgttg cctatgttca cttctctgcg 22726362PRTArabidopsis thaliana 6Met Glu Asp Asp Arg Val Phe Ser Ser Val His Ser Thr Val Phe Lys1 5 10 15Glu Ser Glu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly Tyr Asp 20 25 30Phe Asn Gln Gly Val Asp Tyr Pro Lys Leu Met Arg Ser Met Leu Thr 35 40 45Thr Gly Phe Gln Ala Ser Asn Leu Gly Glu Ala Ile Asp Val Val Asn 50 55 60Gln Met Phe Glu Phe Val Leu Lys Leu Asp Trp Arg Leu Ala Asp Glu65 70 75 80Thr Thr Val Ala Glu Asp Cys Ser Glu Glu Glu Lys Asn Pro Ser Phe 85 90 95Arg Glu Ser Val Lys Cys Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu 100 105 110Val Ser Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln His His 115 120 125Met Val Asp Val Ile Val Thr Thr Thr Gly Gly Val Glu Glu Asp Leu 130 135

140Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ser Leu Pro Gly145 150 155 160Ala Tyr Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val 165 170 175Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Phe 180 185 190Asp Glu Met Leu Lys Glu Gln Lys Glu Glu Asn Val Leu Trp Thr Pro 195 200 205Ser Lys Leu Leu Ala Arg Leu Gly Lys Glu Ile Asn Asn Glu Ser Ser 210 215 220Tyr Leu Tyr Trp Ala Tyr Lys Met Asn Ile Pro Val Phe Cys Pro Gly225 230 235 240Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Arg 245 250 255Thr Ser Gly Leu Ile Ile Asp Val Val Gln Asp Ile Arg Ala Met Asn 260 265 270Gly Glu Ala Val His Ala Asn Pro Lys Lys Thr Gly Met Ile Ile Leu 275 280 285Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg 290 295 300Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Gly Gln Glu Phe Asp305 310 315 320Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys 325 330 335Ile Arg Gly Ser Ala Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser 340 345 350His Pro Asn Leu Tyr Leu Thr Gln Trp Phe 355 360719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7ggtggtgttg aggaagatc 19820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 8ggtgcacgcc ctgatgaagc 2091660DNADianthus caryophyllusCDS(256)..(1374) 9gtcattacaa tgcataggat cattgcacat gctaccttcc tcattgcact tgagcttgcc 60atacttttgt ttttgacgtt tgataataat actatgaaaa tattatgttt tttcttttgt 120gtgttggtgt ttttgaagtt gtttttgata agcagaaccc agttgtttta cacttttacc 180attgaactac tgcaattcta aaactttgtt tacattttaa ttccatcaaa gattgagttc 240agcataggaa aaagg atg gag gat gct aat cat gat agt gtg gca tct gcg 291 Met Glu Asp Ala Asn His Asp Ser Val Ala Ser Ala 1 5 10cac tct gca gca ttc aaa aag tcg gag aat tta gag ggg aaa agc gtt 339His Ser Ala Ala Phe Lys Lys Ser Glu Asn Leu Glu Gly Lys Ser Val 15 20 25aag att gag ggt tat gat ttt aat caa ggt gta aac tat tcc aaa ctc 387Lys Ile Glu Gly Tyr Asp Phe Asn Gln Gly Val Asn Tyr Ser Lys Leu 30 35 40ttg caa tct ttc gct tct aat ggg ttt caa gcc tcg aat ctt gga gat 435Leu Gln Ser Phe Ala Ser Asn Gly Phe Gln Ala Ser Asn Leu Gly Asp45 50 55 60gcc att gaa gta gtt aat cat atg cta gat tgg agt ctg gca gat gag 483Ala Ile Glu Val Val Asn His Met Leu Asp Trp Ser Leu Ala Asp Glu 65 70 75gca cct gtg gac gat tgt agc gag gaa gag agg gat cct aaa ttc aga 531Ala Pro Val Asp Asp Cys Ser Glu Glu Glu Arg Asp Pro Lys Phe Arg 80 85 90gaa tct gtg aag tgc aaa gtg ttc ttg ggc ttt act tca aat ctt att 579Glu Ser Val Lys Cys Lys Val Phe Leu Gly Phe Thr Ser Asn Leu Ile 95 100 105tcc tct ggt gtt cgt gac aca att cgg tat ctc gtg caa cat cat atg 627Ser Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln His His Met 110 115 120gtt gac gtg ata gta acg aca acc gga ggt ata gaa gaa gat cta ata 675Val Asp Val Ile Val Thr Thr Thr Gly Gly Ile Glu Glu Asp Leu Ile125 130 135 140aaa gga aga tcc atc aag tgc ctt gca ccc act ttc aaa ggc gat ttt 723Lys Gly Arg Ser Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe 145 150 155gcc tta cca gga gct caa tta cgc tcc aaa ggg ttg aat cga att ggt 771Ala Leu Pro Gly Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly 160 165 170aat ctg ttg gtt ccg aat gat aac tac tgt aaa ttt gag gat tgg atc 819Asn Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile 175 180 185att cca att tta gat aag atg ttg gaa gag caa att tca gag aaa atc 867Ile Pro Ile Leu Asp Lys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile 190 195 200tta tgg aca cca tcg aag ttg att ggt cga tta gga aga gaa ata aac 915Leu Trp Thr Pro Ser Lys Leu Ile Gly Arg Leu Gly Arg Glu Ile Asn205 210 215 220gat gag agt tca tac ctt tac tgg gcc ttc aag aac aat att cca gta 963Asp Glu Ser Ser Tyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile Pro Val 225 230 235ttt tgc cca ggt tta aca gac ggc tca ctc gga gac atg cta tat ttt 1011Phe Cys Pro Gly Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe 240 245 250cat tct ttt cgc aat ccg ggt tta atc gtc gat gtt gtg caa gat ata 1059His Ser Phe Arg Asn Pro Gly Leu Ile Val Asp Val Val Gln Asp Ile 255 260 265aga gca gta aat ggc gag gct gtg cac gca gcg cct agg aaa aca ggc 1107Arg Ala Val Asn Gly Glu Ala Val His Ala Ala Pro Arg Lys Thr Gly 270 275 280atg att ata ctc ggt gga ggg ttg cct aag cac cac atc tgc aac gca 1155Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala285 290 295 300aac atg atg aga aat ggc gcc gat tat gct gtt ttc atc aac act gcc 1203Asn Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala 305 310 315gaa gag ttt gac ggc agt gat tct ggt gct cgc ccc gat gag gct att 1251Glu Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Ile 320 325 330tca tgg ggc aaa att agc gga tct gct aag act gtg aag gtg cat tgt 1299Ser Trp Gly Lys Ile Ser Gly Ser Ala Lys Thr Val Lys Val His Cys 335 340 345gat gcc acg ata gct ttc cct cta cta gtc gct gag aca ttt gca gca 1347Asp Ala Thr Ile Ala Phe Pro Leu Leu Val Ala Glu Thr Phe Ala Ala 350 355 360aaa aga gaa aaa gag agg aag agc tgt taaaactttt ttgattgttg 1394Lys Arg Glu Lys Glu Arg Lys Ser Cys365 370aaaaatctgt gttatacaag tctcgaaatg cattttagta attgacttga tcttatcatt 1454tcaatgtgtt atctttgaaa atgttggtaa tgaaacatct cacctcttct atacaacatt 1514gttgatccat tgtactccgt atcttgtaat tttggaaaaa aaaaaccgtc tattgttacg 1574agagagtaca tttttgaggt aaaaatatag gatttttgtg cgatgcaaat gctggttatt 1634cccttgaaaa aaaaaaaaaa aaaaaa 166010373PRTDianthus caryophyllus 10Met Glu Asp Ala Asn His Asp Ser Val Ala Ser Ala His Ser Ala Ala1 5 10 15Phe Lys Lys Ser Glu Asn Leu Glu Gly Lys Ser Val Lys Ile Glu Gly 20 25 30Tyr Asp Phe Asn Gln Gly Val Asn Tyr Ser Lys Leu Leu Gln Ser Phe 35 40 45Ala Ser Asn Gly Phe Gln Ala Ser Asn Leu Gly Asp Ala Ile Glu Val 50 55 60Val Asn His Met Leu Asp Trp Ser Leu Ala Asp Glu Ala Pro Val Asp65 70 75 80Asp Cys Ser Glu Glu Glu Arg Asp Pro Lys Phe Arg Glu Ser Val Lys 85 90 95Cys Lys Val Phe Leu Gly Phe Thr Ser Asn Leu Ile Ser Ser Gly Val 100 105 110Arg Asp Thr Ile Arg Tyr Leu Val Gln His His Met Val Asp Val Ile 115 120 125Val Thr Thr Thr Gly Gly Ile Glu Glu Asp Leu Ile Lys Gly Arg Ser 130 135 140Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ala Leu Pro Gly145 150 155 160Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val 165 170 175Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Leu 180 185 190Asp Lys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile Leu Trp Thr Pro 195 200 205Ser Lys Leu Ile Gly Arg Leu Gly Arg Glu Ile Asn Asp Glu Ser Ser 210 215 220Tyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile Pro Val Phe Cys Pro Gly225 230 235 240Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Arg 245 250 255Asn Pro Gly Leu Ile Val Asp Val Val Gln Asp Ile Arg Ala Val Asn 260 265 270Gly Glu Ala Val His Ala Ala Pro Arg Lys Thr Gly Met Ile Ile Leu 275 280 285Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg 290 295 300Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala Glu Glu Phe Asp305 310 315 320Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Ile Ser Trp Gly Lys 325 330 335Ile Ser Gly Ser Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile 340 345 350Ala Phe Pro Leu Leu Val Ala Glu Thr Phe Ala Ala Lys Arg Glu Lys 355 360 365Glu Arg Lys Ser Cys 37011780DNALycopersicon esculentumCDS(43)..(522) 11aaagaatcct agagagagaa agggaatcct agagagagaa gc atg tcg gac gaa 54 Met Ser Asp Glu 1gaa cac cat ttt gag tca aag gca gat gct ggt gcc tca aaa act ttc 102Glu His His Phe Glu Ser Lys Ala Asp Ala Gly Ala Ser Lys Thr Phe5 10 15 20cca cag caa gct gga acc atc cgt aag aat ggt tac atc gtt atc aaa 150Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile Val Ile Lys 25 30 35ggc cgt ccc tgc aag gtt gtt gag gtc tcc act tca aaa act gga aaa 198Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys 40 45 50cac gga cat gct aaa tgt cac ttt gtg gca att gac att ttc aat gga 246His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile Phe Asn Gly 55 60 65aag aaa ctg gaa gat atc gtt ccg tcc tcc cac aat tgt gat gtg cca 294Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys Asp Val Pro 70 75 80cat gtt aac cgt acc gac tat cag ctg att gat atc tct gaa gat ggt 342His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser Glu Asp Gly85 90 95 100ttt gtc tca ctt ctt act gaa agt gga aac acc aag gat gac ctc agg 390Phe Val Ser Leu Leu Thr Glu Ser Gly Asn Thr Lys Asp Asp Leu Arg 105 110 115ctt ccc acc gat gaa aat ctg ctg aag cag gtt aaa gat ggg ttc cag 438Leu Pro Thr Asp Glu Asn Leu Leu Lys Gln Val Lys Asp Gly Phe Gln 120 125 130gaa gga aag gat ctt gtg gtg tct gtt atg tct gcg atg ggc gaa gag 486Glu Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met Gly Glu Glu 135 140 145cag att aac gcc gtt aag gat gtt ggt acc aag aat tagttatgtc 532Gln Ile Asn Ala Val Lys Asp Val Gly Thr Lys Asn 150 155 160atggcagcat aatcactgcc aaagctttaa gacattatca tatcctaatg tggtactttg 592atatcactag attataaact gtgttatttg cactgttcaa aacaaaagaa agaaaactgc 652tgttatggct agagaaagta ttggctttga gcttttgaca gcacagttga actatgtgaa 712aattctactt tttttttttt gggtaaaata ctgctcgttt aatgttttgc aaaaaaaaaa 772aaaaaaaa 78012160PRTLycopersicon esculentum 12Met Ser Asp Glu Glu His His Phe Glu Ser Lys Ala Asp Ala Gly Ala1 5 10 15Ser Lys Thr Phe Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr 20 25 30Ile Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser 35 40 45Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50 55 60Ile Phe Asn Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn65 70 75 80Cys Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile 85 90 95Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Ser Gly Asn Thr Lys 100 105 110Asp Asp Leu Arg Leu Pro Thr Asp Glu Asn Leu Leu Lys Gln Val Lys 115 120 125Asp Gly Phe Gln Glu Gly Lys Asp Leu Val Val Ser Val Met Ser Ala 130 135 140Met Gly Glu Glu Gln Ile Asn Ala Val Lys Asp Val Gly Thr Lys Asn145 150 155 16013812DNADianthus caryophyllusCDS(67)..(546) 13ctcttttaca tcaatcgaaa aaaaattagg gttcttattt tagagtgaga ggcgaaaaat 60cgaacg atg tcg gac gac gat cac cat ttc gag tca tcg gcc gac gcc 108 Met Ser Asp Asp Asp His His Phe Glu Ser Ser Ala Asp Ala 1 5 10gga gca tcc aag act tac cct caa caa gct ggt aca atc cgc aag agc 156Gly Ala Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Ser15 20 25 30ggt cac atc gtc atc aaa aat cgc cct tgc aag gtg gtt gag gtt tct 204Gly His Ile Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser 35 40 45acc tcc aag act ggc aag cac ggt cat gcc aaa tgt cac ttt gtt gcc 252Thr Ser Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala 50 55 60att gac att ttc aac ggc aag aag ctg gaa gat att gtc ccc tca tcc 300Ile Asp Ile Phe Asn Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser 65 70 75cac aat tgt gat gtt cca cat gtc aac cgt gtc gac tac cag ctg ctt 348His Asn Cys Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Leu 80 85 90gat atc act gaa gat ggc ttt gtt agt ctg ctg act gac agt ggt gac 396Asp Ile Thr Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Asp95 100 105 110acc aag gat gat ctg aag ctt cct gct gat gag gcc ctt gtg aag cag 444Thr Lys Asp Asp Leu Lys Leu Pro Ala Asp Glu Ala Leu Val Lys Gln 115 120 125atg aag gag gga ttt gag gcg ggg aaa gac ttg att ctg tca gtc atg 492Met Lys Glu Gly Phe Glu Ala Gly Lys Asp Leu Ile Leu Ser Val Met 130 135 140tgt gca atg gga gaa gag cag atc tgc gcc gtc aag gac gtt agt ggt 540Cys Ala Met Gly Glu Glu Gln Ile Cys Ala Val Lys Asp Val Ser Gly 145 150 155ggc aag tagaagcttt tgatgaatcc aatactacgc ggtgcagttg aagcaatagt 596Gly Lys 160aatctcgaga acattctgaa ccttatatgt tgaattgatg gtgcttagtt tgttttggaa 656atctctttgc aattaagttg taccaaatca atggatgtaa tgtcttgaat ttgttttatt 716tttgttttga tgtttgctgt gattgcatta tgcattgtta tgagttatga cctgttataa 776cacaaggttt tggtaaaaaa aaaaaaaaaa aaaaaa 81214160PRTDianthus caryophyllus 14Met Ser Asp Asp Asp His His Phe Glu Ser Ser Ala Asp Ala Gly Ala1 5 10 15Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Ser Gly His 20 25 30Ile Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser 35 40 45Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50 55 60Ile Phe Asn Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn65 70 75 80Cys Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Leu Asp Ile 85 90 95Thr Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Asp Thr Lys 100 105 110Asp Asp Leu Lys Leu Pro Ala Asp Glu Ala Leu Val Lys Gln Met Lys 115 120 125Glu Gly Phe Glu Ala Gly Lys Asp Leu Ile Leu Ser Val Met Cys Ala 130 135 140Met Gly Glu Glu Gln Ile Cys Ala Val Lys Asp Val Ser Gly Gly Lys145 150 155 16015702DNAArabidopsis thalianaCDS(56)..(529) 15ctgttaccaa aaaatctgta ccgcaaaatc ctcgtcgaag ctcgctgctg caacc atg 58 Met 1tcc gac gag gag cat cac ttt gag tcc agt gac gcc gga gcg tcc aaa 106Ser Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser Lys 5 10 15acc tac cct caa caa gct gga acc atc cgt aag aat ggt tac atc gtc 154Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile Val 20 25 30atc aaa aat cgt ccc tgc aag gtt gtt gag gtt tca acc tcg aag act 202Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr 35 40 45ggc aag cat ggt cat gct aaa tgt cat ttt gta gct att gat atc ttc 250Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile Phe50 55 60 65acc agc aag aaa ctc gaa gat att gtt cct

tct tcc cac aat tgt gat 298Thr Ser Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys Asp 70 75 80gtt cct cat gtc aac cgt act gat tat cag ctg att gac att tct gaa 346Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser Glu 85 90 95gat gga tat gtc agt ttg ttg act gat aac ggt agt acc aag gat gac 394Asp Gly Tyr Val Ser Leu Leu Thr Asp Asn Gly Ser Thr Lys Asp Asp 100 105 110ctt aag ctc cct aat gat gac act ctg ctc caa cag atc aag agt ggg 442Leu Lys Leu Pro Asn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser Gly 115 120 125ttt gat gat gga aaa gat cta gtg gtg agt gta atg tca gct atg gga 490Phe Asp Asp Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met Gly130 135 140 145gag gaa cag atc aat gct ctt aag gac atc ggt ccc aag tgagactaac 539Glu Glu Gln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys 150 155aaagcctccc ctttgttatg agattcttct tcttctgtag gcttccatta ctcgtcggag 599attatcttgt ttttgggtta ctcctatttt ggatatttaa acttttgtta ataatgccat 659cttcttcaac cttttccttc tagatggttt ttatacttct tct 70216158PRTArabidopsis thaliana 16Met Ser Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser1 5 10 15Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile 20 25 30Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60Phe Thr Ser Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95Glu Asp Gly Tyr Val Ser Leu Leu Thr Asp Asn Gly Ser Thr Lys Asp 100 105 110Asp Leu Lys Leu Pro Asn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser 115 120 125Gly Phe Asp Asp Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met 130 135 140Gly Glu Glu Gln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys145 150 1551720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17aaarrycgmc cytgcaaggt 201817DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18aatacgactc actatag 171920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 19tcyttnccyt cmkctaahcc 202017DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20attaaccctc actaaag 172122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21ctgttaccaa aaaatctgta cc 222221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22agaagaagta taaaaaccat c 212323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23aaagaatcct agagagagaa agg 232418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24ttttacatca atcgaaaa 182523DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25accaaaacct gtgttataac tcc 2326581DNAArabidopsis thalianaCDS(1)..(579) 26ggt ggt gtt gag gaa gat ctc ata aaa tgc ctt gca cct aca ttt aaa 48Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala Pro Thr Phe Lys1 5 10 15ggt gat ttc tct cta cct gga gct tat tta agg tca aag gga ttg aac 96Gly Asp Phe Ser Leu Pro Gly Ala Tyr Leu Arg Ser Lys Gly Leu Asn 20 25 30cga att ggg aat ttg ctg gtt cct aat gat aac tac tgc aag ttt gag 144Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu 35 40 45gat tgg atc att ccc atc ttt gac gag atg ttg aag gaa cag aaa gaa 192Asp Trp Ile Ile Pro Ile Phe Asp Glu Met Leu Lys Glu Gln Lys Glu 50 55 60gag aat gtg ttg tgg act cct tct aaa ctg tta gca cgg ctg gga aaa 240Glu Asn Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg Leu Gly Lys65 70 75 80gaa atc aac aat gag agt tca tac ctt tat tgg gca tac aag atg aat 288Glu Ile Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys Met Asn 85 90 95att cca gta ttc tgc cca ggg tta aca gat ggc tct ctt agg gat atg 336Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly Ser Leu Arg Asp Met 100 105 110ctg tat ttt cac tct ttt cgt acc tct ggc ctc atc atc gat gta gta 384Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu Ile Ile Asp Val Val 115 120 125caa gat atc aga gct atg aac ggc gaa gct gtc cat gca aat cct aaa 432Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Asn Pro Lys 130 135 140aag aca ggg atg ata atc ctt gga ggg ggc ttg cca aag cac cac ata 480Lys Thr Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile145 150 155 160tgt aat gcc aat atg atg cgc aat ggt gca gat tac gct gta ttt ata 528Cys Asn Ala Asn Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile 165 170 175aac acc ggg caa gaa ttt gat ggg agc gac tcg ggt gca cgc cct gat 576Asn Thr Gly Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp 180 185 190gaa gc 581Glu27523DNADianthus caryophyllusCDS(1)..(522) 27mga aga tcc atc aag tgc ctt gca ccc act ttc aaa ggc gat ttt gcc 48Arg Arg Ser Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ala1 5 10 15tta cca gga gct caa tta cgc tcc aaa ggg ttg aat cga att ggt aat 96Leu Pro Gly Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn 20 25 30ctg ttg gtt ccg aat gat aac tac tgt aaa ttt gag gat tgg atc att 144Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile 35 40 45cca att tta gat aag atg ttg gaa gag caa att tca gag aaa atc tta 192Pro Ile Leu Asp Lys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile Leu 50 55 60tgg aca cca tcg aag ttg att ggt cga tta gga aga gaa ata aac gat 240Trp Thr Pro Ser Lys Leu Ile Gly Arg Leu Gly Arg Glu Ile Asn Asp65 70 75 80gag agt tca tac ctt tac tgg gcc ttc aag aac aat att cca gta ttt 288Glu Ser Ser Tyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile Pro Val Phe 85 90 95tgc cca ggt tta aca gac ggc tca ctc gga gac atg cta tat ttt cat 336Cys Pro Gly Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His 100 105 110tct ttt cgc aat ccg ggt tta atc atc gat gtt gtg caa gat ata aga 384Ser Phe Arg Asn Pro Gly Leu Ile Ile Asp Val Val Gln Asp Ile Arg 115 120 125gca gta aat ggc gag gct gtg cac gca gcg cct agg aaa aca ggc atg 432Ala Val Asn Gly Glu Ala Val His Ala Ala Pro Arg Lys Thr Gly Met 130 135 140att ata ctc ggt gga ggg ttg cct aag cac cac atc tgc aac gca aac 480Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn145 150 155 160atg atg aga aat ggc gcc gat tat gct gtt ttc atc aac acc g 523Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr 165 1702824DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28ttgargaaga tycatmaart gcct 242923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29ccatcaaayt cytgkgcrgt gtt 2330484DNAArabidopsis thalianaCDS(2)..(112) 30t gca cgc cct gat gaa gct gtg tct tgg ggt aaa att agg ggt tct gct 49 Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Ser Ala 1 5 10 15aaa acc gtt aag gtc tgc ttt tta att tct tca cat cct aat tta tat 97Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser His Pro Asn Leu Tyr 20 25 30ctc act cag tgg ttt tgagtacata tttaatattg gatcattctt gcaggtatac 152Leu Thr Gln Trp Phe 35tgtgatgcta ccatagcctt cccattgttg gttgcagaaa catttgccac aaagagagac 212caaacctgtg agtctaagac ttaagaactg actggtcgtt ttggccatgg attcttaaag 272atcgttgctt tttgatttta cactggagtg accatataac actccacatt gatgtggctg 332tgacgcgaat tgtcttcttg cgaattgtac tttagtttct ctcaacctaa aatgatttgc 392agattgtgtt ttcgtttaaa acacaagagt cttgtagtca ataatccttt gccttataaa 452attattcagt tccaacaaaa aaaaaaaaaa aa 48431559DNALycopersicon esculentumCDS(1)..(156)modified_base(542)a, t, c or g 31ggt gct cgt cct gat gaa gct gta tca tgg gga aag ata cgt ggt ggt 48Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Gly1 5 10 15gcc aag act gtg aag gtg cat tgt gat gca acc att gca ttt ccc ata 96Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe Pro Ile 20 25 30tta gta gct gag aca ttt gca gct aag agt aag gaa ttc tcc cag ata 144Leu Val Ala Glu Thr Phe Ala Ala Lys Ser Lys Glu Phe Ser Gln Ile 35 40 45agg tgc caa gtt tgaacattga ggaagctgtc cttccgacca cacatatgaa 196Arg Cys Gln Val 50ttgctagctt ttgaagccaa cttgctagtg tgcagcacca tttattctgc aaaactgact 256agagagcagg gtatattcct ctaccccgag ttagacgaca tcctgtatgg ttcaaattaa 316ttatttttct ccccttcaca ccatgttatt tagttctctt cctcttcgaa agtgaagagc 376ttagatgttc ataggttttg aattatgttg gaggttggtg ataactgact agtcctctta 436ccatatagat aatgtatcct tgtactatga gattttgggt gtgtttgata ccaaggaaaa 496atgtttattt ggaaaacaat tggattttta atttaaaaaa aattgnttaa aaaaaaaaaa 556aaa 5593222PRTArtificial SequenceDescription of Artificial Sequence Synthetic conserved peptide fragment 32Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 203329DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33gagctcaaga ataacatctc ataagaaac 293427DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34ctcgagtgct cacttctctc tcttagg 273512PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 35Cys Asn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser1 5 103612PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 36Cys Thr Asp Asp Gly Leu Thr Ala Gln Met Arg Leu1 5 103712PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 37Cys Thr Asp Glu Ala Leu Leu Thr Gln Leu Lys Asn1 5 103832DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38aagcttgatc gtggtcaact tcctctgtta cc 323927DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39gagctcagaa gaagtataaa aaccatc 274027DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40ctcgagtgct cacttctctc tcttagg 274129DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41gagctcaaga ataacatctc ataagaaac 294230DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42ctcgagctaa actccattcg ctgacttcgc 304329DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43gagctctagt aaatataaga gtgtcttgc 2944353PRTUnknownDescription of Unknown Fungi sequence 44Met Ala Asp Asn Gln Ile Pro Ser Ser Val Ala Asp Ala Val Leu Val1 5 10 15Lys Ser Ile Glu Met Pro Glu Gly Ser Gln Lys Val Glu Glu Leu Asp 20 25 30Phe Asn Lys Phe Lys Gly Arg Pro Ile Thr Val Asp Asp Leu Leu Gln 35 40 45Gly Met Lys His Met Gly Phe Gln Ala Ser Ser Met Cys Glu Ala Val 50 55 60Arg Ile Ile Asn Glu Met Arg Ala Tyr Arg Asp Pro Thr Thr Ser Glu65 70 75 80Lys Thr Thr Ile Phe Leu Gly Tyr Thr Ser Asn Leu Ile Ser Ser Gly 85 90 95Leu Arg Gly Thr Leu Arg Tyr Leu Val Gln His Lys His Val Ser Ala 100 105 110Ile Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Phe Ile Lys Cys Leu 115 120 125Gly Asp Thr Tyr Met Ser Ser Phe Ser Ala Val Gly Ala Asp Leu Arg 130 135 140Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Val Val Pro Asn Ser Asn145 150 155 160Tyr Cys Ala Phe Glu Asp Trp Val Val Pro Ile Leu Asp Lys Met Leu 165 170 175Glu Glu Gln Glu Ala Ser Arg Gly Thr Glu Asn Glu Ile Asn Trp Thr 180 185 190Pro Ser Lys Val Ile His Arg Leu Gly Lys Glu Ile Asn Asp Glu Arg 195 200 205Ser Val Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro Val Phe Cys Pro 210 215 220Ala Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Thr Phe225 230 235 240Lys Ala Ser Pro Glu Gln Leu Arg Ile Asp Ile Val Glu Asp Ile Arg 245 250 255Lys Ile Asn Thr Ile Ala Val Arg Ala Lys Arg Ala Gly Met Ile Ile 260 265 270Leu Gly Gly Gly Ile Val Lys His His Ile Ala Asn Ala Cys Leu Met 275 280 285Arg Asn Gly Ala Glu Ser Ala Val Tyr Ile Asn Thr Ala Gln Glu Phe 290 295 300Asp Gly Ser Asp Ala Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly305 310 315 320Lys Ile Lys Val Gly Ala Asp Ala Val Lys Val Tyr Met Glu Ala Thr 325 330 335Ala Ala Phe Pro Phe Ile Val Ala Asn Thr Phe Ala Lys Glu Asp Gly 340 345 350Leu45387PRTUnknownDescription of Unknown Yeast sequence 45Met Ser Asp Ile Asn Glu Lys Leu Pro Glu Leu Leu Gln Asp Ala Val1 5 10 15Leu Lys Ala Ser Val Pro Ile Pro Asp Asp Phe Val Lys Val Gln Gly 20 25 30Ile Asp Tyr Ser Lys Pro Glu Ala Thr Asn Met Arg Ala Thr Asp Leu 35 40 45Ile Glu Ala Met Lys Thr Met Gly Phe Gln Ala Ser Ser Val Gly Thr 50 55 60Ala Cys Glu Ile Ile Asp Ser Met Arg Ser Trp Arg Gly Lys His Ile65 70 75 80Asp Glu Leu Asp Asp His Glu Lys Lys Gly Cys Phe Asp Glu Glu Gly 85 90 95Tyr Gln Lys Thr Thr Ile Phe Met Gly Tyr Thr Ser Asn Leu Ile Ser 100 105 110Ser Gly Val Arg Glu Thr Leu Arg Tyr Leu Val Gln His Lys Met Val 115 120 125Asp Ala Val Val Thr Ser Ala Gly Gly Val Glu Glu Asp Leu Ile Lys 130 135 140Cys Leu Ala Pro Thr Tyr Leu Gly Glu Phe Ala Leu Lys Gly Lys Ser145 150 155 160Leu Arg Asp Gln Gly Met Asn Arg Ile Gly Asn Leu Leu Val Pro Asn 165 170 175Asp Asn Tyr Cys Lys Phe Glu Glu Trp Ile Val Pro Ile Leu Asp Lys 180 185 190Met Leu Glu Glu Gln Asp Glu Tyr Val Lys Lys His Gly Ala Asp Cys 195 200 205Leu Glu Ala Asn Gln Asp Val Asp Ser Pro Ile Trp Thr Pro Ser Lys 210 215 220Met Ile Asp Arg Phe Gly Lys Glu Ile Asn Asp Glu Ser Ser Val Leu225 230 235 240Tyr Trp Ala His Lys Asn Lys Ile Pro Ile Phe Cys Pro Ser Leu Thr 245 250 255Asp Gly Ser Ile Gly Asp Met Leu Phe Phe His Thr Phe Lys Ala Ser 260 265 270Pro Lys Gln Leu Arg Val Asp Ile Val Gly Asp Ile Arg Lys Ile Asn 275 280 285Ser Met Ser Met Ala Ala Tyr Arg Ala Gly Met Ile Ile Leu Gly Gly 290 295 300Gly Leu Ile Lys His His Ile Ala Asn Ala Cys Leu Met Arg Asn Gly305 310 315 320Ala Asp Tyr Ala Val Tyr Ile Asn Thr Gly Gln Glu Tyr Asp Gly Ser 325

330 335Asp Ala Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Lys 340 345 350Ala Glu Ala Lys Ser Val Lys Leu Phe Ala Asp Val Thr Thr Val Leu 355 360 365Pro Leu Ile Val Ala Ala Thr Phe Ala Ser Gly Lys Pro Ile Lys Lys 370 375 380Val Lys Asn38546370PRTUnknownDescription of Unknown Archaebacterial sequence 46Met Arg Asp Ile Lys Asp Asn Pro Ile Arg Arg Gly Ile Ala Glu Gln1 5 10 15Ser Glu Ala Met His Pro Gly Tyr Thr Asn Arg Ala Lys Pro Tyr Gly 20 25 30Cys Lys Arg Asp Pro Lys Asp Ile Val Leu Lys Glu Ser Glu Asp Ile 35 40 45Glu Gly Ile Ala Ile Glu Gly Pro Trp Leu Glu Asp Asp Ile Ser Leu 50 55 60Glu Glu Ile Ile Lys Lys Tyr Tyr Leu Lys Ile Gly Phe Gln Ala Ser65 70 75 80His Ile Gly Lys Ala Ile Lys Ile Trp Lys His Ile Glu Glu Lys Arg 85 90 95Lys Lys Gly Asp Glu Ile Thr Val Phe Phe Gly Tyr Thr Ser Asn Ile 100 105 110Val Ser Ser Gly Leu Arg Glu Ile Ile Ala Tyr Leu Val Lys His Lys 115 120 125Lys Ile Asp Ile Ile Val Thr Thr Ala Gly Gly Val Glu Glu Asp Phe 130 135 140Ile Lys Cys Leu Lys Pro Phe Ile Leu Gly Asp Trp Glu Val Asp Gly145 150 155 160Lys Met Leu Arg Glu Lys Gly Ile Asn Arg Ile Gly Asn Ile Phe Val 165 170 175Pro Asn Asp Arg Tyr Ile Ala Phe Glu Glu Tyr Met Met Glu Phe Phe 180 185 190Glu Glu Ile Leu Asn Leu Gln Arg Glu Thr Gly Lys Ile Ile Thr Ala 195 200 205Ser Glu Phe Cys Tyr Lys Leu Gly Glu Phe Met Asp Lys Lys Leu Lys 210 215 220Ser Lys Glu Lys Glu Lys Ser Ile Leu Tyr Trp Ala Tyr Lys Asn Asn225 230 235 240Ile Pro Ile Phe Cys Pro Ala Ile Thr Asp Gly Ser Ile Gly Asp Met 245 250 255Leu Tyr Phe Phe Lys Lys Tyr Asn Lys Asp Glu Glu Leu Lys Ile Asp 260 265 270Val Ala Asn Asp Ile Val Lys Leu Asn Asp Ile Ala Ile Asn Ser Lys 275 280 285Glu Thr Ala Cys Ile Val Leu Gly Gly Ser Leu Pro Lys His Ser Ile 290 295 300Ile Asn Ala Asn Leu Phe Arg Glu Gly Thr Asp Tyr Ala Ile Tyr Val305 310 315 320Thr Thr Ala Leu Pro Trp Asp Gly Ser Leu Ser Gly Ala Pro Pro Glu 325 330 335Glu Gly Val Ser Trp Gly Lys Ile Gly Ala Lys Ala Asp Tyr Val Glu 340 345 350Ile Trp Gly Asp Ala Thr Ile Ile Phe Pro Leu Leu Val Tyr Cys Val 355 360 365Met Lys 3704723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47gcngarttyg ayggntccga yca 234830DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48ccgagctcct gttaccaaaa aatctgtacc 304936DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49acctcgagcg gccgcagaag aagtataaaa accatc 365030DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 50cgtcgacgat atctcttttt atattcaaac 305132DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51cgtctagaca ttgttttagg aaagttaaat ga 32521235DNAArabidopsis thaliana 52accctagatc gctttcttca gtgttctata aaaactaaac tccattcgct gacttcgcaa 60agaagaacac tttctctctg aaatctcaaa ttcatctctt ctcttccgat ttcgctgaat 120catgtcagac gacgagcatc acttcgaatc cagcgacgcc ggagcttcta agacttatcc 180tcaacaagcc ggtaacattc gtaaaggtgg tcacatcgtc atcaagggac gtccctgcaa 240ggttttgtct ctgatttgat tattattgat tttagaggaa tcatcttcat ggattgtatt 300aaagcagtgt tccgttacct gatcgttgtg aatttttgag gtttagtgat tctggattgt 360gatctggtgt ttagtgttga gaaaaacctc tgtttttgaa gtttatggat ttatagggtt 420tttaaatcta taatagggtt taattcaatt ggtgatatgt ggggtttatg atataggtgg 480ttgaggtatc gacttcgaag actgggaagc atggtcacgc caagtgtcac tttgttgcca 540ttgatatctt tacttctaag aagcttgaag atatcgttcc ttcttcccac aattgtgatg 600tgagtcttgt gaatggatta gaaacgttat acaaagtcta taatttttga ctcacaacac 660aaaactgttt cctttttatt ggcacaggtt ccacatgtga atcgtgttga ttatcagttg 720attgatatct ctgaagatgg ctttgtatgt catcttcttt ttcactagtt cagctttgtg 780ttttgtcttt gcccatatgg ttgaattaga gggttttgtt ctttgattac atttacaggt 840tagtcttctt actgataatg gtagcactaa ggatgatctg aagctgccaa cagatgaagc 900tttactcaca cagctcaaga atggatttga ggagggtaag gatattgttg tgtctgtcat 960gtctgcaatg ggagaggagc agatgtgtgc tctcaaggaa gttggtccca agtaataata 1020ataagtaagc attctctctt ttacagaggc tatgtattat caagtttgac agagtcaaat 1080gttataagaa caaagtttgg tccttttttt tggtcttctt agtataattt aagcccacat 1140gtgtttccca tgcaagacac tcttatattt actagtatat cttactattg gttttggttg 1200tggagaagtt actgttgaca gttccaaacc tctac 123553161PRTMycosphaerella fijiensis 53Gly Leu Asn Arg Ile Gly Asn Phe Leu Val Pro Asn Asp Asn Tyr Cys1 5 10 15Arg Phe Glu Asp Trp Val Met Pro Ile Leu Asp Thr Met Leu Glu Glu 20 25 30Gln Glu Ala Cys Lys Gly Ser Gly Glu Ala Ile His Trp Thr Pro Ser 35 40 45Lys Ile Ile Asn Arg Leu Gly Lys Glu Val Asn Asp Glu Ser Ser Val 50 55 60Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro Val Phe Cys Pro Ala Leu65 70 75 80Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Thr Phe Lys Ser 85 90 95Ser Pro Gln Gln Leu Arg Val Asp Ile Val Glu Asp Ile Arg Lys Ile 100 105 110Asn Thr Leu Ala Val Arg Ala Lys Arg Thr Gly Met Ile Ile Leu Gly 115 120 125Gly Gly Ile Val Lys His His Ile Ala Asn Ala Asn Leu Met Arg Asn 130 135 140Gly Ala Glu Ser Ala Val Tyr Ile Asn Thr Ala Glu Phe Asp Gly Ser145 150 155 160Asp54861DNAArabidopsis sp. 54tttttccctt ctcccaatct catcttctcc gaaaaccttt cttctctcaa atttctgtga 60aaacatgtct gacgacgagc accactttga ggccagcgaa tccggagctt ccaagaccta 120tcctcaatca gccggtaaca tccgtaaagg tggtcacatc gtcatcaaaa accgtccctg 180caaggttgtt gaggtttcga cttccaaaac tggcaagcac ggtcacgcca aatgtcactt 240tgttgctatt gatatcttca ctgctaagaa gcttgaagat attgttccat cttcccacaa 300ttgtgatgtt ccacatgtga accgtgttga ttaccagttg attgatatca ctgaggatgg 360cttcgtgagc cttctcactg acagtggtgg caccaaggat gatctcaagc ttcccaccga 420tgatggtctc accgcccaga tgaggcttgg attcgatgag ggaaaggata ttgtggtgtc 480tgtcatgtct tccatgggag aggagcagat ctgtgccgtc aaggaagttg gtggtggcaa 540gtaaacaagt atcattcgat atattattac cagtttgaca acggacgtca atgttataag 600aaccaaaaga tgtttttctt tttcctaatt tagacccttt gtgtgtgttt cttgttgcaa 660gacaaccata tctattggtt ttggattgtt ggaaaagttt gtgttgaaac attcaaagtt 720tcttatgaga tgttattctt aaaaccactt tttgtttgtt cactggatat gtttgttcat 780gaagcttgtt ttaagcaact ctttacatga tattcattgc tatttgcacg attcaagagt 840gaaatataca ttttatttaa c 86155159PRTArabidopsis thaliana 55Met Ser Asp Asp Glu His His Phe Glu Ala Ser Glu Ser Gly Ala Ser1 5 10 15Lys Thr Tyr Pro Gln Ser Ala Gly Asn Ile Arg Lys Gly Gly His Ile 20 25 30Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60Phe Thr Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Ile Asp Ile Thr 85 90 95Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Gly Thr Lys Asp 100 105 110Asp Leu Lys Leu Pro Thr Asp Asp Gly Leu Thr Ala Gln Met Arg Leu 115 120 125Gly Phe Asp Glu Gly Lys Asp Ile Val Val Ser Val Met Ser Ser Met 130 135 140Gly Glu Glu Gln Ile Cys Ala Val Lys Glu Val Gly Gly Gly Lys145 150 15556698DNALycopersicon esculentumCDS(52)..(528) 56cttcctgaat ttttctcctt ctccttctcc gttcaatcga atttttcagc c atg tct 57 Met Ser 1gac gag gag cat caa ttt gag tct aag gct gat gcc gga gca tca aaa 105Asp Glu Glu His Gln Phe Glu Ser Lys Ala Asp Ala Gly Ala Ser Lys 5 10 15act tac cct caa caa gct ggt act att cgt aag aac ggt tat atc gtc 153Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile Val 20 25 30atc aaa ggc cgt cca tgc aag gtt gtg gaa gtc tct aca tcc aaa act 201Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr35 40 45 50ggc aag cac ggt cac gcc aaa tgt cat ttc gtt gct att gac atc ttc 249Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile Phe 55 60 65act ggg aag aag ctt gag gat att gtc ccc tct tca cac aat tgt gat 297Thr Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys Asp 70 75 80gtg ccc cat gtt aat cgt aca gat tat cag ctt att gac atc tct gaa 345Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser Glu 85 90 95gat gga ttt gtg agt ctg ctt act gac aat ggt aac acc aag gat gac 393Asp Gly Phe Val Ser Leu Leu Thr Asp Asn Gly Asn Thr Lys Asp Asp 100 105 110ctc agg ctt cct act gat gaa aat ctg ctt tca ctg atc aag gac ggg 441Leu Arg Leu Pro Thr Asp Glu Asn Leu Leu Ser Leu Ile Lys Asp Gly115 120 125 130ttt gcc gag ggt aag gac ctc gtt gtg tct gtt atg tca gct atg ggt 489Phe Ala Glu Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met Gly 135 140 145gag gaa cag att aat gct ttg aag gat att ggc ccc aag tgatctcttg 538Glu Glu Gln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys 150 155attggatgga ttgcttgacg cgatggttct ttacgacctt gagtgagata gatatttata 598gtcatggaaa aaaattgtga tcttatggaa tattcgtatc atgatttatg gaccattgtg 658agttagattt ttatttatgt tgttttaaat tgtggtattc 69857159PRTLycopersicon esculentum 57Met Ser Asp Glu Glu His Gln Phe Glu Ser Lys Ala Asp Ala Gly Ala1 5 10 15Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr 20 25 30Ile Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser 35 40 45Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50 55 60Ile Phe Thr Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn65 70 75 80Cys Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile 85 90 95Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Asn Gly Asn Thr Lys 100 105 110Asp Asp Leu Arg Leu Pro Thr Asp Glu Asn Leu Leu Ser Leu Ile Lys 115 120 125Asp Gly Phe Ala Glu Gly Lys Asp Leu Val Val Ser Val Met Ser Ala 130 135 140Met Gly Glu Glu Gln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys145 150 15558158PRTArabidopsis thaliana 58Met Ser Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser1 5 10 15Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile 20 25 30Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60Phe Thr Ser Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95Glu Asp Gly Tyr Val Ser Leu Leu Thr Asp Asn Gly Ser Thr Lys Asp 100 105 110Asp Leu Lys Leu Pro Asn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser 115 120 125Gly Phe Asp Asp Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met 130 135 140Gly Glu Glu Gln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys145 150 15559159PRTArabidopsis thaliana 59Met Ser Asp Asp Glu His His Phe Glu Ala Ser Glu Ser Gly Ala Ser1 5 10 15Lys Thr Tyr Pro Gln Ser Ala Gly Asn Ile Arg Lys Gly Gly His Ile 20 25 30Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60Phe Thr Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Ile Asp Ile Thr 85 90 95Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Gly Thr Lys Asp 100 105 110Asp Leu Lys Leu Pro Thr Asp Asp Gly Leu Thr Ala Gln Met Arg Leu 115 120 125Gly Phe Asp Glu Gly Lys Asp Ile Val Val Ser Val Met Ser Ser Met 130 135 140Gly Glu Glu Gln Ile Cys Ala Val Lys Glu Val Gly Gly Gly Lys145 150 15560158PRTArabidopsis thaliana 60Met Ser Asp Asp Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser1 5 10 15Lys Thr Tyr Pro Gln Gln Ala Gly Asn Ile Arg Lys Gly Gly His Ile 20 25 30Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60Phe Thr Ser Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Asn Gly Ser Thr Lys Asp 100 105 110Asp Leu Lys Leu Pro Thr Asp Glu Ala Leu Leu Thr Gln Leu Lys Asn 115 120 125Gly Phe Glu Glu Gly Lys Asp Ile Val Val Ser Val Met Ser Ala Met 130 135 140Gly Glu Glu Gln Met Cys Ala Leu Lys Glu Val Gly Pro Lys145 150 15561477DNAArabidopsis thaliana 61atgtccgacg aggagcatca ctttgagtcc agtgacgccg gagcgtccaa aacctaccct 60caacaagctg gaaccatccg taagaatggt tacatcgtca tcaaaaatcg tccctgcaag 120gttgttgagg tttcaacctc gaagactggc aagcatggtc atgctaaatg tcattttgta 180gctattgata tcttcaccag caagaaactc gaagatattg ttccttcttc ccacaattgt 240gatgttcctc atgtcaaccg tactgattat cagctgattg acatttctga agatggatat 300gtcagtttgt tgactgataa cggtagtacc aaggatgacc ttaagctccc taatgatgac 360actctgctcc aacagatcaa gagtgggttt gatgatggaa aagatctagt ggtgagtgtg 420atgtcagcta tgggagagga acagatcaat gctcttaagg acatcggtcc caagtga 47762480DNAArabidopsis thaliana 62atgtctgacg acgagcacca ctttgaggcc agcgaatccg gagcttccaa gacctatcct 60caatcagccg gtaacatccg taaaggtggt cacatcgtca tcaaaaaccg tccctgcaag 120gttgttgagg tttcgacttc caaaactggc aagcacggtc acgccaaatg tcactttgtt 180gctattgata tcttcactgc taagaagctt gaagatattg ttccatcttc ccacaattgt 240gatgttccac atgtgaaccg tgttgattac cagttgattg atatcactga ggatggcttc 300gtgagccttc tcactgacag tggtggcacc aaggatgatc tcaagcttcc caccgatgat 360ggtctcaccg cccagatgag gcttggattc gatgagggaa aggatattgt ggtgtctgtc 420atgtcttcca tgggagagga gcagatctgt gccgtcaagg aagttggtgg tggcaagtaa 48063477DNAArabidopsis thaliana 63atgtcagacg acgagcatca cttcgaatcc agcgacgccg gagcttctaa gacttatcct 60caacaagccg gtaacattcg taaaggtggt cacatcgtca tcaagggacg tccctgcaag 120gtggttgagg tatcgacttc gaagactggg aagcatggtc acgccaagtg tcactttgtt 180gccattgata tctttacttc taagaagctt gaagatatcg ttccttcttc ccacaattgt 240gatgttccac atgtgaatcg tgttgattat cagttgattg atatctctga agatggcttt 300gttagtcttc ttactgataa tggtagcact aaggatgatc tgaagctgcc aacagatgaa 360gctttactca cacagctcaa gaatggattt gaggagggta aggatattgt tgtgtctgtc 420atgtctgcaa tgggagagga gcagatgtgt gctctcaagg aagttggtcc caagtaa 47764700DNALycopersicon esculentumCDS(56)..(532) 64aaatttctcc ttctccttaa tcctctccac cggcgaaccg gcgaagatca aaacg atg 58 Met

1tcg gac gaa gag cac cac ttc gaa tcc aag gcc gat gcc gga gct tca 106Ser Asp Glu Glu His His Phe Glu Ser Lys Ala Asp Ala Gly Ala Ser 5 10 15aag acg tat cct caa caa gct ggt act att cgt aaa ggt ggt cac atc 154Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Gly Gly His Ile 20 25 30gtc ata aaa aat cgt cct tgc aag gtg gtt gaa gtt tca act tcc aag 202Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45aca ggc aag cac ggt cat gct aaa tgt cac ttc gtg gca att gac att 250Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile50 55 60 65ttc act gga aag aaa ctt gag gat att gtt ccc tct tct cac aat tgt 298Phe Thr Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys 70 75 80gat gtt cct cat gtg aat agg act gat tat caa ctt att gat atc tct 346Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95gag gat ggc ttt gtg agt ctg ttg act gaa aat ggt aac acc aag gat 394Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Asn Gly Asn Thr Lys Asp 100 105 110gac ttg aga ctc cca act gat gat act ctt ctg gct cag gtc aaa gat 442Asp Leu Arg Leu Pro Thr Asp Asp Thr Leu Leu Ala Gln Val Lys Asp 115 120 125ggt ttt gct gag ggg aaa gac ctg gtt cta tca gtg atg tct gcc atg 490Gly Phe Ala Glu Gly Lys Asp Leu Val Leu Ser Val Met Ser Ala Met130 135 140 145gga gag gag cag att tgt ggt atc aag gac att ggc cct aag 532Gly Glu Glu Gln Ile Cys Gly Ile Lys Asp Ile Gly Pro Lys 150 155tagctgcaga tggtattggt gtatgtttac agagtttcta taaaagatgt attaagaacc 592aaaacttctt tactttctct tgcagttgct ctatataact gccatttaac tattattata 652tgtgttgtga ttagattctt gtctcactac agtatttcct ttactctg 70065159PRTLycopersicon esculentum 65Met Ser Asp Glu Glu His His Phe Glu Ser Lys Ala Asp Ala Gly Ala1 5 10 15Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Gly Gly His 20 25 30Ile Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser 35 40 45Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50 55 60Ile Phe Thr Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn65 70 75 80Cys Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile 85 90 95Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Asn Gly Asn Thr Lys 100 105 110Asp Asp Leu Arg Leu Pro Thr Asp Asp Thr Leu Leu Ala Gln Val Lys 115 120 125Asp Gly Phe Ala Glu Gly Lys Asp Leu Val Leu Ser Val Met Ser Ala 130 135 140Met Gly Glu Glu Gln Ile Cys Gly Ile Lys Asp Ile Gly Pro Lys145 150 15566658DNABrassica napusCDS(1)..(477) 66atg tct gac gag gag cac cac ttc gag tcc agc gac gcc gga gct tcc 48Met Ser Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser1 5 10 15aaa acc tac cct cag cag gct ggt aac atc cgc aag ggt ggt cac atc 96Lys Thr Tyr Pro Gln Gln Ala Gly Asn Ile Arg Lys Gly Gly His Ile 20 25 30gtc atc aag ggc cgt ccc tgc aag gtt gtt gag gtt tcg act tcg aag 144Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45act ggg aag cac ggt cac gca aag tgt cac ttt gtt gct atc gac atc 192Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60ttc act gct aag aag ctc gag gat att gtt ccc tct tcc cac aat tgt 240Phe Thr Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80gat gtt ccc cat gtg aac cgt att gac tac cag ttg att gat atc tct 288Asp Val Pro His Val Asn Arg Ile Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95gag aat ggc ttt gtt agc ctt ttg acc gac agt ggt ggc acc aag gac 336Glu Asn Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Gly Thr Lys Asp 100 105 110gac ctc aag ctt ccc acc gat gat aat ctc agc gct ctg atg aag agt 384Asp Leu Lys Leu Pro Thr Asp Asp Asn Leu Ser Ala Leu Met Lys Ser 115 120 125gga ttc gag gag gga aag gat gtg gtg gtg tct gtc atg tct tcc atg 432Gly Phe Glu Glu Gly Lys Asp Val Val Val Ser Val Met Ser Ser Met 130 135 140gga gag gag cag atc tgt gcc gtc aag gaa gtt ggt ggt ggc aag 477Gly Glu Glu Gln Ile Cys Ala Val Lys Glu Val Gly Gly Gly Lys145 150 155taaaacccat tctctgagag aggataatct tattaccagt ggtcaatgtt ataagaacaa 537gaacttgttt tttttccttt ttctaattta gatcatttgt gttgtgtttc tttgttgcaa 597gacaaccatt atctattatt ggttttggat tgtttaaaaa aaaaaaaaaa aaaaaaaaaa 657a 65867159PRTBrassica napus 67Met Ser Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser1 5 10 15Lys Thr Tyr Pro Gln Gln Ala Gly Asn Ile Arg Lys Gly Gly His Ile 20 25 30Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55 60Phe Thr Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys65 70 75 80Asp Val Pro His Val Asn Arg Ile Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95Glu Asn Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Gly Thr Lys Asp 100 105 110Asp Leu Lys Leu Pro Thr Asp Asp Asn Leu Ser Ala Leu Met Lys Ser 115 120 125Gly Phe Glu Glu Gly Lys Asp Val Val Val Ser Val Met Ser Ser Met 130 135 140Gly Glu Glu Gln Ile Cys Ala Val Lys Glu Val Gly Gly Gly Lys145 150 155688PRTMycosphaerella fijiensis 68Gly Leu Asn Arg Ile Gly Asn Leu1 5698PRTMycosphaerella fijiensis 69Ala Glu Phe Asp Gly Ser Asp Gln1 5701335DNABrassica napusCDS(57)..(1160) 70cttgctagaa ccctaaaact ccctcccaaa actctccaca tcttccgaga aagaag atg 59 Met 1gag gag gat cgt gtt ctc tcg tct gtc cac tca acg gtc ttc aag gaa 107Glu Glu Asp Arg Val Leu Ser Ser Val His Ser Thr Val Phe Lys Glu 5 10 15tcc gaa tcg ttg gaa gga aag tgc gac aaa atc gaa gga tac gat ttc 155Ser Glu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly Tyr Asp Phe 20 25 30aac caa gga gta aac tac ccg aag ctc ctc cga tcc atg ctc aca acc 203Asn Gln Gly Val Asn Tyr Pro Lys Leu Leu Arg Ser Met Leu Thr Thr 35 40 45ggc ttc caa gcc tca aac ctc ggc gac gta att gat gtc gtt aat caa 251Gly Phe Gln Ala Ser Asn Leu Gly Asp Val Ile Asp Val Val Asn Gln50 55 60 65atg cta gag tgg aga ctc tct gat gaa act ata gca cct gaa gac tgt 299Met Leu Glu Trp Arg Leu Ser Asp Glu Thr Ile Ala Pro Glu Asp Cys 70 75 80agt gaa gag gag aag gat cca gcg tat aga gag tcc gtg aag tgt aaa 347Ser Glu Glu Glu Lys Asp Pro Ala Tyr Arg Glu Ser Val Lys Cys Lys 85 90 95atc ttt cta ggc ttc act tcg aat ctt gtt tcc tct ggt gtt aga gag 395Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Val Arg Glu 100 105 110act att cga tac ctt gtt cag cat cat atg gtt gat gtt ata gtt act 443Thr Ile Arg Tyr Leu Val Gln His His Met Val Asp Val Ile Val Thr 115 120 125aca act ggt ggc gta gag gaa gat ctc atc aaa tgc ctt gct cct act 491Thr Thr Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala Pro Thr130 135 140 145ttc aaa ggt gat ttc tct cta ccg ggt gcg tat ctt cgg tca aag gga 539Phe Lys Gly Asp Phe Ser Leu Pro Gly Ala Tyr Leu Arg Ser Lys Gly 150 155 160ttg aac cgg atc ggg aac ttg ctt gtt cct aat gat aac tac tgc aag 587Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys 165 170 175ttt gag gat tgg atc att ccc atc ttt gac cag atg ttg aag gaa cag 635Phe Glu Asp Trp Ile Ile Pro Ile Phe Asp Gln Met Leu Lys Glu Gln 180 185 190aaa gaa gag agt gtg ttg tgg acg cct tct aaa ttg tta gcg cgg ctg 683Lys Glu Glu Ser Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg Leu 195 200 205ggg aaa gaa ata aat aat gag agt tca tat ctt tat tgg gca tac aag 731Gly Lys Glu Ile Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys210 215 220 225atg aat att cca gtg ttc tgc cgg ggg tta acc gat ggc tct ctc ggt 779Met Asn Ile Pro Val Phe Cys Arg Gly Leu Thr Asp Gly Ser Leu Gly 230 235 240gat atg ttg tat ttt cac tca ttt cgt acc tct ggc ctt gtc atc gat 827Asp Met Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu Val Ile Asp 245 250 255gtt gtg caa gat att aga gct atg aac ggt gaa gca gtc cat gcg act 875Val Val Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Thr 260 265 270cca aga aag aca ggg atg ata atc ctt gga ggg ggc ttg ccg aag cac 923Pro Arg Lys Thr Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His 275 280 285cac ata tgt aat gcc aac atg atg cgt aac ggt gcg gat tac gct gtg 971His Ile Cys Asn Ala Asn Met Met Arg Asn Gly Ala Asp Tyr Ala Val290 295 300 305ttt atc aac acc ggg caa gag ttt gat gga agt gac tcg ggt gca cgc 1019Phe Ile Asn Thr Gly Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg 310 315 320cct gat gaa gca gtg tct tgg ggt aaa ata agg gga tct gct aaa act 1067Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Ser Ala Lys Thr 325 330 335gtc aag gtg tac tgt gat gct acc ata gcc ttc cct ttg ttg gtt gct 1115Val Lys Val Tyr Cys Asp Ala Thr Ile Ala Phe Pro Leu Leu Val Ala 340 345 350gaa aca ttt gcc tcc aag aga gaa caa agc tgt gag cac aag acc 1160Glu Thr Phe Ala Ser Lys Arg Glu Gln Ser Cys Glu His Lys Thr 355 360 365taagcccaag aaagcttacg tctcttttat cggtttgttc ttccatcttg ttgttgtacc 1220ctttgtcctg ctttacataa cattcatctc taaaacaata ctacctcctt ttgacaaaaa 1280ataaaaaaaa ttggaaaaat ggtttcacaa gaataaaaaa aaaaaaaaaa aaaaa 133571368PRTBrassica napus 71Met Glu Glu Asp Arg Val Leu Ser Ser Val His Ser Thr Val Phe Lys1 5 10 15Glu Ser Glu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly Tyr Asp 20 25 30Phe Asn Gln Gly Val Asn Tyr Pro Lys Leu Leu Arg Ser Met Leu Thr 35 40 45Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp Val Ile Asp Val Val Asn 50 55 60Gln Met Leu Glu Trp Arg Leu Ser Asp Glu Thr Ile Ala Pro Glu Asp65 70 75 80Cys Ser Glu Glu Glu Lys Asp Pro Ala Tyr Arg Glu Ser Val Lys Cys 85 90 95Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Val Arg 100 105 110Glu Thr Ile Arg Tyr Leu Val Gln His His Met Val Asp Val Ile Val 115 120 125Thr Thr Thr Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala Pro 130 135 140Thr Phe Lys Gly Asp Phe Ser Leu Pro Gly Ala Tyr Leu Arg Ser Lys145 150 155 160Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr Cys 165 170 175Lys Phe Glu Asp Trp Ile Ile Pro Ile Phe Asp Gln Met Leu Lys Glu 180 185 190Gln Lys Glu Glu Ser Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg 195 200 205Leu Gly Lys Glu Ile Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr 210 215 220Lys Met Asn Ile Pro Val Phe Cys Arg Gly Leu Thr Asp Gly Ser Leu225 230 235 240Gly Asp Met Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu Val Ile 245 250 255Asp Val Val Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His Ala 260 265 270Thr Pro Arg Lys Thr Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys 275 280 285His His Ile Cys Asn Ala Asn Met Met Arg Asn Gly Ala Asp Tyr Ala 290 295 300Val Phe Ile Asn Thr Gly Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala305 310 315 320Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Ser Ala Lys 325 330 335Thr Val Lys Val Tyr Cys Asp Ala Thr Ile Ala Phe Pro Leu Leu Val 340 345 350Ala Glu Thr Phe Ala Ser Lys Arg Glu Gln Ser Cys Glu His Lys Thr 355 360 365721862DNAMedicago sativaCDS(293)..(1396) 72gaaaccttct tcttctggag caaagtcgcc attccctacc tccttcttca ttcttattct 60ctataacaaa cggtccgacc ggatccaagt tgcaccggtt cgaaccgctt tagttactac 120taacggttcg aaccgttatt tttcaacccg tgacaaacgt ggaaggcttc gttgtttctt 180cttcttcttc ttaattacca tgcgtttttg tttttctttt gagtcattga agtcttgttt 240tttgtcgtgt ttctgtcttg agaccgtgaa agagaaaaca aagagtacga ga atg agt 298 Met Ser 1gaa aca aag caa gaa gat gat aca att atg tcc tca gtt cac tcc act 346Glu Thr Lys Gln Glu Asp Asp Thr Ile Met Ser Ser Val His Ser Thr 5 10 15gtc ttc aaa gaa tcc gaa aat ctc gca gga aag tgt gtc caa atc gag 394Val Phe Lys Glu Ser Glu Asn Leu Ala Gly Lys Cys Val Gln Ile Glu 20 25 30ggt tat gat ttc aac cgc ggc gtc gat tat caa cag ctt ctc aaa tca 442Gly Tyr Asp Phe Asn Arg Gly Val Asp Tyr Gln Gln Leu Leu Lys Ser35 40 45 50atg ctc aca act ggt ttt caa gct tcc aac ttt ggt gat gcc gtt aaa 490Met Leu Thr Thr Gly Phe Gln Ala Ser Asn Phe Gly Asp Ala Val Lys 55 60 65gtc gtt aat caa atg cta gat tgg agg ttg gtt gat gaa cca att gat 538Val Val Asn Gln Met Leu Asp Trp Arg Leu Val Asp Glu Pro Ile Asp 70 75 80gag gat tgt gat gaa gat aag aag gat ttg gag tat agg aaa tct gtt 586Glu Asp Cys Asp Glu Asp Lys Lys Asp Leu Glu Tyr Arg Lys Ser Val 85 90 95aca tgc aaa gtg ttt ttg ggt ttc act tct aat ctt atc tct tct ggt 634Thr Cys Lys Val Phe Leu Gly Phe Thr Ser Asn Leu Ile Ser Ser Gly 100 105 110gtt aga gat gtt gtt cgt tac ctt tgt cag cat cac atg gtt cat gta 682Val Arg Asp Val Val Arg Tyr Leu Cys Gln His His Met Val His Val115 120 125 130gtt gtt aca act aca ggt ggt att gaa gaa gat ctt ata aag tgc ctt 730Val Val Thr Thr Thr Gly Gly Ile Glu Glu Asp Leu Ile Lys Cys Leu 135 140 145gca cca aca tat aaa gga gag ttc tct ttg ccc gga gct tat ctt cgc 778Ala Pro Thr Tyr Lys Gly Glu Phe Ser Leu Pro Gly Ala Tyr Leu Arg 150 155 160tca aaa gga ttg aat cga atc ggt aat tta ttg gtc cct aat gaa aat 826Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Glu Asn 165 170 175tat tgc aaa ttt gag gac tgg att att cct att ttt gat caa atg ttg 874Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Phe Asp Gln Met Leu 180 185 190aag gag caa aag gaa gag aaa gtg ctg tgg aca ccg tct aag tta ata 922Lys Glu Gln Lys Glu Glu Lys Val Leu Trp Thr Pro Ser Lys Leu Ile195 200 205 210gct cga ttg gga aaa gag atc aac aat gaa aac tcc tac ctt tac tgg 970Ala Arg Leu Gly Lys Glu Ile Asn Asn Glu Asn Ser Tyr Leu Tyr Trp 215 220 225gca tat aag aac aat att cca gtt tat tgt cca gga tta acc gat ggc 1018Ala Tyr Lys Asn Asn Ile Pro Val Tyr Cys Pro Gly Leu Thr Asp Gly 230 235 240tca ctg ggt gac atg ctg tac ttc cat tcc ttc cac aac cct ggt ctg 1066Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe His Asn Pro Gly Leu 245 250 255att gtg gac ata gtg caa gat ata agg gcc atg aat ggt gaa gct gta 1114Ile Val Asp Ile Val Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val 260 265 270cat gca aat cct agc aag acg ggc atg att att tta gga ggc ggc ctt 1162His Ala Asn Pro Ser Lys Thr Gly Met Ile Ile Leu Gly Gly Gly Leu275

280 285 290cca aaa cat cac att tgc aat gcc aat atg atg cgc aat ggt gca gac 1210Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg Asn Gly Ala Asp 295 300 305tat gcg gtt ttt att aat act gca caa gaa ttt gat gga agt gat tct 1258Tyr Ala Val Phe Ile Asn Thr Ala Gln Glu Phe Asp Gly Ser Asp Ser 310 315 320gga gct cgt cca gat gag gct gtt tca tgg ggg aaa ata cga gga tct 1306Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Ser 325 330 335gct aaa act gtt aag gta cat tgt gat gca acg ata gca ttc cct ctg 1354Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe Pro Leu 340 345 350ctg gtt gct gaa aca ttt gcc tca aga acg tca ccc ctt aat 1396Leu Val Ala Glu Thr Phe Ala Ser Arg Thr Ser Pro Leu Asn355 360 365tgataaaggt ccaccgtcaa aagtaaaagg tgtggctggg aagtgtttta ccgcagctcc 1456acttgtgagt gccaaatgtt ttggtatgta acttataaga ccaaggtcgg ctgtatgtca 1516tacttgagtt gaggtcaaag ttcatttgca atgcagtgtg tttgaggatc ttgatggacc 1576agtttgccat tgacttttaa tttgactgtc ttgttattcg caaggtccac ataacaagca 1636tttttaccat ttagaaacaa tttattagtc ctgaaggaat tgagagtcat gaattcagat 1696gtaaattatg caatgctaac tatatttttt tggaactgtg gtttctctta gatttgaggt 1756gttgaaaact gtaatatcta gagcaaataa gactagaaaa gtttatcaac tattactgat 1816cagttatagt atcttcaata ttttccagaa aaaaaaaaaa aaaaaa 186273368PRTMedicago sativa 73Met Ser Glu Thr Lys Gln Glu Asp Asp Thr Ile Met Ser Ser Val His1 5 10 15Ser Thr Val Phe Lys Glu Ser Glu Asn Leu Ala Gly Lys Cys Val Gln 20 25 30Ile Glu Gly Tyr Asp Phe Asn Arg Gly Val Asp Tyr Gln Gln Leu Leu 35 40 45Lys Ser Met Leu Thr Thr Gly Phe Gln Ala Ser Asn Phe Gly Asp Ala 50 55 60Val Lys Val Val Asn Gln Met Leu Asp Trp Arg Leu Val Asp Glu Pro65 70 75 80Ile Asp Glu Asp Cys Asp Glu Asp Lys Lys Asp Leu Glu Tyr Arg Lys 85 90 95Ser Val Thr Cys Lys Val Phe Leu Gly Phe Thr Ser Asn Leu Ile Ser 100 105 110Ser Gly Val Arg Asp Val Val Arg Tyr Leu Cys Gln His His Met Val 115 120 125His Val Val Val Thr Thr Thr Gly Gly Ile Glu Glu Asp Leu Ile Lys 130 135 140Cys Leu Ala Pro Thr Tyr Lys Gly Glu Phe Ser Leu Pro Gly Ala Tyr145 150 155 160Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn 165 170 175Glu Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Phe Asp Gln 180 185 190Met Leu Lys Glu Gln Lys Glu Glu Lys Val Leu Trp Thr Pro Ser Lys 195 200 205Leu Ile Ala Arg Leu Gly Lys Glu Ile Asn Asn Glu Asn Ser Tyr Leu 210 215 220Tyr Trp Ala Tyr Lys Asn Asn Ile Pro Val Tyr Cys Pro Gly Leu Thr225 230 235 240Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe His Asn Pro 245 250 255Gly Leu Ile Val Asp Ile Val Gln Asp Ile Arg Ala Met Asn Gly Glu 260 265 270Ala Val His Ala Asn Pro Ser Lys Thr Gly Met Ile Ile Leu Gly Gly 275 280 285Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg Asn Gly 290 295 300Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala Gln Glu Phe Asp Gly Ser305 310 315 320Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg 325 330 335Gly Ser Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe 340 345 350Pro Leu Leu Val Ala Glu Thr Phe Ala Ser Arg Thr Ser Pro Leu Asn 355 360 365741363DNAMusa acuminataCDS(93)..(1220) 74ggcacgagcg cgcggcgccc gcaacgaata ttgcagagag taagaaggat cctcgccttt 60gtcaccaaac ccttggtttc cagcgaggcg ac atg gaa ggc ggc gcc gcg gga 113 Met Glu Gly Gly Ala Ala Gly 1 5ggg cag cga gac cgg gaa acc ctg gac gcg gtg cgg tcg gtg gtg ttt 161Gly Gln Arg Asp Arg Glu Thr Leu Asp Ala Val Arg Ser Val Val Phe 10 15 20aag cct tcc gta tcc ttg gag gag aag cgg ttc ccg agg gtc cag ggg 209Lys Pro Ser Val Ser Leu Glu Glu Lys Arg Phe Pro Arg Val Gln Gly 25 30 35tac gac ttc aac cgg ggt tgt gac ctc atc ggc ctc ctc gat tcc atc 257Tyr Asp Phe Asn Arg Gly Cys Asp Leu Ile Gly Leu Leu Asp Ser Ile40 45 50 55tcc tct acc ggg ttc caa gct tcc aac ctc ggc gac gcc atc gat gtc 305Ser Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp Ala Ile Asp Val 60 65 70atc aat cag atg att gac tgg agg ctc tcc cat gat gcg ccc acg gaa 353Ile Asn Gln Met Ile Asp Trp Arg Leu Ser His Asp Ala Pro Thr Glu 75 80 85gat tgc agc gag gaa gag cgc aat ctg gct tac agg caa tcg gtc acg 401Asp Cys Ser Glu Glu Glu Arg Asn Leu Ala Tyr Arg Gln Ser Val Thr 90 95 100tgc aag atc ttt ctg ggc ttc act tcg aac ctt gta tct tct ggc atc 449Cys Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Ile 105 110 115agg gag ata att cgg ttt ctt gtg cag cac cga atg gtt gaa gtt tta 497Arg Glu Ile Ile Arg Phe Leu Val Gln His Arg Met Val Glu Val Leu120 125 130 135gtc aca act gct ggc ggc att gaa gaa gat tta atc aaa tgc ctt gct 545Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu Ile Lys Cys Leu Ala 140 145 150cca aca tat aag ggt gac ttt tct ttg cct gga tcg tat ctg cgt tca 593Pro Thr Tyr Lys Gly Asp Phe Ser Leu Pro Gly Ser Tyr Leu Arg Ser 155 160 165aaa gga ttg aat cgg ata gga aac ctt ctt gtc cct aat gac aat tac 641Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr 170 175 180tgc aaa ttc gag gac tgg atc atg cca att ctg gac cag atg tta ctt 689Cys Lys Phe Glu Asp Trp Ile Met Pro Ile Leu Asp Gln Met Leu Leu 185 190 195gaa cag act aca gag aat gta gtt tgg aca cca tct aaa gtg att gcg 737Glu Gln Thr Thr Glu Asn Val Val Trp Thr Pro Ser Lys Val Ile Ala200 205 210 215cgc ctt gga aaa gaa ata aat gat gaa agt tca tac ctg tac tgg gca 785Arg Leu Gly Lys Glu Ile Asn Asp Glu Ser Ser Tyr Leu Tyr Trp Ala 220 225 230tac aag aac aat gtt tct gtc tat tgc ccg gca ttg act gat gga tca 833Tyr Lys Asn Asn Val Ser Val Tyr Cys Pro Ala Leu Thr Asp Gly Ser 235 240 245ttg ggg gat atg ttg tac tgc cat tca gtg cgg aat cct ggt tta ctt 881Leu Gly Asp Met Leu Tyr Cys His Ser Val Arg Asn Pro Gly Leu Leu 250 255 260att gat att gtg caa gac ata cga gca atg aat gga gaa gct gta cat 929Ile Asp Ile Val Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His 265 270 275gtg ggt ctg aga aag act ggg gtc ata att ctt ggt ggg ggc ctc cca 977Val Gly Leu Arg Lys Thr Gly Val Ile Ile Leu Gly Gly Gly Leu Pro280 285 290 295aag cac cat ata tgt aat gcc aac atg ttt cgg aat ggt gca gat tat 1025Lys His His Ile Cys Asn Ala Asn Met Phe Arg Asn Gly Ala Asp Tyr 300 305 310gct gtt tat gtc aac act gca cag gaa ttt gat gga agt gat tct gga 1073Ala Val Tyr Val Asn Thr Ala Gln Glu Phe Asp Gly Ser Asp Ser Gly 315 320 325gca gag cct gat gag gcg att tca tgg gga aag ata aaa ggt tct gcg 1121Ala Glu Pro Asp Glu Ala Ile Ser Trp Gly Lys Ile Lys Gly Ser Ala 330 335 340aag act att aaa gtt cat tgt gat gca act att gct ttt cct cta ttg 1169Lys Thr Ile Lys Val His Cys Asp Ala Thr Ile Ala Phe Pro Leu Leu 345 350 355gta gct gca aca ttt gca aga aag ttt cag gaa aga aac aac aaa tta 1217Val Ala Ala Thr Phe Ala Arg Lys Phe Gln Glu Arg Asn Asn Lys Leu360 365 370 375gcc tgatgggggt gcaaaaggtg atcatcttat ttggattcaa ataccttaat 1270Alagtaatctgct aacatctgca gatgctgtat tcttgcaaac caaaaattta atattagata 1330accgagagcc tacagagggt cctttcaaaa aaa 136375376PRTMusa acuminata 75Met Glu Gly Gly Ala Ala Gly Gly Gln Arg Asp Arg Glu Thr Leu Asp1 5 10 15Ala Val Arg Ser Val Val Phe Lys Pro Ser Val Ser Leu Glu Glu Lys 20 25 30Arg Phe Pro Arg Val Gln Gly Tyr Asp Phe Asn Arg Gly Cys Asp Leu 35 40 45Ile Gly Leu Leu Asp Ser Ile Ser Ser Thr Gly Phe Gln Ala Ser Asn 50 55 60Leu Gly Asp Ala Ile Asp Val Ile Asn Gln Met Ile Asp Trp Arg Leu65 70 75 80Ser His Asp Ala Pro Thr Glu Asp Cys Ser Glu Glu Glu Arg Asn Leu 85 90 95Ala Tyr Arg Gln Ser Val Thr Cys Lys Ile Phe Leu Gly Phe Thr Ser 100 105 110Asn Leu Val Ser Ser Gly Ile Arg Glu Ile Ile Arg Phe Leu Val Gln 115 120 125His Arg Met Val Glu Val Leu Val Thr Thr Ala Gly Gly Ile Glu Glu 130 135 140Asp Leu Ile Lys Cys Leu Ala Pro Thr Tyr Lys Gly Asp Phe Ser Leu145 150 155 160Pro Gly Ser Tyr Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu 165 170 175Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Met Pro 180 185 190Ile Leu Asp Gln Met Leu Leu Glu Gln Thr Thr Glu Asn Val Val Trp 195 200 205Thr Pro Ser Lys Val Ile Ala Arg Leu Gly Lys Glu Ile Asn Asp Glu 210 215 220Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys Asn Asn Val Ser Val Tyr Cys225 230 235 240Pro Ala Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Cys His Ser 245 250 255Val Arg Asn Pro Gly Leu Leu Ile Asp Ile Val Gln Asp Ile Arg Ala 260 265 270Met Asn Gly Glu Ala Val His Val Gly Leu Arg Lys Thr Gly Val Ile 275 280 285Ile Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met 290 295 300Phe Arg Asn Gly Ala Asp Tyr Ala Val Tyr Val Asn Thr Ala Gln Glu305 310 315 320Phe Asp Gly Ser Asp Ser Gly Ala Glu Pro Asp Glu Ala Ile Ser Trp 325 330 335Gly Lys Ile Lys Gly Ser Ala Lys Thr Ile Lys Val His Cys Asp Ala 340 345 350Thr Ile Ala Phe Pro Leu Leu Val Ala Ala Thr Phe Ala Arg Lys Phe 355 360 365Gln Glu Arg Asn Asn Lys Leu Ala 370 375761451DNAPopulus deltoidesCDS(8)..(1126) 76gggattt atg aca ggc aaa aaa caa tgg gag gaa gat ttg cta tca tca 49 Met Thr Gly Lys Lys Gln Trp Glu Glu Asp Leu Leu Ser Ser 1 5 10gta cgg acc aca gtg ttt aaa gaa tca gaa gct ctt gat ggg aaa tgc 97Val Arg Thr Thr Val Phe Lys Glu Ser Glu Ala Leu Asp Gly Lys Cys15 20 25 30att aaa att gaa ggt tat gat ttt aat caa gga gtg aac tac tct aag 145Ile Lys Ile Glu Gly Tyr Asp Phe Asn Gln Gly Val Asn Tyr Ser Lys 35 40 45ctt ctc aaa tcc atg gtc tct acc ggg ttt caa gct tcc aac ctt gga 193Leu Leu Lys Ser Met Val Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly 50 55 60gat gcc att caa gtt gtt aat aac atg cta gac tgg agg ctt gct gat 241Asp Ala Ile Gln Val Val Asn Asn Met Leu Asp Trp Arg Leu Ala Asp 65 70 75gaa gag ata aca gaa gat tgt agt gat gag gag agg gag ttg gcc tat 289Glu Glu Ile Thr Glu Asp Cys Ser Asp Glu Glu Arg Glu Leu Ala Tyr 80 85 90aga gag tct gtg aga tgc aaa ctg ttc ttg ggt ttt aca tca aat ctt 337Arg Glu Ser Val Arg Cys Lys Leu Phe Leu Gly Phe Thr Ser Asn Leu95 100 105 110gtt tct tca ggt gtc aga gat aca att cga tat ctt gtt cag cat cat 385Val Ser Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln His His 115 120 125atg gtt gat gta gtg gtt aca acg gca ggt ggc ata gaa gaa gat ctt 433Met Val Asp Val Val Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu 130 135 140ata aaa tgc ctg gca cca aca tac aaa ggt gac ttt tct cta ccc ggg 481Ile Lys Cys Leu Ala Pro Thr Tyr Lys Gly Asp Phe Ser Leu Pro Gly 145 150 155gct caa tta cga tca aaa ggg ttg aat cga att ggt aac ttg ttg gta 529Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val 160 165 170cct aat gac aac tac tgc aaa ttt gag gat tgg atc att cca atc ttt 577Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Phe175 180 185 190gac caa atg ttg aag gaa caa att gaa gag aat atc acc tgg aca cct 625Asp Gln Met Leu Lys Glu Gln Ile Glu Glu Asn Ile Thr Trp Thr Pro 195 200 205tct aaa tta ata gct cgc atg ggg aaa gaa ata aat aat gag agt tca 673Ser Lys Leu Ile Ala Arg Met Gly Lys Glu Ile Asn Asn Glu Ser Ser 210 215 220tac ctt tat tgg gca tat aag aac gac att cca gta ttc tgt cca ggc 721Tyr Leu Tyr Trp Ala Tyr Lys Asn Asp Ile Pro Val Phe Cys Pro Gly 225 230 235tta aca gat ggt tct cta ggg gac atg cta tac ttt cat tcc ttc cac 769Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe His 240 245 250aac cct ggt cta att gtt gcc ata gtc caa gat att aga gcc atg aat 817Asn Pro Gly Leu Ile Val Ala Ile Val Gln Asp Ile Arg Ala Met Asn255 260 265 270ggt gaa gct gtc cac gca agt cct aga aaa act ggt atc atc att ctt 865Gly Glu Ala Val His Ala Ser Pro Arg Lys Thr Gly Ile Ile Ile Leu 275 280 285gga ggt ggg ctt cct aag cat cat ata tgc aat gcc aat atg atg cgt 913Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg 290 295 300aac ggt gca gat tat gct gta ttc atc aat aca gca caa gaa ttt gat 961Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala Gln Glu Phe Asp 305 310 315ggg agt gat tct gga gct cat cct gat gag gct gta tcg tgg ggg aaa 1009Gly Ser Asp Ser Gly Ala His Pro Asp Glu Ala Val Ser Trp Gly Lys 320 325 330ata cga ggt tct gct aaa act gtt aag gtc cac tgt gat gca act att 1057Ile Arg Gly Ser Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile335 340 345 350gct ttt cct ctc cta gtt gct gaa aca ttt gcc cct agg agg aac aga 1105Ala Phe Pro Leu Leu Val Ala Glu Thr Phe Ala Pro Arg Arg Asn Arg 355 360 365ttc tgc agc agt act caa agc tagggctgtg tgcagttctt ggccagaaaa 1156Phe Cys Ser Ser Thr Gln Ser 370ttgattcatt tttatttgta ttatgactga acgatccgca ggatgggtag tgggctccat 1216tgatgccata aacttctttt tttttcccct cagaattaag ggatccgcca gaacacactg 1276ctctcagccc caaaccattg ttgcctctac tgggagtagc ataaccaatt gaattgcgct 1336cctccaagca gcgcctctta gttgcgttat ttattgtaag tagcgcaacc aactaaatta 1396tgctagttcc cacatttatt gactgctatt ttcaaaagaa aaaaaaaaaa aaaaa 145177373PRTPopulus deltoides 77Met Thr Gly Lys Lys Gln Trp Glu Glu Asp Leu Leu Ser Ser Val Arg1 5 10 15Thr Thr Val Phe Lys Glu Ser Glu Ala Leu Asp Gly Lys Cys Ile Lys 20 25 30Ile Glu Gly Tyr Asp Phe Asn Gln Gly Val Asn Tyr Ser Lys Leu Leu 35 40 45Lys Ser Met Val Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp Ala 50 55 60Ile Gln Val Val Asn Asn Met Leu Asp Trp Arg Leu Ala Asp Glu Glu65 70 75 80Ile Thr Glu Asp Cys Ser Asp Glu Glu Arg Glu Leu Ala Tyr Arg Glu 85 90 95Ser Val Arg Cys Lys Leu Phe Leu Gly Phe Thr Ser Asn Leu Val Ser 100 105 110Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln His His Met Val 115 120 125Asp Val Val Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu Ile Lys 130 135 140Cys Leu Ala Pro Thr Tyr Lys Gly Asp Phe Ser Leu Pro Gly Ala Gln145 150 155 160Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn 165 170 175Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro

Ile Phe Asp Gln 180 185 190Met Leu Lys Glu Gln Ile Glu Glu Asn Ile Thr Trp Thr Pro Ser Lys 195 200 205Leu Ile Ala Arg Met Gly Lys Glu Ile Asn Asn Glu Ser Ser Tyr Leu 210 215 220Tyr Trp Ala Tyr Lys Asn Asp Ile Pro Val Phe Cys Pro Gly Leu Thr225 230 235 240Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe His Asn Pro 245 250 255Gly Leu Ile Val Ala Ile Val Gln Asp Ile Arg Ala Met Asn Gly Glu 260 265 270Ala Val His Ala Ser Pro Arg Lys Thr Gly Ile Ile Ile Leu Gly Gly 275 280 285Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg Asn Gly 290 295 300Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala Gln Glu Phe Asp Gly Ser305 310 315 320Asp Ser Gly Ala His Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg 325 330 335Gly Ser Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe 340 345 350Pro Leu Leu Val Ala Glu Thr Phe Ala Pro Arg Arg Asn Arg Phe Cys 355 360 365Ser Ser Thr Gln Ser 370781488DNAArabidopsis thaliana 78acaataaggc tttaaagccc ataaaaccct taaatatatc aaagcccaaa agaaacgcct 60tttgcgcttt cccgatcgtg gtcaacttcc tctgttacca aaaaatctgt accgcaaaat 120cctcgtcgaa gctcgctgct gcaaccatgt ccgacgagga gcatcacttt gagtccagtg 180acgccggagc gtccaaaacc taccctcaac aagctggaac catccgtaag aatggttaca 240tcgtcatcaa aaatcgtccc tgcaaggttt cgttctcaaa catttctcca ctctcttcct 300ctgatcttat tagatctgtt cattacttag attcctcaga ttcttttttt tgtcacctcc 360acgatgttcg actgatattt gttcttgtca tcattgttaa attcacattt tattgcactt 420ttgttttagc gaaattatta aattggtcat cttcagtttt gttcgattag ataagtttta 480ggattttttc ttacacaagt tactggatca gctgctaaat gtcattttgt gtcgcaggtt 540gttgaggttt caacctcgaa gactggcaag catggtcatg ctaaatgtca ttttgtagct 600attgatatct tcaccagcaa gaaactcgaa gatattgttc cttcttccca caattgtgat 660gtatgtgaaa aaagctcctt tgatcacttt catttcttgt ttgtttcttt caagtcccat 720ttgagatttt gtttttgttg aattgggttt caggttcctc atgtcaaccg tactgattat 780cagctgattg acatttctga agatggatat gtatgtgttc ttaaatagca cttgttcctt 840tatatggttt agttacttgt tctgttttgt aatcattttg caggtcagtt tgttgactga 900taacggtagt accaaggatg accttaagct ccctaatgat gacactctgc tccaacaggt 960taagttttgc atgttcatca cattaaatgt tgctagttaa ttaaaatcaa ctctatgtcg 1020atttctgaaa atggaagaaa aagtgcagag taatgagtga cctgattgtg ttaatgaaac 1080agatcaagag tgggtttgat gatggaaaag atctagtggt gagtgtgatg tcagctatgg 1140gagaggaaca gatcaatgct cttaaggaca tcggtcccaa gtgagactaa caaagcctcc 1200cctttgttat gagattcttc ttcttcttct gtaggcttcc attactcatc ggagattatc 1260ttgtttttgg gtgactccta ttttggatat ttaaactttt gttaataatg ccatcttctt 1320caaccttttc cttctagatg gtttttatac ttcttctaat tgattgattc tttatggttg 1380tccaagtgtc aaagtgttcc acccatatga ttctaacctt ttgatgagcg aagtctttac 1440tcgtgcgtta tgtagagacg tagaagcaat accacaaaag agtataat 1488791566DNAArabidopsis thaliana 79aggataataa tacagtaacc ctagaaaggt ttcctccacc ttcctcttcc cctcctatat 60aaaaaaaatc gacatcgctt ttgctcactt ctctctctta ggtttttttt cccttctccc 120aatctcatct tctccgaaaa cctttcttct ctcaaatttc tgtgaaaaca tgtctgacga 180cgagcaccac tttgaggcca gcgaatccgg agcttccaag acctatcctc aatcagccgg 240taacatccgt aaaggtggtc acatcgtcat caaaaaccgt ccctgcaagg tctgatttct 300atttcatcat caaacatcgt tctcgatctc tttttcctga ttctagatct cgtctctgta 360tagtagctcc ttgattttgt ttttatcctc ggatttgacc tggttctgtt tagtttgaat 420ttttcttata gatcgctact tagatgaata tgatgaatct tatcctgtta ttttgatggt 480ggtacctctc tagattcgtg gaattttggg aaatgaaaat gaaaaatgga tagaaatcaa 540gcaatatcag acgacgcctt ttgtgatttt gaatctaagt agtctattga ttgatttgat 600ttaaacgttt atggagaaca tagatttgat tttgatattt tggttttgat taggttgttg 660aggtttcgac ttccaaaact ggcaagcacg gtcacgccaa atgtcacttt gttgctattg 720atatcttcac tgctaagaag cttgaagata ttgttccatc ttcccacaat tgtgatgtaa 780gttactacac aaactatgta gattcatttt cacagtattt gatatgattg tgtgatctga 840ctcaaatatt gttcctttct ctttttttct caggttccac atgtgaaccg tgttgattac 900cagttgattg atatcactga ggatggcttc gtatgttttt ctttatactc actttcctca 960tcactccagc tttatttatc tattcttgcc ataacttttg tacttgttta cattataggt 1020gagccttctc actgacagtg gtggcaccaa ggatgatctc aagcttccca ccgatgatgg 1080tctcaccgcc caggttattt tcttgtcttt tcatactcgc acacaaatga cttgactttg 1140tattcatctc tcgaattgtg atattgaaaa cagttgttgt gttttgttaa tgcagatgag 1200gcttggattc gatgagggaa aggatattgt ggtgtctgtc atgtcttcca tgggagagga 1260gcagatctgt gccgtcaagg aagttggtgg tggcaagtaa acaagtatca ttcgatatat 1320tattaccagt ttgacaacgg acgtcaatgt tataagaacc aaaagatgtt tttctttttc 1380ctaatttaga ccctttgtgt gtgtttcttg ttgcaagaca accatatcta ttggttttgg 1440attgttggaa aagtttgtgt tgaaacattc aaagtttctt atgagatgtt attcttaaaa 1500ccactttttg tttgttcact ggatatgttt gttcatgaag cttgttttaa gcaactcttt 1560acatga 156680319DNAArtificial SequenceDescription of Artificial Sequence Promoter sequence 80aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa 60gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 120ttcatttgga gaggacacgc tgaaatcacc agtctctctc tcaagcttgg atcctcgagt 180actagttcag ggagctcgaa ttgatcctct agagctttcg ttcgtatcat cggtttcgac 240aacgttcgtc aagttcaatg catcagtttc attgcgcaca caccagaatc ctactgagtt 300tgagtattat ggcattggg 3198122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 81gggagggact agtgtgcacg cc 228238DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 82gcgaagcggc catggctcga gttttttttt tttttttt 3883521DNAArabidopsis thaliana 83gggagggact agtgtgcacg ccctgatgaa gctgtgtctt ggggtaaaat taggggttct 60gctaaaaccg ttaaggtata ctgtgatgct accatagcct tcccattgtt ggttgcagaa 120acatttgcca caaagagaga ccaaacctgt gagtctaaga cttaagaact gactggttcg 180tacctctggc ctcatcatcg atgtagtaca agatatcaga gctatgaacg gcgaagctgt 240ccatgcaaat cctaaaaaga caggcgtttt ggccatggat tcttaaagat cgttgctttt 300tgattttaca ctggagtgac catataacac tccacattga tgtggctgtg acgcgaattg 360tcttcttgcg aattgtactt tagtttctct caacctaaaa tgatttgcag attgtgtttt 420cgtttaaaac acaagagtct tgtagtcaat aatcctttgc cttataaaat tattcagttc 480caacaaaaaa aaaaaaaaaa ctcgagccat ggccgcttcg c 52184497DNAArabidopsis thaliana 84ctagtgtgca cgccctgatg aagctgtgtc ttggggtaaa attaggggtt ctgctaaaac 60cgttaaggta tactgtgatg ctaccatagc cttcccattg ttggttgcag aaacatttgc 120cacaaagaga gaccaaacct gtgagtctaa gacttaagaa ctgactggtt cgtacctctg 180gcctcatcat cgatgtagta caagatatca gagctatgaa cggcgaagct gtccatgcaa 240atcctaaaaa gacaggcgtt ttggccatgg attcttaaag atcgttgctt tttgatttta 300cactggagtg accatataac actccacatt gatgtggctg tgacgcgaat tgtcttcttg 360cgaattgtac tttagtttct ctcaacctaa aatgatttgc agattgtgtt ttcgtttaaa 420acacaagagt cttgtagtca ataatccttt gccttataaa attattcagt tccaacaaaa 480aaaaaaaaaa aactcga 4978529DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 85ctcgagaaga ataacatctc ataagaaac 298629DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 86gagctcggca agtaaacaag tatcattcg 2987906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 87cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 90688205DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 88aagaataaca tctcataaga aactttgaat gtttcaacac aaacttttcc aacaatccaa 60aaccaataga tatggttgtc ttgcaacaag aaacacacac aaagggtcta aattaggaaa 120aagaaaaaca tcttttggtt cttataacat tgacgtccgt tgtcaaactg gtaataatat 180atcgaatgat acttgtttac ttgcc 20589662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 89gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 240cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 300ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 360ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat 420tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 6629037PRTArabidopsis thaliana 90Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Ser Ala1 5 10 15Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser His Pro Asn Leu Tyr 20 25 30Leu Thr Gln Trp Phe 359152PRTLycopersicon esculentum 91Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Gly1 5 10 15Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe Pro Ile 20 25 30Leu Val Ala Glu Thr Phe Ala Ala Lys Ser Lys Glu Phe Ser Gln Ile 35 40 45Arg Cys Gln Val 5092193PRTArabidopsis thaliana 92Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala Pro Thr Phe Lys1 5 10 15Gly Asp Phe Ser Leu Pro Gly Ala Tyr Leu Arg Ser Lys Gly Leu Asn 20 25 30Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu 35 40 45Asp Trp Ile Ile Pro Ile Phe Asp Glu Met Leu Lys Glu Gln Lys Glu 50 55 60Glu Asn Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg Leu Gly Lys65 70 75 80Glu Ile Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys Met Asn 85 90 95Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly Ser Leu Arg Asp Met 100 105 110Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu Ile Ile Asp Val Val 115 120 125Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Asn Pro Lys 130 135 140Lys Thr Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile145 150 155 160Cys Asn Ala Asn Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile 165 170 175Asn Thr Gly Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp 180 185 190Glu93174PRTDianthus caryophyllus 93Arg Arg Ser Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ala1 5 10 15Leu Pro Gly Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn 20 25 30Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile 35 40 45Pro Ile Leu Asp Lys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile Leu 50 55 60Trp Thr Pro Ser Lys Leu Ile Gly Arg Leu Gly Arg Glu Ile Asn Asp65 70 75 80Glu Ser Ser Tyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile Pro Val Phe 85 90 95Cys Pro Gly Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His 100 105 110Ser Phe Arg Asn Pro Gly Leu Ile Ile Asp Val Val Gln Asp Ile Arg 115 120 125Ala Val Asn Gly Glu Ala Val His Ala Ala Pro Arg Lys Thr Gly Met 130 135 140Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn145 150 155 160Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr 165 1709422PRTArabidopsis thaliana 94Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 209522PRTArabidopsis thaliana 95Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 209622PRTArabidopsis thaliana 96Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 209722PRTBrassica napus 97Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 209822PRTDianthus caryophyllus 98Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 209922PRTLycopersicon esculentum 99Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010022PRTLycopersicon esculentum 100Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010122PRTLycopersicon esculentum 101Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010222PRTMedicago sativa 102Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010322PRTMedicago sativa 103Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010422PRTMedicago sativa 104Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010522PRTLactuca sativa 105Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010622PRTUnknownDescription of Unknown Tree sequence 106Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010722PRTUnknownDescription of Unknown Tree sequence 107Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010822PRTUnknownDescription of Unknown Tree sequence 108Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2010922PRTUnknownDescription of Unknown Tree sequence 109Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His1 5 10 15Ala Lys Cys His Phe Val 2011066DNAArabidopsis thaliana 110tgcaaggttg ttgaggtttc aacctcgaag actggcaagc atggtcatgc taaatgtcat 60tttgta 6611166DNAArabidopsis thaliana 111tgcaaggttg ttgaggtttc gacttccaaa actggcaagc acggtcacgc caaatgtcac 60tttgtt 6611266DNAArabidopsis thaliana 112tgcaaggtgg ttgaggtatc gacttcgaag actgggaagc atggtcacgc caagtgtcac 60tttgtt 6611366DNABrassica napus 113tgcaaggttg ttgaggtttc gacttcgaag actgggaagc acggtcacgc aaagtgtcac 60tttgtt 6611466DNADianthus caryophyllus 114tgcaaggtgg ttgaggtttc tacctccaag actggcaagc acggtcatgc caaatgtcac 60tttgta 6611566DNALycopersicon esculentum 115tgcaaggtgg ttgaagtttc aacctccaag acaggcaagc acggtcatgc taaatgtcac 60ttcgtg 6611666DNALycopersicon esculentum 116tgcaaggttg tggaagtctc tacatccaaa actggcaagc acggtcacgc caaatgtcat

60ttcgtt 6611766DNALycopersicon esculentum 117tgcaaggttg ttgaggtctc cacttccaaa actggcaagc atggacatgc aaaatgtcac 60tttgtg 6611866DNAMedicago sativa 118tgcaaggtgg ttgaagtttc gacttcgaag accgggaagc atggacatgc caagtgtcat 60tttgtt 6611966DNAMedicago sativa 119tgcaaggttg ttgaggtttc tacttcaaaa acaggaaaac atggacatgc aaagtgtcac 60tttgtt 6612066DNAMedicago sativa 120tgcaaggtag ttgaagtttc aacttctaaa actggaaagc atggacatgc aaagtgtcac 60tttgtt 6612166DNALactuca sativa 121tgcaaggttg ttgaagtttc tacctccaag actgggaagc atgggcatgc taagtgtcac 60tttgtc 6612266DNAUnknownDescription of Unknown Tree sequence 122tgcaaggtcg tggaggtttc aacctctaaa actggcaagc atggccatgc taaatgtcac 60tttgtt 6612366DNAUnknownDescription of Unknown Tree sequence 123tgcaaggttg ttgaggtttc cacctcaaag acaggcaagc acggacatgc taagtgccac 60tttgtg 6612466DNAUnknownDescription of Unknown Tree sequence 124tgcaaggttg tggaggtttc tacctctaaa actggcaagc acggccatgc caaatgtcac 60tttgtt 6612566DNAUnknownDescription of Unknown Tree sequence 125tgcaaggttg ttgaggtttc aacctcaaag acaggcaagc atggacatgc taagtgccac 60tttgtg 66126745DNALycopersicon esculentumCDS(55)..(534) 126ttctccacag caaacacaga gaagttcata gcagaagaag agagagattt agct atg 57 Met 1tct gat gaa gaa cac cat ttt gag tcc aaa gct gat gct ggt gcc tca 105Ser Asp Glu Glu His His Phe Glu Ser Lys Ala Asp Ala Gly Ala Ser 5 10 15aaa act tac cct caa caa gct ggt act att cgc aag aat ggt tat ata 153Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile 20 25 30gtt atc aaa ggc aga cct tgc aag gtt gtt gag gtc tcc act tcc aaa 201Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40 45act ggc aag cat gga cat gca aaa tgt cac ttt gtg gca atc gac att 249Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile50 55 60 65ttc aat gca aaa aag ctt gaa gat att gtt cct tca tcc cac aat tgt 297Phe Asn Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys 70 75 80gat gtg cca cat gtc aat cgt act gac tat cag ctg att gac ata tct 345Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95gaa gat ggt ttt gtg tct ctt ctt act gaa aat gga aac acc aaa gac 393Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Asn Gly Asn Thr Lys Asp 100 105 110gac ctc aga ctt ccc acc gat gac acc ctg ttg aac cag gtt aaa ggt 441Asp Leu Arg Leu Pro Thr Asp Asp Thr Leu Leu Asn Gln Val Lys Gly 115 120 125gga ttt gag gaa gga aag gat ctc gtt ctg tct gtg atg tct gca atg 489Gly Phe Glu Glu Gly Lys Asp Leu Val Leu Ser Val Met Ser Ala Met130 135 140 145ggt gaa gag cag atc tgt gct gtg aag gac att ggt acc aag acc 534Gly Glu Glu Gln Ile Cys Ala Val Lys Asp Ile Gly Thr Lys Thr 150 155 160tagttgtgtg cattctgcag cataaataat tgctttttag cgaagacgtt ttatatcttg 594ttatcgtggt acctttgcaa tctgttttat cgtgaaaaca ccttatatct attggcatgg 654cttgaatagt tgaaactcta atagtttctg tttggcataa ggcaatgaac tggatttgat 714agcagaagta atctacatgt cacttttttt t 745127160PRTLycopersicon esculentum 127Met Ser Asp Glu Glu His His Phe Glu Ser Lys Ala Asp Ala Gly Ala1 5 10 15Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr 20 25 30Ile Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser 35 40 45Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50 55 60Ile Phe Asn Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn65 70 75 80Cys Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile 85 90 95Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Asn Gly Asn Thr Lys 100 105 110Asp Asp Leu Arg Leu Pro Thr Asp Asp Thr Leu Leu Asn Gln Val Lys 115 120 125Gly Gly Phe Glu Glu Gly Lys Asp Leu Val Leu Ser Val Met Ser Ala 130 135 140Met Gly Glu Glu Gln Ile Cys Ala Val Lys Asp Ile Gly Thr Lys Thr145 150 155 16012825PRTArtificial SequenceDescription of Artificial Sequence Synthetic primer 128Gly Ala Ala Gly Cys Thr Cys Gly Ala Gly Gly Cys Thr Gly Cys Ala1 5 10 15Ala Cys Cys Ala Thr Gly Thr Cys Cys 20 2512926DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 129ggggagctct tgttagtctc acttgg 26130906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 130cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcaccaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 906131495DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 131gctgcaacca tgtccgacga ggagcatcac tttgagtcca gtgacgccgg agcgtccaaa 60acctaccctc aacaagctgg aaccatccgt aagaatggtt acatcgtcat caaaaatcgt 120ccctgcaagg ttgttgaggt ttcaacctcg aagactggca agcatggtca tgctaaatgt 180cattttgtag ctattgatat cttcaccagc aagaaactcg aagatattgt tccttcttcc 240cacaattgtg atgttcctca tgtcaaccgt actgattatc agctgattga catttctgaa 300gatggatatg tcagtttgtt gactgataac ggtagtacca aggatgacct taagctccct 360aatgatgaca ctctgctcca acagatcaag agtgggtttg atgatggaaa agatctagtg 420gtgagtgtaa tgtcagctat gggagaggaa cagatcaatg ctcttaagga catcggtccc 480aagtgagact aacaa 495132662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 132gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 240cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 300ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 360ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat 420tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 66213330DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 133gcgctcgagc tatgtctgat gaagaacacc 3013428DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 134tttgagctcc agaatgcaca caactagg 28135906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 135cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 906136499DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 136ctatgtctga tgaagaacac cattttgagt ccaaagctga tgctggtgcc tcaaaaactt 60accctcaaca agctggtact attcgcaaga atggttatat agttatcaaa ggcagacctt 120gcaaggttgt tgaggtctcc acttccaaaa ctggcaagca tggacatgca aaatgtcact 180ttgtggcaat cgacattttc aatgcaaaaa agcttgaaga tattgttcct tcatcccaca 240attgtgatgt gccacatgtc aatcgtactg actatcagct gattgacata tctgaagatg 300gttttgtgtc tcttcttact gaaaatggaa acaccaaaga cgacctcaga cttcccaccg 360atgacaccct gttgaaccag gttaaaggtg gatttgagga aggaaaggat ctcgttctgt 420ctgtgatgtc tgcaatgggt gaagagcaga tctgtgctgt gaaggacatt ggtaccaaga 480cctagttgtg tgcattctg 499137662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 137gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 240cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 300ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 360ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat 420tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 66213829DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 138gcatgtcgac atgtctgacg aggagcacc 29139906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 139cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 906140661DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 140tcgacatgtc tgacgaggag caccacttcg agtccagcga cgccggagct tccaaaacct 60accctcagca ggctggtaac atccgcaagg gtggtcacat cgtcatcaag ggccgtccct 120gcaaggttgt tgaggtttcg acttcgaaga ctgggaagca cggtcacgca aagtgtcact 180ttgttgctat tgacatcttc actgctaaga agctcgagga tattgttccc tcttcccaca 240attgtgatgt tccccatgtg aaccgtattg actaccagtt gattgatatc tctgagaatg 300gctttgttag ccttttgacc gacagtggtg gcaccaagga cgacctcaag cttcccaccg 360atgataatct cagcgctctg atgaagagtg gattcgagga gggaaaggat gtggtggtgt 420ctgtcatgtc ttccatggga gaggagcaga tctgtgccgt caaggaagtt ggtggtggca 480agtaaaaccc attctctgag agaggataat cttattacca gtggtcaatg ttataagaac 540aagaacttgt tttttttcct ttttctaatt tagatcattt gtgttgtgtt tctttgttgc 600aagacaacca ttatctatta ttggttttgg attgtttaaa aaaaaaaaaa aaaaaaaaaa 660a 661141662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 141gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 240cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 300ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 360ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat 420tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 662142325DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 142ggtgcacgcc ctgatgaagc agtgtcttgg ggtaaaataa ggggatctgc taaaactgtc 60aaggtgtact gtgatgctac catagccttc cctttgttgg ttgctgaaac atttgcctcc 120aagagagaac aaagctgtga gcacaagacc taagcccaag aaagcttacg tctcttttat 180cggtttgttc ttccatcttg ttgttgtacc ctttgtcctg ctttacataa cattcatctc 240taaaacaata ctacctcctt ttgacaaaaa ataaaaaaaa ttggaaaaat ggtttcacaa 300gaataaaaaa aaaaaaaaaa aaaaa 325143906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 143cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 906144325DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 144tttttttttt tttttttttt tattcttgtg aaaccatttt tccaattttt tttatttttt 60gtcaaaagga ggtagtattg ttttagagat gaatgttatg taaagcagga caaagggtac 120aacaacaaga tggaagaaca aaccgataaa agagacgtaa gctttcttgg gcttaggtct 180tgtgctcaca gctttgttct ctcttggagg caaatgtttc agcaaccaac aaagggaagg 240ctatggtagc atcacagtac accttgacag ttttagcaga tccccttatt ttaccccaag 300acactgcttc atcagggcgt gcacc 325145662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 145gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtgc

aaatggaaat ggatggagaa gagttaatga atgatatggt 240ccttttgttc attctcaaat taatattatt tgttttttct cttatttgtt gtgtgttgaa 300tttgaaatta taagagatat gcaaacattt tgttttgagt aaaaatgtgt caaatcgtgg 360cctctaatga ccgaagttaa tatgaggagt aaaacacttg tagttgtacc attatgctta 420ttcactaggc aacaaatata ttttcagacc tagaaaagct gaaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 66214628DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 146aagctcgaga tgtcggacga agagcacc 2814728DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 147gtagagctcc accaatacca tctgcagc 28148906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 148cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 906149497DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 149atgtcggacg aagagcacca cttcgaatcc aaggccgatg ccggagcttc aaagacgtat 60cctcaacaag ctggtactat tcgtaaaggt ggtcacatcg tcataaaaaa tcgtccttgc 120aaggtggttg aagtttcaac ttccaagaca ggcaagcacg gtcatgctaa atgtcactcg 180tggcaattga cattttcact ggaaagaaac ttgaggatat tgttccctct tctcacaatt 240gtgatgttcc tcatgtgaat aggactgatt atcaacttat tgatatctct gaggatggct 300ttgtgagtct gttgactgaa aatggtaaca ccaaggatga cttgagactc ccaactgatg 360atactcttct ggctcaggtc aaagatggtt ttgctgaggg gaaagacctg gttctatcag 420tgatgtctgc catgggagag gagcagattt gtggtatcaa ggacattggc cctaagtagc 480tgcagatggt attggtg 497150662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 150gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 240cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 300ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 360ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat 420tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 66215127DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 151cgactcgagc agccatgtct gacgagg 2715229DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 152atcgagctca tcacttgggg ccaatatcc 29153906DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 153cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 60aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 120cgtcaacatg gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct 180cagaagacca aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg 240attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 300ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 360tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 420cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca cgcttgtcta 480cctccaaaaa tatcaaagat acagtctcag aagaccaaag ggaattgaga cttttcaaca 540aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 600gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 660catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 720catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 780ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 840ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc tctctaagct 900tggatc 906154486DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 154cagccatgtc tgacgaggag catcaatttg agtctaaggc tgatgccgga gcatcaaaaa 60cttaccctca acaagctggt actattcgta agaacggtta tatcgtcatc aaaggccgtc 120catgcaaggt tgtggaagtc tctacatcca aaactggcaa gcacggtcac gccaaatgtc 180atttcgttgc tattgacatc ttcactggga agaagcttga ggatattgtc ccctcttcac 240acaattgtga tgtgccccat gttaatcgta cagattatca gcttattgac atctctgaag 300atggatttgt gagtctgctt actgacaatg gtaacaccaa ggatgacctc aggcttccta 360ctgatgaaaa tctgctttca ctgatcaagg acgggtttgc cgagggtaag gacctcgttg 420tgtctgttat gtcagctatg ggtgaggaac agattaatgc tttgaaggat attggcccca 480agtgat 486155662DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 155gaattgatcc tctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca 60atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat tatggcattg 120ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg 180ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc 240cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat 300ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc 360ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat 420tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag 480tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct 540tgtcagattc taatcattgc tttataatta tagttatact catggatttg tagttgagta 600tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg 660at 66215622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 156cactgctcac tagtttgatg gc 2215738DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 157gcgaagcggc catggctcga gttttttttt tttttttt 38158413DNALactuca sativa 158cactgctcac tagtttgatg gcagtgattc tggtgctcga cctgatgaag ctgtctcctg 60ggggaaaata cgtggttctg ctaaatctgt caaggtgcac tgtgatgcaa ctatcgcgtt 120ccctttactt gttgcagaaa catttgctgc aaagagagag ggggagatga aaaatgttga 180gtcaaccaaa gctttggttt aaaaaggtgg aacagtgtag gacagggact catttttgat 240attttgtttg ctaaaaaatg gtctttggaa gaatattgat gcacacaaac aaggagacaa 300tgttactgat cttggagagt gtaatgtaaa atgtctaaat aatttcaaag cttctcacaa 360caaatcaaac tttaaaaaaa aaaaaaaaaa aactcgagcc atggccgctt cgc 413159388DNALactuca sativa 159ctagtttgat ggcagtgatt ctggtgctcg acctgatgaa gctgtctcct gggggaaaat 60acgtggttct gctaaatctg tcaaggtgca ctgtgatgca actatcgcgt tccctttact 120tgttgcagaa acatttgctg caaagagaga gggggagatg aaaaatgttg agtcaaccaa 180agctttggtt taaaaaggtg gaacagtgta ggacagggac tcatttttga tattttgttt 240gctaaaaaat ggtctttgga agaatattga tgcacacaaa caaggagaca atgttactga 300tcttggagag tgtacatgta aaatgtctaa ataatttcaa agcttctcac aacaaatcaa 360acttaaaaaa aaaaaaaaaa aaactcga 38816023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 160ggnttraayc gnathggnaa ytt 2316123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 161tgrtcgganc crtcraaytc ngc 23162108DNAMycosphaerella fijiensis 162cgccaagcta tttaggtgac actatagaat actcaagcta tgcatccaac gcgttgggag 60ctctcccata tggtcgacct gcaggcggcc gcgaattcac tagtgatt 108163487DNAMycosphaerella fijiensisCDS(1)..(486) 163ggg tta aat cgt att gga aac ttc tta gtg cca aac gac aat tac tgc 48Gly Leu Asn Arg Ile Gly Asn Phe Leu Val Pro Asn Asp Asn Tyr Cys1 5 10 15cgc ttt gaa gac tgg gtg atg cca atc ctc gac aca atg ctc gaa gaa 96Arg Phe Glu Asp Trp Val Met Pro Ile Leu Asp Thr Met Leu Glu Glu 20 25 30cag gaa gca tgc aag ggt tcg ggc gaa gca atc cac tgg act ccc agc 144Gln Glu Ala Cys Lys Gly Ser Gly Glu Ala Ile His Trp Thr Pro Ser 35 40 45aaa atc atc aac cgg ctt ggc aag gag gtc aac gac gaa tcg tcc gtg 192Lys Ile Ile Asn Arg Leu Gly Lys Glu Val Asn Asp Glu Ser Ser Val 50 55 60tac tac tgg gca tgg aag aac gac att cca gtg ttc tgt ccg gcg ctt 240Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro Val Phe Cys Pro Ala Leu65 70 75 80act gat ggc agt ctc gga gac atg ctg tac ttc cac acg ttc aaa tcc 288Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Thr Phe Lys Ser 85 90 95tca ccg cag cag ctt cga gtc gac att gtg gaa gac atc cga aag atc 336Ser Pro Gln Gln Leu Arg Val Asp Ile Val Glu Asp Ile Arg Lys Ile 100 105 110aac acc ctc gcc gtc cga gcc aag cgc act ggc atg atc att ctc gga 384Asn Thr Leu Ala Val Arg Ala Lys Arg Thr Gly Met Ile Ile Leu Gly 115 120 125ggc ggc att gtc aag cac cac atc gca aat gcc aac ctg atg cgc aat 432Gly Gly Ile Val Lys His His Ile Ala Asn Ala Asn Leu Met Arg Asn 130 135 140ggc gcg gaa agc gca gtg tac atc aat acc gcg ccg aat tcg acg gat 480Gly Ala Glu Ser Ala Val Tyr Ile Asn Thr Ala Pro Asn Ser Thr Asp145 150 155 160ccg acc a 487Pro Thr164162PRTMycosphaerella fijiensis 164Gly Leu Asn Arg Ile Gly Asn Phe Leu Val Pro Asn Asp Asn Tyr Cys1 5 10 15Arg Phe Glu Asp Trp Val Met Pro Ile Leu Asp Thr Met Leu Glu Glu 20 25 30Gln Glu Ala Cys Lys Gly Ser Gly Glu Ala Ile His Trp Thr Pro Ser 35 40 45Lys Ile Ile Asn Arg Leu Gly Lys Glu Val Asn Asp Glu Ser Ser Val 50 55 60Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro Val Phe Cys Pro Ala Leu65 70 75 80Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Thr Phe Lys Ser 85 90 95Ser Pro Gln Gln Leu Arg Val Asp Ile Val Glu Asp Ile Arg Lys Ile 100 105 110Asn Thr Leu Ala Val Arg Ala Lys Arg Thr Gly Met Ile Ile Leu Gly 115 120 125Gly Gly Ile Val Lys His His Ile Ala Asn Ala Asn Leu Met Arg Asn 130 135 140Gly Ala Glu Ser Ala Val Tyr Ile Asn Thr Ala Pro Asn Ser Thr Asp145 150 155 160Pro Thr165214DNAMycosphaerella fijiensis 165aatcgaattc ccgcggccgc catggcggcc gggagcatgc gacgtcgggc ccaattcgcc 60ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa 120cctggcggta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 180agcgaagagg cccgcaccga tcgcccttcc aaca 214

Claims

1-25. (canceled)

26: An isolated growth eIF-5A polypeptide with at least 95% sequence identity to SEQ ID NO: 67.

27: The isolated polypeptide of claim 26, wherein the polypeptide comprises SEQ ID NO: 67.

28: The isolated polypeptide of claim 26, wherein the polypeptide consists of SEQ ID NO: 67.

29: The isolated polypeptide of claim 26, wherein the growth eIF-5A polypeptide is from canola.

30: An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 26.

31: The polynucleotide of claim 30 wherein the polynucleotide sequence comprises SEQ ID NO: 66.

32: The polynucleotide of claim 30 wherein the polynucleotide sequence consists of SEQ ID NO: 66.

33: A vector comprising the polynucleotide of claim 31 or 32 and regulatory sequences operatively linked to the polynucleotide to provide transcription of the polynucleotide in a plant.

34: The vector of claim 33, wherein the plant is canola.

35: A bacterial cell transformed with the vector according to claim 33.

36: A plant cell transformed with the vector according to claim 33.

37: A plant and progeny thereof, wherein the plant is generated from the plant cell of claim 36 and wherein said plant and progeny thereof comprise said vector.

38: A method of increasing expression of endogenous growth eIF-5A in a plant comprising incorporating into the genome of at least one plant cell the vector of claim 33, whereby transcription of the polynucleotide results in increase of expression of growth eIF-5A in the plant.

39: A method of claim 38 wherein the plant is canola.

40: A plant produced by the method of claim 38.

41: Progeny, plant parts or seeds of the plant of claim 40.

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