Patent application title: Isolated eIF-5A and Polynucleotides Encoding Same
Inventors:
John E. Thompson (Waterloo, CA)
Tzann-Wei Wang (Waterloo, CA)
Tzann-Wei Wang (Waterloo, CA)
Assignees:
Senesco Technologies, Inc.
IPC8 Class: AC12N1582FI
USPC Class:
800286
Class name: Method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide encodes an inhibitory rna molecule the rna is antisense
Publication date: 2014-06-26
Patent application number: 20140182014
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.Claims:
1-25. (canceled)
26. An isolated polynucleotide comprising a nucleotide sequence encoding an isolated deoxyhypusine synthase (DHS) polypeptide with at least 95% sequence identity to SEQ ID NO: 73
27. The polynucleotide of claim 26 wherein the polynucleotide sequence comprises or consists of SEQ ID NO: 72.
28. A vector comprising the polynucleotide of claim 26 and regulatory sequences operatively linked to the polynucleotide to provide transcription of the polynucleotide in a plant.
29. An antisense polynucleotide which hybridizes to the polynucleotide of claim 26.
30. A vector comprising the polynucleotide of claim 29 and regulatory sequences operatively linked to the polynucleotide to provide transcription of the polynucleotide in a plant.
31. A method of inhibiting expression of endogenous DHS in a plant, comprising incorporating into the genome of at least one plant cell the vector of claim 30 whereby said transcription of said polynucleotide inhibits expression of endogenous DHS in the plant.
32. A method of claim 31 wherein said inhibition results in a plant having an increased tolerance to environmental stress as compared to a wild type plant.
33. A method of claim 31 wherein said inhibition results in a plant having an increased seed yield and/or increased seed size as compared to a wild type plant.
34. A plant produced by the method of any one of claims 31-33.
35. Progeny, plant parts or seeds of the plant of claim 34.
36. A bacterial cell comprising the vector of claim 28 or 30.
37. A plant cell comprising the vector of claim 28 or 30.
38. A plant or progeny thereof, wherein the plant is generated from the plant cell of claim 37 and wherein said plant and progeny thereof comprise said vector.
39. The polynucleotide of claim 26 wherein the polynucleotide sequence comprises a nucleotide sequence encoding an isolated DHS polypeptide with at least 98% sequence identity to SEQ ID NO: 73
40. The polynucleotide of claim 26 wherein the polynucleotide sequence comprises a nucleotide sequence encoding an isolated DHS polypeptide with at least 99% sequence identity to SEQ ID NO: 73
Description:
RELATED APPLICATIONS
[0001] This application is a continuation-in-part 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 "061945-5019-02-SequenceListing.txt" created on or about Feb. 27, 2014 with a file size of about 180 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, 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 Ampr 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, F1 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-SA (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 catagorized based on the level of expression as determined by Westem 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] FIGS. 45A and B 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. 46A 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] FIGS. 53A-C 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] FIGS. 88A and B 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] FIGS. 89A and B 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. 90A and B and 91A and B 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] FIGS. 92A and B 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] FIGS. 97A and B 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] FIGS. 102A and B 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 apperance).
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 (TM) of the duplex. The TM 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 isoforn 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 by 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 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.
[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'-GGTGGTGT5TGAGGAAGATC (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 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
[0231] Reaction Parameters:
[0232] 94° C. for 3 min
[0233] 94° C./1 min, 58° C./1 min, 72° C./2 min, for 45 cycles
[0234] 72° C. for 15 min.
Example 7
Isolation of Genomic DNA and Southern Analysis
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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
[0239] 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
[0240] 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
[0241] 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
[0242] 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
[0243] 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
[0244] 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.
[0245] 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
[0246] 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.
[0247] 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
[0248] 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.
[0249] 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.
[0250] 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
[0251] 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 μmol 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
[0252] 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.
[0253] 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.
[0254] 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
[0255] 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
[0256] 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
[0257] 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
[0258] 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
[0259] Eukaryotic translation initiation factor 5A (e1F-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
[0260] 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
[0261] 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
[0262] 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 transformned 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
[0263] 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
[0264] 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
[0265] 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 Forma 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.
[0266] For electroporation of the DNA constructs into the competent Agrobacterium cells the 100 μl aliquots were each mixed well with 500 μg 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
[0267] 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 Forma 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.
[0268] 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
[0269] 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/ml kanamycin (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.
[0270] 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.
[0271] 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.
[0272] 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-SA 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.
[0273] 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
[0274] 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
[0275] 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
[0276] 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
[0277] 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 αATeIF-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 αAT-eIF5A was imbibed seeds, keeping with the notion that this is the isoform involved in cell division.
[0278] 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 αAteIF-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 αAteIF-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.
[0279] 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
[0280] 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)
[0281] 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.
[0282] 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)
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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 Methodology for Obtaining Full-Length Arabidopsis Senescence-Induced eIF-5A
[0292] 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.
[0293] 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.
[0294] 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
[0295] T3: (SEQ ID NO: 20) 5'-ATT AAC CCT CAC TAA AG-3' T7: (SEQ ID NO: 18) 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)
[0296] 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).
[0297] 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)
[0298] 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
[0299] 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-5Arabidopsis thaliana Plants Under Control of the SAG 12 Promoter Inserting the SAG 12-Antisense-Full-Length Senescence-Induced eIF-5A Construct in pKYLX71
[0300] 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.
[0301] 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.
[0302] The forward SAG 12 Primer was 5'-GGC CGTCGACGATATCTCTTTTTATATTCAAAC-3' (SEQ ID NO: 50) (underlined portion is SalI recognition site, used for ligating the 5'-end of the PCR fragment into the SalI 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).
[0303] 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).
[0304] 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).
[0305] 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 SalI and SacI.
[0306] The XhoI and SalI 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
[0307] The pKYLX71-SAG 12:antisense-eIF-SA construct was proliferated in E.coli DHa 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 (1/2 MS salt and 50 μg kanamycin/mL) with wildtype as a control; only those seeds containing pKYLX71-SAG 12-antisense-eIF-SA 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
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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 2nd 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
[0314] 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.
[0315] 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 15 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.
[0316] 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/1)]×[reaction volume (ml)/103 (ml/1)]×[106(μmole/mole)]μ[60 (min/hr)/reaction time (min)]×[1/sample volume (ml)].
[0317] 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
[0318] 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 (HC104, 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
[0319] 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.
[0320] 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.
[0321] 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
[0322] 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
[0323] 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
[0324] 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
[0325] 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
[0326] 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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Sequence CWU
1
1
16711584DNALycopersicon 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 15
ttc 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 30 tac 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
45 gtt 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 80 gat 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 95 tgc 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
110 aga 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 115 120 125
gtt 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
155 160 aaa 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
175 tgc 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 180 185 190 gag
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
200 205 cgt 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
240 ctt 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
245 250 255 aat 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
265 270 agg 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
285 atg 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
310 315 320 caa 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
330 335 tca 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
350 gat 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 355 360 365 aag
agt aag gaa ttc tcc cag ata agg tgc caa gtt tgaacattga 1206Lys
Ser Lys Glu Phe Ser Gln Ile Arg Cys Gln Val 370
375 380 ggaagctgtc 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 Val 1
5 10 15 Val Phe Lys Glu Ser
Glu Asn Leu Glu Gly Ser Cys Thr Lys Ile Glu 20
25 30 Gly Tyr Asp Phe Asn Lys Gly Val Asn
Tyr Ala Glu Leu Ile Lys Ser 35 40
45 Met Val Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp
Ala Ile Ala 50 55 60
Ile Val Asn Gln Met Leu Asp Trp Arg Leu Ser His Glu Leu Pro Thr 65
70 75 80 Glu Asp Cys Ser
Glu Glu Glu Arg Asp Val Ala Tyr Arg Glu Ser Val 85
90 95 Thr Cys Lys Ile Phe Leu Gly Phe
Thr Ser Asn Leu Val Ser Ser Gly 100 105
110 Val Arg Asp Thr Val Arg Tyr Leu Val Gln His Arg
Met Val Asp Val 115 120 125
Val Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu Ile Lys Cys Leu
130 135 140 Ala Pro
Thr Tyr Lys Gly Asp Phe Ser Leu Pro Gly Ala Ser Leu Arg 145
150 155 160 Ser Lys Gly Leu Asn Arg
Ile Gly Asn Leu Leu Val Pro Asn Asp Asn 165
170 175 Tyr Cys Lys Phe Glu Asn Trp Ile Ile Pro
Val Phe Asp Gln Met Tyr 180 185
190 Glu Glu Gln Ile Asn Glu Lys Val Leu Trp Thr Pro Ser Lys
Val Ile 195 200 205
Ala Arg Leu Gly Lys Glu Ile Asn Asp Glu Thr Ser Tyr Leu Tyr Trp 210
215 220 Ala Tyr Lys Asn
Arg Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly 225 230
235 240 Ser Leu Gly Asp Met Leu Tyr Phe
His Ser Phe Lys Lys Gly Asp Pro 245 250
255 Asp Asn Pro Asp Leu Asn Pro Gly Leu Val Ile Asp
Ile Val Gly Asp 260 265 270
Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Gly Leu Arg Lys Thr
275 280 285 Gly Met
Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Val Cys Asn 290
295 300 Ala Asn Met Met Arg Asn
Gly Ala Asp Phe Ala Val Phe Ile Asn Thr 305 310
315 320 Ala Gln Glu Phe Asp Gly Ser Asp Ser Gly
Ala Arg Pro Asp Glu Ala 325 330
335 Val Ser Trp Gly Lys Ile Arg Gly Gly Ala Lys Thr Val Lys
Val His 340 345 350
Cys Asp Ala Thr Ile Ala Phe Pro Ile Leu Val Ala Glu Thr Phe Ala
355 360 365 Ala Lys Ser Lys
Glu Phe Ser Gln Ile Arg Cys Gln Val 370
375 380 3369PRTHomo sapiens 3Met Glu Gly
Ser Leu Glu Arg Glu Ala Pro Ala Gly Ala Leu Ala Ala 1 5
10 15 Val Leu Lys His Ser Ser Thr Leu
Pro Pro Glu Ser Thr Gln Val Arg 20 25
30 Gly Tyr Asp Phe Asn Arg Gly Val Asn Tyr Arg Ala Leu
Leu Glu Ala 35 40 45
Phe Gly Thr Thr Gly Phe Gln Ala Thr Asn Phe Gly Arg Ala Val Gln 50
55 60 Gln Val Asn Ala
Met Ile Glu Lys Lys Leu Glu Pro Leu Ser Gln Asp 65 70
75 80Glu Asp Gln His Ala Asp Leu Thr Gln
Ser Arg Arg Pro Leu Thr Ser 85 90
95 Cys Thr Ile Phe Leu Gly Tyr Thr Ser Asn Leu Ile Ser Ser
Gly Ile 100 105 110
Arg Glu Thr Ile Arg Tyr Leu Val Gln His Asn Met Val Asp Val Leu
115 120 125 Val Thr Thr Ala
Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala 130
135 140 Pro Thr Tyr Leu Gly Glu Phe Ser
Leu Arg Gly Lys Glu Leu Arg Glu 145 150
155 160Asn Gly Ile Asn Arg Ile Gly Asn Leu Leu Val Pro
Asn Glu Asn Tyr 165 170
175 Cys Lys Phe Glu Asp Trp Leu Met Pro Ile Leu Asp Gln Met Val Met
180 185 190 Glu Gln Asn
Thr Glu Gly Val Lys Trp Thr Pro Ser Lys Met Ile Ala 195
200 205 Arg Leu Gly Lys Glu Ile Asn Asn
Pro Glu Ser Val Tyr Tyr Trp Ala 210 215
220 Gln Lys Asn His Ile Pro Val Phe Ser Pro Ala Leu Thr
Asp Gly Ser 225 230 235
240Leu Gly Asp Met Ile Phe Phe His Ser Tyr Lys Asn Pro Gly Leu Val
245 250 255 Leu Asp Ile Val
Glu Asp Leu Arg Leu Ile Asn Thr Gln Ala Ile Phe 260
265 270 Ala Lys Cys Thr Gly Met Ile Ile Leu
Gly Gly Gly Val Val Lys His 275 280
285 His Ile Ala Asn Ala Asn Leu Met Arg Asn Gly Ala Asp Tyr
Ala Val 290 295 300
Tyr Ile Asn Thr Ala Gln Glu Phe Asp Gly Ser Asp Ser Gly Ala Arg 305
310 315 320Pro Asp Glu Ala Val
Ser Trp Gly Lys Ile Arg Val Asp Ala Gln Pro 325
330 335 Val Lys Val Tyr Ala Asp Ala Ser Leu Val
Phe Pro Leu Leu Val Ala 340 345
350 Glu Thr Phe Ala Gln Lys Met Asp Ala Phe Met His Glu Lys Asn
Glu 355 360 365 Asp
46PRTArtificial SequenceDescription of Artificial Sequence Conserved
peptide fragment 4Thr Gly Lys His Gly His
1 5 52272DNAArabidopsis
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 10
ttc 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 45 ctc 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
60 gtc aat caa atg gttcgtttct cgaattcatc aaaaataaaa
attccttctt 305Val Asn Gln Met
65
tttgttttcc 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 85 tgt 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 95 100
aaa 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
105 110 115 gat act att cgt
tat ctt gtt cag cat cat atg gtttgtgatt tttgctttat 556Asp Thr Ile Arg
Tyr Leu Val Gln His His Met 120
125 caccctgctt 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
140 ctc 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 145 150 155
gga 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
185 190 ttt gac gag atg ttg aag gaa cag aaa
gaa gag gtattgcttt atctttcctt 862Phe Asp Glu Met Leu Lys Glu Gln Lys
Glu Glu 195 200
tttatatgat 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 205 210 215
aac aat gag agt tca tac ctt tat tgg gca tac aag gtatccaaaa
1075Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys 220
225 230 ttttaacctt 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
235 240 gat 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
255 ggc ctc atc atc gat gta gta caa ggtacttctt ttactcaata
agtcagtgtg 1288Gly Leu Ile Ile Asp Val Val Gln
260 265
ataaatattc 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
295 300 atg 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
315 gaa 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 320 325 330 tgg 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
340 345 att 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 Phe 350 355
360 tttaatattg 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 Lys 1 5
10 15 Glu Ser Glu Ser Leu Glu Gly Lys
Cys Asp Lys Ile Glu Gly Tyr Asp 20 25
30 Phe Asn Gln Gly Val Asp Tyr Pro Lys Leu Met Arg
Ser Met Leu Thr 35 40 45
Thr Gly Phe Gln Ala Ser Asn Leu Gly Glu Ala Ile Asp Val Val Asn
50 55 60 Gln Met Phe
Glu Phe Val Leu Lys Leu Asp Trp Arg Leu Ala Asp Glu 65
70 75 80 Thr Thr Val Ala Glu Asp Cys
Ser Glu Glu Glu Lys Asn Pro Ser Phe 85
90 95 Arg Glu Ser Val Lys Cys Lys Ile Phe Leu Gly
Phe Thr Ser Asn Leu 100 105
110 Val Ser Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln His
His 115 120 125 Met
Val Asp Val Ile Val Thr Thr Thr Gly Gly Val Glu Glu Asp Leu 130
135 140 Ile Lys Cys Leu Ala
Pro Thr Phe Lys Gly Asp Phe Ser Leu Pro Gly 145 150
155 160 Ala Tyr Leu Arg Ser Lys Gly Leu Asn
Arg Ile Gly Asn Leu Leu Val 165 170
175 Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile
Pro Ile Phe 180 185 190
Asp Glu Met Leu Lys Glu Gln Lys Glu Glu Asn Val Leu Trp Thr Pro
195 200 205 Ser Lys Leu
Leu Ala Arg Leu Gly Lys Glu Ile Asn Asn Glu Ser Ser 210
215 220 Tyr Leu Tyr Trp Ala Tyr Lys
Met Asn Ile Pro Val Phe Cys Pro Gly 225 230
235 240 Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr
Phe His Ser Phe Arg 245 250
255 Thr Ser Gly Leu Ile Ile Asp Val Val Gln Asp Ile Arg Ala Met
Asn 260 265 270 Gly
Glu Ala Val His Ala Asn Pro Lys Lys Thr Gly Met Ile Ile Leu 275
280 285 Gly Gly Gly Leu Pro
Lys His His Ile Cys Asn Ala Asn Met Met Arg 290 295
300 Asn Gly Ala Asp Tyr Ala Val Phe Ile
Asn Thr Gly Gln Glu Phe Asp 305 310 315
320 Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser
Trp Gly Lys 325 330 335
Ile Arg Gly Ser Ala Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser
340 345 350 His Pro Asn
Leu Tyr Leu Thr Gln Trp Phe 355
360 719DNAArtificial
SequenceDescription of Artificial Sequence Primer 7ggtggtgttg aggaagatc
19820DNAArtificial
SequenceDescription of Artificial Sequence 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
10 cac 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 25 aag 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 40 ttg 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 75
gca 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 90 gaa 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
105 tcc 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 110 115 120
gtt 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
145 150 155 gcc 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
165 170 aat 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
185 att 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 190 195 200 tta
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
230 235 ttt 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
250 cat 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 255 260 265 aga 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
275 280 atg 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
315 gaa 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
320 325 330 tca 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
340 345 gat 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
360 aaa aga gaa aaa gag agg aag agc tgt taaaactttt
ttgattgttg 1394Lys Arg Glu Lys Glu Arg Lys Ser Cys
365 370
aaaaatctgt 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 Ala 1 5 10
15 Phe Lys Lys Ser Glu Asn Leu Glu Gly Lys Ser Val Lys
Ile Glu Gly 20 25 30
Tyr Asp Phe Asn Gln Gly Val Asn Tyr Ser Lys Leu Leu Gln Ser Phe
35 40 45 Ala Ser Asn Gly
Phe Gln Ala Ser Asn Leu Gly Asp Ala Ile Glu Val 50
55 60 Val Asn His Met Leu Asp Trp Ser
Leu Ala Asp Glu Ala Pro Val Asp 65 70
75 80 Asp Cys Ser Glu Glu Glu Arg Asp Pro Lys Phe Arg
Glu Ser Val Lys 85 90
95 Cys Lys Val Phe Leu Gly Phe Thr Ser Asn Leu Ile Ser Ser Gly Val
100 105 110 Arg Asp
Thr Ile Arg Tyr Leu Val Gln His His Met Val Asp Val Ile 115
120 125 Val Thr Thr Thr Gly Gly
Ile Glu Glu Asp Leu Ile Lys Gly Arg Ser 130 135
140 Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly
Asp Phe Ala Leu Pro Gly 145 150 155
160 Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu
Leu Val 165 170 175
Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Leu
180 185 190 Asp Lys Met Leu
Glu Glu Gln Ile Ser Glu Lys Ile Leu Trp Thr Pro 195
200 205 Ser Lys Leu Ile Gly Arg Leu Gly
Arg Glu Ile Asn Asp Glu Ser Ser 210 215
220 Tyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile Pro Val
Phe Cys Pro Gly 225 230 235
240 Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Arg
245 250 255 Asn Pro
Gly Leu Ile Val Asp Val Val Gln Asp Ile Arg Ala Val Asn 260
265 270 Gly Glu Ala Val His Ala
Ala Pro Arg Lys Thr Gly Met Ile Ile Leu 275 280
285 Gly Gly Gly Leu Pro Lys His His Ile Cys
Asn Ala Asn Met Met Arg 290 295 300
Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala Glu Glu
Phe Asp 305 310 315 320
Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Ile Ser Trp Gly Lys
325 330 335 Ile Ser Gly Ser
Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile 340
345 350 Ala Phe Pro Leu Leu Val Ala Glu
Thr Phe Ala Ala Lys Arg Glu Lys 355 360
365 Glu Arg Lys Ser Cys
370
11780DNALycopersicon esculentumCDS(43)..(522) 11aaagaatcct
agagagagaa agggaatcct agagagagaa gc atg tcg gac gaa 54
Met Ser Asp Glu
1 gaa 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 35 ggc
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 50 cac 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
65 aag 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 80
cat 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
110 115 ctt 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
130 gaa 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 135 140 145 cag
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
155 160 atggcagcat 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 Ala 1
5 10 15 Ser Lys Thr Phe
Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr 20
25 30 Ile Val Ile Lys Gly Arg Pro Cys
Lys Val Val Glu Val Ser Thr Ser 35 40
45 Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe
Val Ala Ile Asp 50 55 60
Ile Phe Asn Gly Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn
65 70 75 80 Cys Asp
Val Pro His Val Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile
85 90 95 Ser Glu Asp Gly Phe Val
Ser Leu Leu Thr Glu Ser Gly Asn Thr Lys 100
105 110 Asp Asp Leu Arg Leu Pro Thr Asp Glu Asn
Leu Leu Lys Gln Val Lys 115 120
125 Asp Gly Phe Gln Glu Gly Lys Asp Leu Val Val Ser Val Met
Ser Ala 130 135 140
Met Gly Glu Glu Gln Ile Asn Ala Val Lys Asp Val Gly Thr Lys Asn 145
150 155 160 13812DNADianthus
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 10
gga 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 45 acc 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
60 att 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 75 cac
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 90 gat 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
125 atg 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
130 135 140 tgt 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
150 155 ggc aag tagaagcttt tgatgaatcc
aatactacgc ggtgcagttg aagcaatagt 596Gly Lys
160
aatctcgaga 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 Ala 1 5
10 15 Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr
Ile Arg Lys Ser Gly His 20 25
30 Ile Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser
Thr Ser 35 40 45
Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50
55 60 Ile Phe Asn Gly
Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn 65 70
75 80 Cys Asp Val Pro His Val Asn Arg
Val Asp Tyr Gln Leu Leu Asp Ile 85 90
95 Thr Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Ser
Gly Asp Thr Lys 100 105 110
Asp Asp Leu Lys Leu Pro Ala Asp Glu Ala Leu Val Lys Gln Met Lys
115 120 125 Glu Gly
Phe Glu Ala Gly Lys Asp Leu Ile Leu Ser Val Met Cys Ala 130
135 140 Met Gly Glu Glu Gln Ile
Cys Ala Val Lys Asp Val Ser Gly Gly Lys 145 150
155 160 15702DNAArabidopsis
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 15 acc 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 30 atc 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 45
ggc 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 80 gtt 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
95 gat 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 100 105 110
ctt 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
115 120 125 ttt 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 150 155
aaagcctccc 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 Ser 1 5
10 15 Lys Thr Tyr Pro Gln Gln Ala
Gly Thr Ile Arg Lys Asn Gly Tyr Ile 20 25
30 Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu
Val Ser Thr Ser Lys 35 40 45
Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp
Ile 50 55 60 Phe
Thr Ser Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys 65
70 75 80 Asp Val Pro His Val
Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser 85
90 95 Glu Asp Gly Tyr Val Ser Leu Leu Thr
Asp Asn Gly Ser Thr Lys Asp 100 105
110 Asp Leu Lys Leu Pro Asn Asp Asp Thr Leu Leu Gln Gln
Ile Lys Ser 115 120 125
Gly Phe Asp Asp Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met
130 135 140 Gly Glu Glu
Gln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys 145
150 155 1720DNAArtificial
SequenceDescription of Artificial Sequence Primer 17aaarrycgmc cytgcaaggt
201817DNAArtificial
SequenceDescription of Artificial Sequence Primer 18aatacgactc actatag
171920DNAArtificial
SequenceDescription of Artificial Sequence Primer 19tcyttnccyt cmkctaahcc
202017DNAArtificial
SequenceDescription of Artificial Sequence Primer 20attaaccctc actaaag
172122DNAArtificial
SequenceDescription of Artificial Sequence Primer 21ctgttaccaa aaaatctgta
cc 222221DNAArtificial
SequenceDescription of Artificial Sequence Primer 22agaagaagta taaaaaccat
c 212323DNAArtificial
SequenceDescription of Artificial Sequence Primer 23aaagaatcct agagagagaa
agg 232418DNAArtificial
SequenceDescription of Artificial Sequence Primer 24ttttacatca atcgaaaa
182523DNAArtificial
SequenceDescription of Artificial Sequence 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 15
ggt 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 30 cga 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
45 gat 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 60
gag 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 95 att 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
110 ctg 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 115 120 125
caa 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
135 140 aag 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
175 aac 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 180 185 190 gaa
gc 581Glu
27523DNADianthus 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
15 tta 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 30
ctg 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 45 cca 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
60 tgg 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 95 tgc 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
105 110 tct 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
125 gca 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 130 135 140
att 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
170 2824DNAArtificial SequenceDescription of
Artificial Sequence Primer 28ttgargaaga tycatmaart gcct
242923DNAArtificial SequenceDescription of
Artificial Sequence 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 15 aaa 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 30 ctc act cag tgg ttt tgagtacata tttaatattg
gatcattctt gcaggtatac 152Leu Thr Gln Trp Phe
35
tgtgatgcta 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 15 gcc 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 30 tta 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
45 agg tgc caa gtt tgaacattga ggaagctgtc cttccgacca
cacatatgaa 196Arg Cys Gln Val
50
ttgctagctt 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 Conserved
peptide fragment 32Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly
Lys His Gly His 1 5 10
15 Ala Lys Cys His Phe Val
20 3329DNAArtificial SequenceDescription of Artificial
Sequence Primer 33gagctcaaga ataacatctc ataagaaac
293427DNAArtificial SequenceDescription of Artificial
Sequence Primer 34ctcgagtgct cacttctctc tcttagg
273512PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 35Cys Asn Asp Asp Thr Leu Leu Gln Gln Ile
Lys Ser 1 5 10
3612PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 36Cys Thr Asp Asp Gly Leu Thr Ala Gln Met Arg Leu
1 5 10 3712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Cys
Thr Asp Glu Ala Leu Leu Thr Gln Leu Lys Asn 1
5 10 3832DNAArtificial SequenceDescription of
Artificial Sequence Primer 38aagcttgatc gtggtcaact tcctctgtta cc
323927DNAArtificial SequenceDescription of
Artificial Sequence Primer 39gagctcagaa gaagtataaa aaccatc
274027DNAArtificial SequenceDescription of
Artificial Sequence Primer 40ctcgagtgct cacttctctc tcttagg
274129DNAArtificial SequenceDescription of
Artificial Sequence Primer 41gagctcaaga ataacatctc ataagaaac
294230DNAArtificial SequenceDescription of
Artificial Sequence Primer 42ctcgagctaa actccattcg ctgacttcgc
304329DNAArtificial SequenceDescription of
Artificial Sequence Primer 43gagctctagt aaatataaga gtgtcttgc
2944353PRTUnknownFungi sequence 44Met Ala Asp
Asn Gln Ile Pro Ser Ser Val Ala Asp Ala Val Leu Val 1 5
10 15 Lys Ser Ile Glu Met Pro Glu Gly
Ser Gln Lys Val Glu Glu Leu Asp 20 25
30 Phe Asn Lys Phe Lys Gly Arg Pro Ile Thr Val Asp Asp
Leu Leu Gln 35 40 45
Gly Met Lys His Met Gly Phe Gln Ala Ser Ser Met Cys Glu Ala Val 50
55 60 Arg Ile Ile Asn Glu
Met Arg Ala Tyr Arg Asp Pro Thr Thr Ser Glu 65 70
75 80Lys Thr Thr Ile Phe Leu Gly Tyr Thr Ser
Asn Leu Ile Ser Ser Gly 85 90
95 Leu Arg Gly Thr Leu Arg Tyr Leu Val Gln His Lys His Val Ser
Ala 100 105 110 Ile
Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Phe Ile Lys Cys Leu 115
120 125 Gly Asp Thr Tyr Met Ser
Ser Phe Ser Ala Val Gly Ala Asp Leu Arg 130 135
140 Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Val
Val Pro Asn Ser Asn 145 150 155
160Tyr Cys Ala Phe Glu Asp Trp Val Val Pro Ile Leu Asp Lys Met Leu
165 170 175 Glu Glu
Gln Glu Ala Ser Arg Gly Thr Glu Asn Glu Ile Asn Trp Thr 180
185 190 Pro Ser Lys Val Ile His Arg
Leu Gly Lys Glu Ile Asn Asp Glu Arg 195 200
205 Ser Val Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro
Val Phe Cys Pro 210 215 220
Ala Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Thr Phe
225 230 235 240Lys Ala
Ser Pro Glu Gln Leu Arg Ile Asp Ile Val Glu Asp Ile Arg
245 250 255 Lys Ile Asn Thr Ile Ala
Val Arg Ala Lys Arg Ala Gly Met Ile Ile 260
265 270 Leu Gly Gly Gly Ile Val Lys His His Ile
Ala Asn Ala Cys Leu Met 275 280
285 Arg Asn Gly Ala Glu Ser Ala Val Tyr Ile Asn Thr Ala Gln
Glu Phe 290 295 300
Asp Gly Ser Asp Ala Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly 305
310 315 320Lys Ile Lys Val Gly
Ala Asp Ala Val Lys Val Tyr Met Glu Ala Thr 325
330 335 Ala Ala Phe Pro Phe Ile Val Ala Asn Thr
Phe Ala Lys Glu Asp Gly 340 345
350 Leu
45387PRTUnknownYeast sequence 45Met Ser Asp Ile Asn Glu Lys Leu Pro
Glu Leu Leu Gln Asp Ala Val 1 5 10
15 Leu Lys Ala Ser Val Pro Ile Pro Asp Asp Phe Val Lys Val
Gln Gly 20 25 30
Ile Asp Tyr Ser Lys Pro Glu Ala Thr Asn Met Arg Ala Thr Asp Leu
35 40 45 Ile Glu Ala Met Lys
Thr Met Gly Phe Gln Ala Ser Ser Val Gly Thr 50 55
60 Ala Cys Glu Ile Ile Asp Ser Met Arg Ser
Trp Arg Gly Lys His Ile 65 70 75
80Asp Glu Leu Asp Asp His Glu Lys Lys Gly Cys Phe Asp Glu Glu
Gly 85 90 95 Tyr
Gln Lys Thr Thr Ile Phe Met Gly Tyr Thr Ser Asn Leu Ile Ser
100 105 110 Ser Gly Val Arg Glu
Thr Leu Arg Tyr Leu Val Gln His Lys Met Val 115
120 125 Asp Ala Val Val Thr Ser Ala Gly Gly
Val Glu Glu Asp Leu Ile Lys 130 135
140 Cys Leu Ala Pro Thr Tyr Leu Gly Glu Phe Ala Leu Lys
Gly Lys Ser 145 150 155
160Leu Arg Asp Gln Gly Met Asn Arg Ile Gly Asn Leu Leu Val Pro Asn
165 170 175 Asp Asn Tyr Cys
Lys Phe Glu Glu Trp Ile Val Pro Ile Leu Asp Lys 180
185 190 Met Leu Glu Glu Gln Asp Glu Tyr Val
Lys Lys His Gly Ala Asp Cys 195 200
205 Leu Glu Ala Asn Gln Asp Val Asp Ser Pro Ile Trp Thr Pro
Ser Lys 210 215 220
Met Ile Asp Arg Phe Gly Lys Glu Ile Asn Asp Glu Ser Ser Val Leu 225
230 235 240Tyr Trp Ala His Lys
Asn Lys Ile Pro Ile Phe Cys Pro Ser Leu Thr 245
250 255 Asp Gly Ser Ile Gly Asp Met Leu Phe Phe
His Thr Phe Lys Ala Ser 260 265
270 Pro Lys Gln Leu Arg Val Asp Ile Val Gly Asp Ile Arg Lys Ile
Asn 275 280 285 Ser
Met Ser Met Ala Ala Tyr Arg Ala Gly Met Ile Ile Leu Gly Gly 290
295 300 Gly Leu Ile Lys His His
Ile Ala Asn Ala Cys Leu Met Arg Asn Gly 305 310
315 320Ala Asp Tyr Ala Val Tyr Ile Asn Thr Gly Gln
Glu Tyr Asp Gly Ser 325 330
335 Asp Ala Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Lys
340 345 350 Ala Glu
Ala Lys Ser Val Lys Leu Phe Ala Asp Val Thr Thr Val Leu 355
360 365 Pro Leu Ile Val Ala Ala Thr
Phe Ala Ser Gly Lys Pro Ile Lys Lys 370 375
380 Val Lys Asn
385 46370PRTUnknownArchaebacterial sequence 46Met
Arg Asp Ile Lys Asp Asn Pro Ile Arg Arg Gly Ile Ala Glu Gln 1
5 10 15 Ser Glu Ala Met His Pro
Gly Tyr Thr Asn Arg Ala Lys Pro Tyr Gly 20
25 30 Cys Lys Arg Asp Pro Lys Asp Ile Val Leu Lys
Glu Ser Glu Asp Ile 35 40 45
Glu Gly Ile Ala Ile Glu Gly Pro Trp Leu Glu Asp Asp Ile Ser Leu
50 55 60 Glu Glu Ile
Ile Lys Lys Tyr Tyr Leu Lys Ile Gly Phe Gln Ala Ser 65
70 75 80His Ile Gly Lys Ala Ile Lys Ile
Trp Lys His Ile Glu Glu Lys Arg 85 90
95 Lys Lys Gly Asp Glu Ile Thr Val Phe Phe Gly Tyr Thr
Ser Asn Ile 100 105 110
Val Ser Ser Gly Leu Arg Glu Ile Ile Ala Tyr Leu Val Lys His Lys
115 120 125 Lys Ile Asp Ile
Ile Val Thr Thr Ala Gly Gly Val Glu Glu Asp Phe 130
135 140 Ile Lys Cys Leu Lys Pro Phe Ile
Leu Gly Asp Trp Glu Val Asp Gly 145 150
155 160Lys Met Leu Arg Glu Lys Gly Ile Asn Arg Ile Gly
Asn Ile Phe Val 165 170
175 Pro Asn Asp Arg Tyr Ile Ala Phe Glu Glu Tyr Met Met Glu Phe Phe
180 185 190 Glu Glu Ile
Leu Asn Leu Gln Arg Glu Thr Gly Lys Ile Ile Thr Ala 195
200 205 Ser Glu Phe Cys Tyr Lys Leu Gly
Glu Phe Met Asp Lys Lys Leu Lys 210 215
220 Ser Lys Glu Lys Glu Lys Ser Ile Leu Tyr Trp Ala Tyr
Lys Asn Asn 225 230 235
240Ile Pro Ile Phe Cys Pro Ala Ile Thr Asp Gly Ser Ile Gly Asp Met
245 250 255 Leu Tyr Phe Phe
Lys Lys Tyr Asn Lys Asp Glu Glu Leu Lys Ile Asp 260
265 270 Val Ala Asn Asp Ile Val Lys Leu Asn
Asp Ile Ala Ile Asn Ser Lys 275 280
285 Glu Thr Ala Cys Ile Val Leu Gly Gly Ser Leu Pro Lys His
Ser Ile 290 295 300
Ile Asn Ala Asn Leu Phe Arg Glu Gly Thr Asp Tyr Ala Ile Tyr Val 305
310 315 320Thr Thr Ala Leu Pro
Trp Asp Gly Ser Leu Ser Gly Ala Pro Pro Glu 325
330 335 Glu Gly Val Ser Trp Gly Lys Ile Gly Ala
Lys Ala Asp Tyr Val Glu 340 345
350 Ile Trp Gly Asp Ala Thr Ile Ile Phe Pro Leu Leu Val Tyr Cys
Val 355 360 365 Met
Lys
3704723DNAArtificial SequenceDescription of Artificial Sequence Primer
47gcngarttyg ayggntccga yca
234830DNAArtificial SequenceDescription of Artificial Sequence Primer
48ccgagctcct gttaccaaaa aatctgtacc
304936DNAArtificial SequenceDescription of Artificial Sequence Primer
49acctcgagcg gccgcagaag aagtataaaa accatc
365030DNAArtificial SequenceDescription of Artificial Sequence Primer
50cgtcgacgat atctcttttt atattcaaac
305132DNAArtificial SequenceDescription of Artificial Sequence 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 Cys 1
5 10 15 Arg Phe Glu Asp
Trp Val Met Pro Ile Leu Asp Thr Met Leu Glu Glu 20
25 30 Gln Glu Ala Cys Lys Gly Ser Gly Glu
Ala Ile His Trp Thr Pro Ser 35 40
45 Lys Ile Ile Asn Arg Leu Gly Lys Glu Val Asn Asp Glu Ser
Ser Val 50 55 60
Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro Val Phe Cys Pro Ala Leu 65
70 75 80Thr Asp Gly Ser Leu
Gly Asp Met Leu Tyr Phe His Thr Phe Lys Ser 85
90 95 Ser Pro Gln Gln Leu Arg Val Asp Ile Val
Glu Asp Ile Arg Lys Ile 100 105
110 Asn Thr Leu Ala Val Arg Ala Lys Arg Thr Gly Met Ile Ile Leu
Gly 115 120 125 Gly
Gly Ile Val Lys His His Ile Ala Asn Ala Asn Leu Met Arg Asn 130
135 140 Gly Ala Glu Ser Ala Val
Tyr Ile Asn Thr Ala Glu Phe Asp Gly Ser 145 150
155 160Asp
54861DNAArabidopsis 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
Ser 1 5 10 15 Lys
Thr Tyr Pro Gln Ser Ala Gly Asn Ile Arg Lys Gly Gly His Ile
20 25 30 Val Ile Lys Asn Arg Pro
Cys Lys Val Val Glu Val Ser Thr Ser Lys 35 40
45 Thr Gly Lys His Gly His Ala Lys Cys His Phe
Val Ala Ile Asp Ile 50 55 60
Phe Thr Ala Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys
65 70 75 80Asp Val Pro
His Val Asn Arg Val Asp Tyr Gln Leu Ile Asp Ile Thr 85
90 95 Glu Asp Gly Phe Val Ser Leu Leu
Thr Asp Ser Gly Gly Thr Lys Asp 100 105
110 Asp Leu Lys Leu Pro Thr Asp Asp Gly Leu Thr Ala Gln
Met Arg Leu 115 120 125
Gly Phe Asp Glu Gly Lys Asp Ile Val Val Ser Val Met Ser Ser Met
130 135 140 Gly Glu Glu
Gln Ile Cys Ala Val Lys Glu Val Gly Gly Gly Lys 145
150 155 56698DNALycopersicon
esculentumCDS(52)..(528) 56cttcctgaat ttttctcctt ctccttctcc gttcaatcga
atttttcagc c atg tct 57
Met Ser
1 gac 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 15 act 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
30 atc 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 65 act
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 80 gtg 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
95 gat 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 100 105 110
ctc 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
140 145 gag 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 150 155
attggatgga 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 Ala 1 5
10 15 Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr
Ile Arg Lys Asn Gly Tyr 20 25
30 Ile Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser
Thr Ser 35 40 45
Lys Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50
55 60 Ile Phe Thr Gly
Lys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn 65 70
75 80 Cys Asp Val Pro His Val Asn Arg
Thr Asp Tyr Gln Leu Ile Asp Ile 85 90
95 Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Asn
Gly Asn Thr Lys 100 105 110
Asp Asp Leu Arg Leu Pro Thr Asp Glu Asn Leu Leu Ser Leu Ile Lys
115 120 125 Asp Gly
Phe Ala Glu Gly Lys Asp Leu Val Val Ser Val Met Ser Ala 130
135 140 Met Gly Glu Glu Gln Ile
Asn Ala Leu Lys Asp Ile Gly Pro Lys 145 150
155 58158PRTArabidopsis thaliana 58Met Ser
Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser 1
5 10 15 Lys Thr Tyr Pro Gln Gln Ala
Gly Thr Ile Arg Lys Asn Gly Tyr Ile 20 25
30 Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val
Ser Thr Ser Lys 35 40 45
Thr Gly Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile
50 55 60 Phe Thr Ser Lys
Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys 65 70
75 80Asp Val Pro His Val Asn Arg Thr Asp
Tyr Gln Leu Ile Asp Ile Ser 85 90
95 Glu Asp Gly Tyr Val Ser Leu Leu Thr Asp Asn Gly Ser Thr
Lys Asp 100 105 110
Asp Leu Lys Leu Pro Asn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser
115 120 125 Gly Phe Asp Asp
Gly Lys Asp Leu Val Val Ser Val Met Ser Ala Met 130
135 140 Gly Glu Glu Gln Ile Asn Ala Leu
Lys Asp Ile Gly Pro Lys 145 150
155 59159PRTArabidopsis thaliana 59Met Ser Asp Asp Glu His His
Phe Glu Ala Ser Glu Ser Gly Ala Ser 1 5
10 15 Lys Thr Tyr Pro Gln Ser Ala Gly Asn Ile Arg Lys
Gly Gly His Ile 20 25 30
Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys
35 40 45 Thr Gly Lys His
Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50
55 60 Phe Thr Ala Lys Lys Leu Glu Asp Ile
Val Pro Ser Ser His Asn Cys 65 70 75
80Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Ile Asp
Ile Thr 85 90 95
Glu Asp Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Gly Thr Lys Asp
100 105 110 Asp Leu Lys Leu Pro
Thr Asp Asp Gly Leu Thr Ala Gln Met Arg Leu 115
120 125 Gly Phe Asp Glu Gly Lys Asp Ile Val
Val Ser Val Met Ser Ser Met 130 135
140 Gly Glu Glu Gln Ile Cys Ala Val Lys Glu Val Gly Gly
Gly Lys 145 150 155
60158PRTArabidopsis thaliana 60Met Ser Asp Asp Glu His His Phe Glu Ser
Ser Asp Ala Gly Ala Ser 1 5 10
15 Lys Thr Tyr Pro Gln Gln Ala Gly Asn Ile Arg Lys Gly Gly His
Ile 20 25 30 Val
Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35
40 45 Thr Gly Lys His Gly His
Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55
60 Phe Thr Ser Lys Lys Leu Glu Asp Ile Val Pro
Ser Ser His Asn Cys 65 70 75
80Asp Val Pro His Val Asn Arg Val Asp Tyr Gln Leu Ile Asp Ile Ser
85 90 95 Glu Asp Gly
Phe Val Ser Leu Leu Thr Asp Asn Gly Ser Thr Lys Asp 100
105 110 Asp Leu Lys Leu Pro Thr Asp Glu
Ala Leu Leu Thr Gln Leu Lys Asn 115 120
125 Gly Phe Glu Glu Gly Lys Asp Ile Val Val Ser Val Met
Ser Ala Met 130 135 140
Gly Glu Glu Gln Met Cys Ala Leu Lys Glu Val Gly Pro Lys 145
150 155 61477DNAArabidopsis
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 15 aag 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 30
gtc 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 45 aca 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
80 gat 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 95
gag 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
100 105 110 gac 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
120 125 ggt 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 150 155 tagctgcaga
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 Ala 1 5 10
15 Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys
Gly Gly His 20 25 30
Ile Val Ile Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser
35 40 45 Lys Thr Gly Lys
His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50
55 60 Ile Phe Thr Gly Lys Lys Leu Glu
Asp Ile Val Pro Ser Ser His Asn 65 70
75 80 Cys Asp Val Pro His Val Asn Arg Thr Asp Tyr Gln
Leu Ile Asp Ile 85 90
95 Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Asn Gly Asn Thr Lys
100 105 110 Asp Asp
Leu Arg Leu Pro Thr Asp Asp Thr Leu Leu Ala Gln Val Lys 115
120 125 Asp Gly Phe Ala Glu Gly
Lys Asp Leu Val Leu Ser Val Met Ser Ala 130 135
140 Met Gly Glu Glu Gln Ile Cys Gly Ile Lys
Asp Ile Gly Pro Lys 145 150 155
66658DNABrassica 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 15 aaa 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
30 gtc 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 45 act 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 60 ttc 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 95
gag 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
100 105 110 gac 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
120 125 gga 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
140 gga 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 150 155
taaaacccat 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 Ser 1 5 10
15 Lys Thr Tyr Pro Gln Gln Ala Gly Asn Ile Arg Lys Gly Gly His
Ile 20 25 30 Val
Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys 35
40 45 Thr Gly Lys His Gly
His Ala Lys Cys His Phe Val Ala Ile Asp Ile 50 55
60 Phe Thr Ala Lys Lys Leu Glu Asp Ile
Val Pro Ser Ser His Asn Cys 65 70 75
80 Asp Val Pro His Val Asn Arg Ile Asp Tyr Gln Leu Ile
Asp Ile Ser 85 90 95
Glu Asn Gly Phe Val Ser Leu Leu Thr Asp Ser Gly Gly Thr Lys Asp
100 105 110 Asp Leu Lys
Leu Pro Thr Asp Asp Asn Leu Ser Ala Leu Met Lys Ser 115
120 125 Gly Phe Glu Glu Gly Lys Asp
Val Val Val Ser Val Met Ser Ser Met 130 135
140 Gly Glu Glu Gln Ile Cys Ala Val Lys Glu Val
Gly Gly Gly Lys 145 150 155
688PRTMycosphaerella fijiensis 68Gly Leu Asn Arg Ile Gly Asn
Leu 1 5
698PRTMycosphaerella fijiensis 69Ala Glu Phe Asp Gly Ser Asp Gln
1 5 701335DNABrassica
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 15 tcc 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 30 aac 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 45
ggc 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 80 agt 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
95 atc 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 100 105 110
act 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
115 120 125 aca 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
155 160 ttg 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
175 ttt 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 180 185 190 aaa 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
200 205 ggg 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
240 gat 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
245 250 255 gtt 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
265 270 cca 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
285 cac 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
310 315 320 cct 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
330 335 gtc 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
350 gaa 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 355 360 365 taagcccaag
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 Lys 1 5 10
15 Glu Ser Glu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly Tyr
Asp 20 25 30 Phe
Asn Gln Gly Val Asn Tyr Pro Lys Leu Leu Arg Ser Met Leu Thr 35
40 45 Thr Gly Phe Gln Ala
Ser Asn Leu Gly Asp Val Ile Asp Val Val Asn 50 55
60 Gln Met Leu Glu Trp Arg Leu Ser Asp
Glu Thr Ile Ala Pro Glu Asp 65 70 75
80 Cys Ser Glu Glu Glu Lys Asp Pro Ala Tyr Arg Glu Ser
Val Lys Cys 85 90 95
Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Val Arg
100 105 110 Glu Thr Ile
Arg Tyr Leu Val Gln His His Met Val Asp Val Ile Val 115
120 125 Thr Thr Thr Gly Gly Val Glu
Glu Asp Leu Ile Lys Cys Leu Ala Pro 130 135
140 Thr Phe Lys Gly Asp Phe Ser Leu Pro Gly Ala
Tyr Leu Arg Ser Lys 145 150 155
160 Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr
Cys 165 170 175 Lys
Phe Glu Asp Trp Ile Ile Pro Ile Phe Asp Gln Met Leu Lys Glu
180 185 190 Gln Lys Glu Glu Ser
Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg 195
200 205 Leu Gly Lys Glu Ile Asn Asn Glu Ser
Ser Tyr Leu Tyr Trp Ala Tyr 210 215
220 Lys Met Asn Ile Pro Val Phe Cys Arg Gly Leu Thr Asp
Gly Ser Leu 225 230 235
240 Gly Asp Met Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu Val Ile
245 250 255 Asp Val Val
Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His Ala 260
265 270 Thr Pro Arg Lys Thr Gly Met
Ile Ile Leu Gly Gly Gly Leu Pro Lys 275 280
285 His His Ile Cys Asn Ala Asn Met Met Arg Asn
Gly Ala Asp Tyr Ala 290 295 300
Val Phe Ile Asn Thr Gly Gln Glu Phe Asp Gly Ser Asp Ser Gly
Ala 305 310 315 320 Arg
Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly Ser Ala Lys
325 330 335 Thr Val Lys Val Tyr
Cys Asp Ala Thr Ile Ala Phe Pro Leu Leu Val 340
345 350 Ala Glu Thr Phe Ala Ser Lys Arg Glu
Gln Ser Cys Glu His Lys Thr 355 360
365 721862DNAMedicago 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
1 gaa 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
15 gtc 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 30
ggt 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 65 gtc 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
80 gag 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 95 aca 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
105 110 gtt 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
145 gca 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
150 155 160 tca 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
170 175 tat 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
190 aag 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
215 220 225 gca 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
235 240 tca 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
255 att 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 260 265 270 cat
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
300 305 tat 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
320 gga 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 325 330 335 gct 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
345 350 ctg 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 360 365
tgataaaggt 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 His 1 5 10
15 Ser Thr Val Phe Lys Glu Ser Glu Asn Leu Ala Gly Lys Cys Val
Gln 20 25 30 Ile
Glu Gly Tyr Asp Phe Asn Arg Gly Val Asp Tyr Gln Gln Leu Leu 35
40 45 Lys Ser Met Leu Thr
Thr Gly Phe Gln Ala Ser Asn Phe Gly Asp Ala 50 55
60 Val Lys Val Val Asn Gln Met Leu Asp
Trp Arg Leu Val Asp Glu Pro 65 70 75
80 Ile Asp Glu Asp Cys Asp Glu Asp Lys Lys Asp Leu Glu
Tyr Arg Lys 85 90 95
Ser Val Thr Cys Lys Val Phe Leu Gly Phe Thr Ser Asn Leu Ile Ser
100 105 110 Ser Gly Val
Arg Asp Val Val Arg Tyr Leu Cys Gln His His Met Val 115
120 125 His Val Val Val Thr Thr Thr
Gly Gly Ile Glu Glu Asp Leu Ile Lys 130 135
140 Cys Leu Ala Pro Thr Tyr Lys Gly Glu Phe Ser
Leu Pro Gly Ala Tyr 145 150 155
160 Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu Val Pro
Asn 165 170 175 Glu
Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile Phe Asp Gln
180 185 190 Met Leu Lys Glu Gln
Lys Glu Glu Lys Val Leu Trp Thr Pro Ser Lys 195
200 205 Leu Ile Ala Arg Leu Gly Lys Glu Ile
Asn Asn Glu Asn Ser Tyr Leu 210 215
220 Tyr Trp Ala Tyr Lys Asn Asn Ile Pro Val Tyr Cys Pro
Gly Leu Thr 225 230 235
240 Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe His Asn Pro
245 250 255 Gly Leu Ile
Val Asp Ile Val Gln Asp Ile Arg Ala Met Asn Gly Glu 260
265 270 Ala Val His Ala Asn Pro Ser
Lys Thr Gly Met Ile Ile Leu Gly Gly 275 280
285 Gly Leu Pro Lys His His Ile Cys Asn Ala Asn
Met Met Arg Asn Gly 290 295 300
Ala Asp Tyr Ala Val Phe Ile Asn Thr Ala Gln Glu Phe Asp Gly
Ser 305 310 315 320 Asp
Ser Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg
325 330 335 Gly Ser Ala Lys Thr
Val Lys Val His Cys Asp Ala Thr Ile Ala Phe 340
345 350 Pro Leu Leu Val Ala Glu Thr Phe Ala
Ser Arg Thr Ser Pro Leu Asn 355 360
365 741363DNAMusa 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 5 ggg 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
20 aag 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 35 tac
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 70 atc 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 85
gat 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 95 100 tgc 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
110 115 agg 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
150 cca 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
155 160 165 aaa 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
175 180 tgc 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
195 gaa 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
220 225 230 tac 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
240 245 ttg 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
260 att 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 265 270 275 gtg
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
305 310 gct 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
325 gca 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 330 335 340 aag 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
350 355 gta 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 1270Ala gtaatctgct 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 Asp 1 5
10 15 Ala Val Arg Ser Val Val Phe Lys Pro
Ser Val Ser Leu Glu Glu Lys 20 25
30 Arg Phe Pro Arg Val Gln Gly Tyr Asp Phe Asn Arg Gly
Cys Asp Leu 35 40 45
Ile Gly Leu Leu Asp Ser Ile Ser Ser Thr Gly Phe Gln Ala Ser Asn
50 55 60 Leu Gly Asp
Ala Ile Asp Val Ile Asn Gln Met Ile Asp Trp Arg Leu 65
70 75 80 Ser His Asp Ala Pro Thr Glu
Asp Cys Ser Glu Glu Glu Arg Asn Leu 85
90 95 Ala Tyr Arg Gln Ser Val Thr Cys Lys Ile Phe
Leu Gly Phe Thr Ser 100 105
110 Asn Leu Val Ser Ser Gly Ile Arg Glu Ile Ile Arg Phe Leu Val
Gln 115 120 125 His
Arg Met Val Glu Val Leu Val Thr Thr Ala Gly Gly Ile Glu Glu 130
135 140 Asp Leu Ile Lys Cys
Leu Ala Pro Thr Tyr Lys Gly Asp Phe Ser Leu 145 150
155 160 Pro Gly Ser Tyr Leu Arg Ser Lys Gly
Leu Asn Arg Ile Gly Asn Leu 165 170
175 Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp
Ile Met Pro 180 185 190
Ile Leu Asp Gln Met Leu Leu Glu Gln Thr Thr Glu Asn Val Val Trp
195 200 205 Thr Pro Ser
Lys Val Ile Ala Arg Leu Gly Lys Glu Ile Asn Asp Glu 210
215 220 Ser Ser Tyr Leu Tyr Trp Ala
Tyr Lys Asn Asn Val Ser Val Tyr Cys 225 230
235 240 Pro Ala Leu Thr Asp Gly Ser Leu Gly Asp Met
Leu Tyr Cys His Ser 245 250
255 Val Arg Asn Pro Gly Leu Leu Ile Asp Ile Val Gln Asp Ile Arg
Ala 260 265 270 Met
Asn Gly Glu Ala Val His Val Gly Leu Arg Lys Thr Gly Val Ile 275
280 285 Ile Leu Gly Gly Gly
Leu Pro Lys His His Ile Cys Asn Ala Asn Met 290 295
300 Phe Arg Asn Gly Ala Asp Tyr Ala Val
Tyr Val Asn Thr Ala Gln Glu 305 310 315
320 Phe Asp Gly Ser Asp Ser Gly Ala Glu Pro Asp Glu Ala
Ile Ser Trp 325 330 335
Gly Lys Ile Lys Gly Ser Ala Lys Thr Ile Lys Val His Cys Asp Ala
340 345 350 Thr Ile Ala
Phe Pro Leu Leu Val Ala Ala Thr Phe Ala Arg Lys Phe 355
360 365 Gln Glu Arg Asn Asn Lys Leu
Ala 370 375
761451DNAPopulus 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 10 gta 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
45 ctt 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 60 gat 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 75 gaa 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
90 aga 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
115 120 125 atg 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
135 140 ata 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
155 gct 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 160 165 170 cct
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
200 205 tct 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
220 tac 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 225 230 235 tta 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
245 250 aac 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
285 gga 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
290 295 300 aac 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
310 315 ggg 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
330 ata 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
355 360 365 ttc tgc agc agt
act caa agc tagggctgtg tgcagttctt ggccagaaaa 1156Phe Cys Ser Ser
Thr Gln Ser 370
ttgattcatt 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 Arg 1 5
10 15 Thr Thr Val Phe Lys Glu Ser Glu
Ala Leu Asp Gly Lys Cys Ile Lys 20 25
30 Ile Glu Gly Tyr Asp Phe Asn Gln Gly Val Asn Tyr
Ser Lys Leu Leu 35 40 45
Lys Ser Met Val Ser Thr Gly Phe Gln Ala Ser Asn Leu Gly Asp Ala
50 55 60 Ile Gln Val
Val Asn Asn Met Leu Asp Trp Arg Leu Ala Asp Glu Glu 65
70 75 80 Ile Thr Glu Asp Cys Ser Asp
Glu Glu Arg Glu Leu Ala Tyr Arg Glu 85
90 95 Ser Val Arg Cys Lys Leu Phe Leu Gly Phe Thr
Ser Asn Leu Val Ser 100 105
110 Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln His His Met
Val 115 120 125 Asp
Val Val Val Thr Thr Ala Gly Gly Ile Glu Glu Asp Leu Ile Lys 130
135 140 Cys Leu Ala Pro Thr
Tyr Lys Gly Asp Phe Ser Leu Pro Gly Ala Gln 145 150
155 160 Leu Arg Ser Lys Gly Leu Asn Arg Ile
Gly Asn Leu Leu Val Pro Asn 165 170
175 Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile
Phe Asp Gln 180 185 190
Met Leu Lys Glu Gln Ile Glu Glu Asn Ile Thr Trp Thr Pro Ser Lys
195 200 205 Leu Ile Ala
Arg Met Gly Lys Glu Ile Asn Asn Glu Ser Ser Tyr Leu 210
215 220 Tyr Trp Ala Tyr Lys Asn Asp
Ile Pro Val Phe Cys Pro Gly Leu Thr 225 230
235 240 Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His
Ser Phe His Asn Pro 245 250
255 Gly Leu Ile Val Ala Ile Val Gln Asp Ile Arg Ala Met Asn Gly
Glu 260 265 270 Ala
Val His Ala Ser Pro Arg Lys Thr Gly Ile Ile Ile Leu Gly Gly 275
280 285 Gly Leu Pro Lys His
His Ile Cys Asn Ala Asn Met Met Arg Asn Gly 290 295
300 Ala Asp Tyr Ala Val Phe Ile Asn Thr
Ala Gln Glu Phe Asp Gly Ser 305 310 315
320 Asp Ser Gly Ala His Pro Asp Glu Ala Val Ser Trp Gly
Lys Ile Arg 325 330 335
Gly Ser Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe
340 345 350 Pro Leu Leu
Val Ala Glu Thr Phe Ala Pro Arg Arg Asn Arg Phe Cys 355
360 365 Ser Ser Thr Gln Ser
370
781488DNAArabidopsis 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 Primer
81gggagggact agtgtgcacg cc
228238DNAArtificial SequenceDescription of Artificial Sequence 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 Primer
85ctcgagaaga ataacatctc ataagaaac
298629DNAArtificial SequenceDescription of Artificial Sequence 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 Ala 1 5 10
15 Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser His Pro Asn
Leu Tyr 20 25 30
Leu Thr Gln Trp Phe
35
9152PRTLycopersicon esculentum 91Gly Ala Arg Pro Asp Glu Ala Val Ser Trp
Gly Lys Ile Arg Gly Gly 1 5 10
15 Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile Ala Phe
Pro Ile 20 25 30
Leu Val Ala Glu Thr Phe Ala Ala Lys Ser Lys Glu Phe Ser Gln Ile
35 40 45 Arg Cys Gln Val
50
92193PRTArabidopsis thaliana 92Gly
Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala Pro Thr Phe Lys 1
5 10 15 Gly Asp Phe Ser Leu
Pro Gly Ala Tyr Leu Arg Ser Lys Gly Leu Asn 20
25 30 Arg Ile Gly Asn Leu Leu Val Pro Asn
Asp Asn Tyr Cys Lys Phe Glu 35 40
45 Asp Trp Ile Ile Pro Ile Phe Asp Glu Met Leu Lys Glu
Gln Lys Glu 50 55 60
Glu Asn Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg Leu Gly Lys 65
70 75 80 Glu Ile Asn Asn
Glu Ser Ser Tyr Leu Tyr Trp Ala Tyr Lys Met Asn 85
90 95 Ile Pro Val Phe Cys Pro Gly Leu
Thr Asp Gly Ser Leu Arg Asp Met 100 105
110 Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu Ile
Ile Asp Val Val 115 120 125
Gln Asp Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Asn Pro Lys
130 135 140 Lys Thr
Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile 145
150 155 160 Cys Asn Ala Asn Met Met
Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile 165
170 175 Asn Thr Gly Gln Glu Phe Asp Gly Ser Asp
Ser Gly Ala Arg Pro Asp 180 185
190 Glu
93174PRTDianthus caryophyllus 93Arg Arg Ser Ile Lys Cys Leu Ala
Pro Thr Phe Lys Gly Asp Phe Ala 1 5 10
15 Leu Pro Gly Ala Gln Leu Arg Ser Lys Gly Leu Asn
Arg Ile Gly Asn 20 25 30
Leu Leu Val Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile
35 40 45 Pro Ile Leu
Asp Lys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile Leu 50
55 60 Trp Thr Pro Ser Lys Leu Ile
Gly Arg Leu Gly Arg Glu Ile Asn Asp 65 70
75 80 Glu Ser Ser Tyr Leu Tyr Trp Ala Phe Lys Asn
Asn Ile Pro Val Phe 85 90
95 Cys Pro Gly Leu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe
His 100 105 110 Ser
Phe Arg Asn Pro Gly Leu Ile Ile Asp Val Val Gln Asp Ile Arg 115
120 125 Ala Val Asn Gly Glu
Ala Val His Ala Ala Pro Arg Lys Thr Gly Met 130 135
140 Ile Ile Leu Gly Gly Gly Leu Pro Lys
His His Ile Cys Asn Ala Asn 145 150 155
160 Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn
Thr 165 170
9422PRTArabidopsis thaliana 94Cys Lys Val Val Glu Val Ser Thr Ser Lys
Thr Gly Lys His Gly His 1 5 10
15 Ala Lys Cys His Phe Val
20 9522PRTArabidopsis thaliana 95Cys Lys Val Val
Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His 1 5
10 15 Ala Lys Cys His Phe Val
20 9622PRTArabidopsis
thaliana 96Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly
His 1 5 10 15 Ala
Lys Cys His Phe Val
20 9722PRTBrassica napus 97Cys Lys Val Val Glu Val Ser Thr Ser Lys
Thr Gly Lys His Gly His 1 5 10
15 Ala Lys Cys His Phe Val
20 9822PRTDianthus caryophyllus 98Cys Lys Val Val
Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His 1 5
10 15 Ala Lys Cys His Phe Val
20 9922PRTLycopersicon
esculentum 99Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly
His 1 5 10 15 Ala
Lys Cys His Phe Val
20 10022PRTLycopersicon esculentum 100Cys Lys Val Val Glu Val Ser
Thr Ser Lys Thr Gly Lys His Gly His 1 5
10 15 Ala Lys Cys His Phe Val
20 10122PRTLycopersicon esculentum
101Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His 1
5 10 15 Ala Lys Cys His
Phe Val 20
10222PRTMedicago sativa 102Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr
Gly Lys His Gly His 1 5 10
15 Ala Lys Cys His Phe Val
20 10322PRTMedicago sativa 103Cys Lys Val Val Glu Val
Ser Thr Ser Lys Thr Gly Lys His Gly His 1 5
10 15 Ala Lys Cys His Phe Val
20 10422PRTMedicago sativa 104Cys
Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His 1
5 10 15 Ala Lys Cys His Phe Val
20
10522PRTLactuca sativa 105Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly
Lys His Gly His 1 5 10
15 Ala Lys Cys His Phe Val
20 10622PRTUnknownTree sequence peptide 106Cys Lys Val
Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His 1 5
10 15 Ala Lys Cys His Phe Val
20 10722PRTUnknownTree
sequence peptide 107Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys
His Gly His 1 5 10 15
Ala Lys Cys His Phe Val
20 10822PRTUnknownTree sequence peptide 108Cys Lys Val Val Glu
Val Ser Thr Ser Lys Thr Gly Lys His Gly His 1 5
10 15 Ala Lys Cys His Phe Val
20 10922PRTUnknownTree
sequence peptide 109Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys
His Gly His 1 5 10 15
Ala Lys Cys His Phe Val
20 11066DNAArabidopsis 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
6612266DNAUnknownTree
oligonucleotide sequence 122tgcaaggtcg tggaggtttc aacctctaaa actggcaagc
atggccatgc taaatgtcac 60tttgtt
6612366DNAUnknownTree oligonucleotide sequence
123tgcaaggttg ttgaggtttc cacctcaaag acaggcaagc acggacatgc taagtgccac
60tttgtg
6612466DNAUnknownTree oligonucleotide sequence 124tgcaaggttg tggaggtttc
tacctctaaa actggcaagc acggccatgc caaatgtcac 60tttgtt
6612566DNAUnknownTree
oligonucleotide 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 15 aaa 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 30
gtt 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 45 act 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
80 gat 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 95
gaa 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
100 105 110 gac 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
120 125 gga 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 Ala 1 5 10
15 Ser Lys Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg
Lys Asn Gly Tyr 20 25 30
Ile Val Ile Lys Gly Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser
35 40 45 Lys Thr Gly
Lys His Gly His Ala Lys Cys His Phe Val Ala Ile Asp 50
55 60 Ile Phe Asn Ala Lys Lys Leu
Glu Asp Ile Val Pro Ser Ser His Asn 65 70
75 80 Cys Asp Val Pro His Val Asn Arg Thr Asp Tyr
Gln Leu Ile Asp Ile 85 90
95 Ser Glu Asp Gly Phe Val Ser Leu Leu Thr Glu Asn Gly Asn Thr
Lys 100 105 110 Asp
Asp Leu Arg Leu Pro Thr Asp Asp Thr Leu Leu Asn Gln Val Lys 115
120 125 Gly Gly Phe Glu Glu
Gly Lys Asp Leu Val Leu Ser Val Met Ser Ala 130 135
140 Met Gly Glu Glu Gln Ile Cys Ala Val
Lys Asp Ile Gly Thr Lys Thr 145 150 155
160 12825PRTArtificial SequenceDescription of Artificial
Sequence Primer 128Gly Ala Ala Gly Cys Thr Cys Gly Ala Gly Gly Cys Thr
Gly Cys Ala 1 5 10 15
Ala Cys Cys Ala Thr Gly Thr Cys Cys
20 2512926DNAArtificial SequenceDescription of
Artificial Sequence 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 Primer 133gcgctcgagc tatgtctgat gaagaacacc
3013428DNAArtificial SequenceDescription of
Artificial Sequence 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 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 Primer 146aagctcgaga
tgtcggacga agagcacc
2814728DNAArtificial SequenceDescription of Artificial Sequence 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 Primer
151cgactcgagc agccatgtct gacgagg
2715229DNAArtificial SequenceDescription of Artificial Sequence 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 Primer
156cactgctcac tagtttgatg gc
2215738DNAArtificial SequenceDescription of Artificial Sequence 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 Primer
160ggnttraayc gnathggnaa ytt
2316123DNAArtificial SequenceDescription of Artificial Sequence 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 15
cgc 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 30 cag 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
45 aaa 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 60
tac 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 95 tca 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
110 aac 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 115 120 125
ggc 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
135 140 ggc 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 Thr 164162PRTMycosphaerella
fijiensis 164Gly Leu Asn Arg Ile Gly Asn Phe Leu Val Pro Asn Asp Asn Tyr
Cys 1 5 10 15 Arg
Phe Glu Asp Trp Val Met Pro Ile Leu Asp Thr Met Leu Glu Glu
20 25 30 Gln Glu Ala Cys Lys
Gly Ser Gly Glu Ala Ile His Trp Thr Pro Ser 35
40 45 Lys Ile Ile Asn Arg Leu Gly Lys Glu
Val Asn Asp Glu Ser Ser Val 50 55
60 Tyr Tyr Trp Ala Trp Lys Asn Asp Ile Pro Val Phe Cys
Pro Ala Leu 65 70 75
80 Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Thr Phe Lys Ser
85 90 95 Ser Pro Gln
Gln Leu Arg Val Asp Ile Val Glu Asp Ile Arg Lys Ile 100
105 110 Asn Thr Leu Ala Val Arg Ala
Lys Arg Thr Gly Met Ile Ile Leu Gly 115 120
125 Gly Gly Ile Val Lys His His Ile Ala Asn Ala
Asn Leu Met Arg Asn 130 135 140
Gly Ala Glu Ser Ala Val Tyr Ile Asn Thr Ala Pro Asn Ser Thr
Asp 145 150 155 160 Pro
Thr
165214DNAMycosphaerella fijiensis 165aatcgaattc ccgcggccgc catggcggcc
gggagcatgc gacgtcgggc ccaattcgcc 60ctatagtgag tcgtattaca attcactggc
cgtcgtttta caacgtcgtg actgggaaaa 120cctggcggta cccaacttaa tcgccttgca
gcacatcccc ctttcgccag ctggcgtaat 180agcgaagagg cccgcaccga tcgcccttcc
aaca 21416622PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
motif 166Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly Lys His Gly His
1 5 10 15 Ala Lys Cys
His Phe Val 20
16722PRTArtificial SequenceDescription of Artificial Sequence Synthetic
amino acid motif 167Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr Gly
Xaa His Gly His 1 5 10
15 Ala Lys Cys His Phe Val
20
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