CN1329619A - Hypersensitive response elicitor fragments which are active but do not elicit hypersensitive response - Google Patents

Hypersensitive response elicitor fragments which are active but do not elicit hypersensitive response Download PDF

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CN1329619A
CN1329619A CN99814028A CN99814028A CN1329619A CN 1329619 A CN1329619 A CN 1329619A CN 99814028 A CN99814028 A CN 99814028A CN 99814028 A CN99814028 A CN 99814028A CN 1329619 A CN1329619 A CN 1329619A
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Z·－M·魏
H·范
J·L·尼格梅耶
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EDEN BIASCIENCE CORP
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Abstract

The present invention is directed to isolated active fragments of a hypersensitive response elicitor protein or polypeptide which fragment does not elicit a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode such fragments. Isolated fragments of hypersensitive response elicitor proteins or polypeptides in accordance with the present invention and the isolated DNA molecules that encode them have the following activities: imparting disease resistance to plants, enhancing plant growth, and/or controlling insects on plants. This can be achieved by applying the fragments of a hypersensitive response elicitor in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding the fragment can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.

Description

Activity is arranged but do not bring out anaphylactoid anaphylaxis exciton fragment
It is the rights and interests of 60/103,050 U.S. Provisional Patent Application that the application requires on October 5th, 1998 application, sequence number.
Invention field
The present invention relates to the active fragments of anaphylaxis exciton, described fragment is not brought out anaphylaxis.
Background of invention
Interaction between bacterial pathogens and their plant host generally belongs to two classes: (1) affinity is done (pathogenic agent-host) mutually, causes intercellular bacterial growth, symptom in the host plant to take place and the disease generation; (2) non-affinity is made (pathogenic agent-non-host) mutually, causes anaphylaxis, and a kind of non-affinity of specific type produces mutually, does not have and carries out venereal disease evil symptom.During the affinity of host plant was done mutually, bacterial population increased significantly and carrying out property symptom produces.During non-affinity was done mutually, bacterial population did not increase, and carrying out property symptom does not produce.
Anaphylaxis is with effective defence relevant a kind of quick, the local necrosis (Kiraly of plant at many pathogenic agent, Z., " defence that the invader triggers: supersensitivity; " Plant Disease:AnAdvanced Treatise, the 5th volume, J.G.Horsfall and E.B.Cowling write, 201-224 page or leaf, Academic Press New York (1980); Klement, Z., " supersensitivity, " Phytopathogenic Prokaryotes.The 2nd volume, M.S.Mount and G.H.Lacy write, 149-177 page or leaf, Academic Press, New York (1982)).If with high density (〉=10 7The pathogenic agent of the limited host range of cell/ml) is as pseudomonas syringae (Pseudomonassyrngae) or separate the blade that starch Erwinia (Erwinia amylovora) is soaked into non-host plant, then observe anaphylaxis (only in isolating vegetable cell, the producing) (Klement that brings out as the bacterium of tissue collapse easily in necrosis under the inoculum of lower level, Z., " causing the pathogenic rapid detection of phytopathy pseudomonas, " Nature 199:299-300; Klement etc., " phytopathogenic bacterium inductive anaphylaxis in the tobacco leaf, " Phytopathology 54:474-477 (1963); Tumer etc., " plant and participate in quantitative relation between the anaphylactoid bacterial cell, " Phytopathology 64:885-890 (1974); Klement, Z., " supersensitivity, " Phytopathogenic Prokaryotes → the 2nd volume, M.S.Mount and G.H.Lacy write, 149-177 page or leaf, Academic Press, New Yorlk (1982)).In non-host, bring out anaphylactoid ability and in the host morbific ability be associated.As Klement, Z. described (" supersensitivity; " Phytopathogenic Prokaryotes, the 2nd volume, M.S.Mount and GH.Lacy write, the 149-177 page or leaf, Academic Press, New York), these pathogenic agent also cause on the physiology in they and affinity host's mutual work and similarly necrose, although this downright bad the delay takes place.In addition, produce the collaborating genes (Lindgren that anaphylaxis or morbific ability depend on one group of called after hrp, P.B. etc., " pseudomonas syringae ' phaseolicola ' cause a disease the supersensitivity of pathogenic and non-host plant of mutation (Pseudomonas syringae pv. ' phaseolicola ') gene cluster control bean plant, " J Bacteriol168:512-22 (1986); Willis, D.K. etc., " the hrp gene of phytopathogenic bacterium, " Mol.Plant-Microbe Interact.4:132-138 (1991)).Therefore, anaphylaxis may keep the clue of this two aspect, the pathogenic basis of plant defense feature bacterium.
The hrp gene is distributed widely in the Gram-negative phytopathogen, wherein they are clusters, conservative and be interchangeable (Willis in some cases, D.K. etc., " the hrp gene of phytopathogenic bacterium, " Mol.Plant-Microbe Interact.4:132-138 (1991); Bonas, U., " the hrp gene of phytopathogenic bacterium; " Current Topics in Microbiology andImmunology:Bacterial Pathogenesis of Plants and Animals-Molecular andCellular Mechanisms, J.L.Dangl writes, the 79-98 page or leaf, Springer-Verlag, Berlin (1994)).Several hrp genes encodings are similar to component (the Van Gijsegem etc. that Yersinia (Yersinia), Shigella (Shigella) and Salmonellas (Salmonella spp.) are used for secreting the protein excretion approach of indispensable protein in the Animal diseases, " evolution conservative of the pathogenic determiner in the plant and animal malignant bacteria, " Trends Microbiol.1:175-180 (1993)).Separating the starch Erwinia, in pseudomonas syringae and the aeruginosa eggplant (P.solanacearum), the hrp gene has shown controls generation and the secretion (He that the glycine albumen exciton is rich in anaphylaxis, S.Y. etc., " pseudomonas syringae cloves mutation (the Pseudomonas syrmgae pv.syrmgae) HarpinPss that causes a disease: by the anaphylactoid albumen in secretion of Hrp approach and the inducing plant; " Cell 73:1255-1266 (1993), Wei, Z.-H. etc., " HrpI that separates the starch Erwinia works in secretion Harpin and HrpI is a member of new protein family, " J Bacterial 175:7958-7967 (1993); Arlat, M. etc., " by the Hrp approach secretion albumen that PopAl-the induced hypersensitivity sample reacts on specific petunia genotype of aeruginosa eggplant " EMBO is (1994) J.13:543-553).
In these albumen first is to be found in the bacterium of separating starch Erwinia Ea321-cause rosaceous plant fire blast, and called after harpin (Wei, Z.-M. etc., " Harpin: phytopathogen is separated the anaphylactoid exciton that the starch Erwinia produces, " Science257:85-88 (1992)).Sudden change in the coding hrpN gene has disclosed, and separates the starch Erwinia and brings out anaphylaxis and cause that in highly susceptible the operatic circle the disease symptom all needs hatpin in non-host's tobacco leaf.Aeruginosa eggplant GMI1000 PopAl albumen has similar physical property and also bring out anaphylaxis in tobacco leaf; and tobacco leaf is not the host (Arlat etc. of this bacterial strain; " by the albumen of aeruginosa eggplant Hrp approach secretion PopAl-induced hypersensitivity sample reaction on specific petunia genotype, " EMBO is (1994) J.13:543-53).Yet aeruginosa eggplant popA mutant also brings out anaphylaxis and cause disease in tomato in tobacco.Therefore, in the Gram-negative phytopathogen, these effects of being rich in glycine anaphylaxis exciton can differ widely.
The anaphylaxis exciton with other phytopathogen separates, clones and order-checking.These excitons comprise: chrysanthemum Erwinia (Erwinia chrysanthemi) (Bauer etc., " and chrysanthemum Erwinia HarpinEch: soft rotten pathogenic effects, " MPMI8 (4): 484-91 (1995)); Carrot soft rot Erwinia (Erwinia carotovora) (Cui etc., " the RsmA mutant of carrot soft rot Erwinia carrot soft rot subspecies (Erwinia carotovora subsp.Carotovora) Ecc71 bacterial strain overexpression hrpNEcc and bring out anaphylaxis sample reaction in tobacco leaf, " MPMI 9 (7): 565-73 (1996)); Si Shi Erwinia (Erwinia stewartii) (Ahmad etc., " Hatpin is to Si Shi Erwinia pathogenic optional in corn; " 8th Int ' l.Cong.Molec.Plant-Microb.Inter.1996 14-19 in July day and Ahmad etc., " Harpin is to Si Shi Erwinia pathogenic optional in corn; " Ann.Mtg.Am.Phytopath.Soc.1996 27-31 in July day) and the mutation (Comell Research Foundation, the WO 94/26782 of Inc.) of causing a disease of pseudomonas syringae cloves.
The present invention tries hard to identify the fragment of anaphylaxis exciton albumen or polypeptide, and described fragment is not brought out anaphylaxis, if but and plant use together then be activated.
Summary of the invention
The present invention relates to the isolating fragment of erwinia anaphylaxis exciton albumen or polypeptide, described fragment is not brought out anaphylaxis in plant, if but and plant use together then have the activity of others.The invention also discloses this segmental isolated DNA molecule of coding.
If use together according to the fragment of anaphylaxis exciton of the present invention and plant then have following activity: conferring disease resistance in plants, promote plant-growth and/or pest control.This relates to, and is giving plant or is effectively being given disease resistance, promoted under the condition of plant-growth and/or pest control by the plant that described plant seed is cultivated, and described fragment is applied to plant or plant seed with the form of non-infection.
As described fragment being applied to plant or plant seed, can use transgenic plant or plant seed to give the alternative method of disease resistance, promotion plant-growth and/or pest control to plant.When using transgenic plant, this relates to, the dna molecular transgenic plant transformed of using according to coding anaphylaxis exciton albumen of the present invention or polypeptide fragment is provided, and giving described plant or effectively giving disease resistance, promote to cultivate described plant under the condition of plant-growth and/or pest control by the plant that described plant seed is cultivated.Perhaps, can provide with the such segmental dna molecular transgenic plant transformed seed of coding, and with described planting seed in soil.Giving plant or effectively giving disease resistance by the plant that described plant seed is cultivated, promote breeding plant under the condition of plant-growth and/or pest control then.
The accompanying drawing summary
Fig. 1 shows the albumen of the brachymemma of anaphylaxis exciton albumen or polypeptide.
Fig. 2 shows a series of proteic synthetic oligonucleotide primer things of harpin that are used to make up brachymemma.N represents N-terminal (5 ' district), and C represents C-terminal (3 ' district).Described primer is corresponding to recognition sequence shown in the application number: N1 (SEQ.ID.No.1), N76 (SEQ.ID.No.2), N99 (SEQ.ID.No.3), N105 (SEQ.ID.No.4), N110 (SEQ.ID.No.5), N137 (SEQ.ID.No.6), N150 (SEQ.ID.No.7), N169 (SEQ.ID.No.8), N210 (SEQ ID.No.9), N267 (SEQ.ID.No.10), N343 (SEQ.ID.No.11), C75 (SEQ.ID.No.12), C104 (SEQ.ID.No.13), C168 (SEQ.ID.No.14), C180 (SEQ.ID.No.15), C204 (SEQ.ID.No.16), C209 (SEQ.ID.No.17), C266 (SEQ.ID.No.18), C342 (SEQ.ID.No.19) and C403 (SEQ.ID.No.20).
Detailed Description Of The Invention
The present invention relates to the fragment of the separation of allergic reaction exciton albumen or polypeptide, wherein said fragment is not brought out allergic reaction but is had other activity in plant. The invention also discloses the dna molecular of this fragment of coding and expression system, host cell and the plant that contains this molecule. The invention discloses the purposes of the dna molecular of the purposes of described fragment self and the described fragment of encoding.
Derive from allergic reaction exciton polypeptide or the albumen of various fungus and bacterium pathogen according to the fragment of allergic reaction exciton polypeptide of the present invention or albumen. Such polypeptide or albumen can bring out the local necrosis in the plant tissue of described exciton contact. The example in the suitable bacteria source of polypeptide or albumen exciton comprises the kind (following bacterium for example: separate starch Erwinia, Erwinia chrysanthemi, Si Shi Erwinia, carrot soft rot Erwinia, pseudomonas syringae, aeruginosa eggplant, xanthomonas campestris and their mixture) of Erwinia, pseudomonas and xanthomonas (Xanthomonas).
The example of the fungal source of allergic reaction exciton albumen or polypeptide is Phytophthora (Phytophthora). Suitable Phytophthora bacterial classification comprises phytophthora parasitica (Phytophthora parasitica), hidden ground epidemic disease mould (Phytophthora cryptogea), camphor tree epidemic disease mould (Phytophthora cinnamomi), Phytophthora capsici (Phytophthora capsici), big male epidemic disease mould (phytophthora megasperma) and oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora).
Derive from the anaphylaxis exciton polypeptide of chrysanthemum Erwinia or albumen and have aminoacid sequence: Met Gln Ile Thr Ile Lys Aia His Ile Gly Gly Asp Leu Gly Val Ser1 5 10 15Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Ash Ser Ala Ala Ser Ser corresponding to following SEQ ID.No.21
20??????????????????25??????????????????30Leu?Gly?Ser?Ser?Val?Asp?Lys?Leu?Ser?Ser?Thr?Ile?Asp?Lys?Leu?Thr
35???????????????????40?????????????????45Ser?Ala?Leu?Thr?Ser?Met?Met?Phe?Gly?Gly?Ala?Leu?Ala?Gln?Gly?Leu
50??????????????????55??????????????????60Gly?Ala?Ser?Ser?Lys?Gly?Leu?Gly?Met?Ser?Asn?Gln?Leu?Gly?Gln?Ser65??????????????????70??????????????????75??????????????????80Phe?Gly?Asn?Gly?Ala?Gln?Gly?Ala?Ser?Asn?Leu?Leu?Ser?val?Pro?Lys
85??????????????????90???????????????????95Ser?Gly?Gly?Asp?Ala?Leu?Ser?Lys?Met?Phe?Asp?Lys?Ala?Leu?Asp?Asp
100??????????????????105????????????????110Leu?Leu?Gly?His?Asp?Thr?Val?Thr?Lys?Leu?Thr?Asn?Gln?Ser?Asn?Gln
115?????????????????120??????????????????125Leu?Ala?Asn?Ser?Met?Leu?Asn?Ala?Ser?Gln?Met?Thr?Gln?Gly?Asn?Met
130?????????????????135?????????????????140Asn?Ala?Phe?Gly?Ser?Gly?Val?Asn?Asn?Ala?Leu?Ser?Ser?Ile?Leu?Gly145?????????????????150?????????????????155?????????????????160Asn?Gly?Leu?Gly?Gln?Ser?Met?Ser?Gly?Phe?Ser?Gln?Pro?Ser?Leu?Gly
165??????????????????170?????????????????175Ala?Gly?Gly?Leu?Gln?Gly?Leu?Ser?Gly?Ala?Gly?Ala?Phe?Asn?Gln?Leu
180??????????????????185??????????????????190Gly?Asn?Ala?Ile?Gly?Met?Gly?Val?Gly?Gln?Asn?Ala?Ala?Leu?Ser?Ala
195?????????????????200?????????????????205Leu?Ser?Asn?Val?Ser?Thr?His?Val?Asp?Gly?Asn?Asn?Arg?His?Phe?Val
210?????????????????215?????????????????220Asp?Lys?Glu?Asp?Arg?Gly?Met?Ala?Lys?Glu?Ile?Gly?Gln?Phe?Met?Asp225?????????????????230?????????????????235?????????????????240Gln?Tyr?Pro?Glu?Ile?Phe?Gly?Lys?Pro?Glu?Tyr?Gln?Lys?Asp?Gly?Trp
245?????????????????250?????????????????255Ser?Ser?Pro?Lys?Thr?Asp?Asp?Lys?Ser?Trp?A?la?Lys?Ala?Leu?Ser?Lys
260?????????????????265??????????????????270Pro?Asp?Asp?Asp?Gly?Met?Thr?Gly?Ala?Ser?Met?Asp?Lys?Phe?Arg?Gln
275?????????????????280?????????????????285Ala?Met?Gly?Met?Ile?Lys?Ser?Ala?Va1?Ala?Gly?Asp?Thr?Gly?Asn?Thr
290?????????????????295?????????????????300Asn?Leu?Asn?Leu?Arg?Gly?Ala?Gly?Gly?Ala?Ser?Leu?Gly?Ile?Asp?Ala305?????????????????310?????????????????315?????????????????320Ala?Val?Val?Gly?Asp?Lys?Ile?Ala?Asn?Met?Ser?Leu?Gly?Lys?Leu?Ala
325?????????????????330?????????????????335Asn?Ala
This anaphylaxis exciton polypeptide or proteic molecular weight are that 34kDa; Thermally-stabilised; glycine content are higher than 16%, and are substantially free of halfcystine.SEQ.ID.No.22DNA：CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG 60GCGTTTATGG CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC 120GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATGCAGAT TCAGCCGGGG 180CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG 240TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG 300CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GGCGGGAATG 360ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CGGACGCGCC 420CGATCATTAA GATAAAGGCG GCTTTTTTTA TTGCAAAACG GTAACGGTGA GGAACCGTTT 480CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG 540GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA 600AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC 660TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TGGATAAACT 720GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT 780GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TGGGCCAGTC 840TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCGA 900TGCGTTGTCA AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG ACACCGTGAC 960CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC 1020 CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CCATTCTCGG 1080CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT 1140GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GCATGGGCGT 1200GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA 1260CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA 1320TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA 1380GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GTATGACCGG 1440CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TGGCGGGTGA 1500TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GTATCGATGC 1560GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA 1620ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC 1680TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA 1740ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CATCTTCCTC 1800GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA ACTCGCCGGC 1860CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GCCCCTTTAG 1920CAGATAGATT GCGGTTTCGT AATCAACATG GTAATGCGGT TCCGCCTGTG CGCCGGCCGG 1980GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC 2040AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG 2100GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T 2141
Must explain the anaphylaxis exciton polypeptide of starch Erwinia or albumen by oneself and have aminoacid sequence: Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser1 5 10 15Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln corresponding to following SEQ.ID.No.23
20??????????????????25??????????????????30Asn?Ala?Gly?Leu?Gly?Gly?Asn?Ser?Ala?Leu?Gly?Leu?Gly?Gly?Gly?Asn
35??????????????????40??????????????????45Gln?Asn?Asp?Thr?Val?Asn?Gln?Leu?Ala?Gly?Leu?Leu?Thr?Gly?Met?Met
50??????????????????55??????????????????60Met?Met?Met?Ser?Met?Met?Gly?Gly?Gly?Gly?Leu?Met?Gly?Gly?Gly?Leu65??????????????????70??????????????????75??????????????????80Gly?Gly?Gly?Leu?Gly?Asn?Gly?Leu?Gly?Gly?Ser?Gly?Gly?Leu?Gly?Glu
85??????????????????90??????????????????95Gly?Leu?Ser?Asn?Ala?Leu?Asn?Asp?Met?Leu?Gly?Gly?Ser?Leu?Asn?Thr
100?????????????????105?????????????????110Leu?Gly?Ser?Lys?Gly?Gly?Asn?Asn?Thr?Thr?Ser?Thr?Thr?Asn?Ser?Pro
115?????????????????120?????????????????125Leu?Asp?Gln?Ala?Leu?Gly?Ile?Asn?Ser?Thr?Ser?Gln?Asn?Asp?Asp?Ser
130?????????????????135?????????????????140Thr?Ser?Gly?Thr?Asp?Ser?Thr?Ser?Asp?Ser?Ser?Asp?Pro?Met?Gln?Gln145?????????????????150?????????????????155?????????????????160Leu?Leu?Lys?Met?Phe?Ser?Glu?Ile?Met?Gln?Ser?Leu?Phe?Gly?Asp?Gly
165?????????????????170?????????????????175Gln?Asp?Gly?Thr?Gln?Gly?Ser?Ser?Ser?Gly?Gly?Lys?Gln?Pro?Thr?Glu
180?????????????????185?????????????????190Gly?Glu?Gln?Asn?Ala?Tyr?Lys?Lys?Gly?Val?Thr?Asp?Ala?Leu?Ser?Gly
195?????????????????200?????????????????205Leu?Met?Gly?Asn?Gly?Leu?Ser?Gln?Leu?Leu?Gly?Asn?Gly?Gly?Leu?Gly
210?????????????????215?????????????????220Gly?Gly?Gln?Gly?Gly?Asn?Ala?Gly?Thr?Gly?Leu?Asp?Gly?Ser?Ser?Leu225?????????????????230?????????????????235?????????????????240Gly?Gly?Lys?Gly?Leu?Gln?Asn?Leu?Ser?Gly?Pro?Val?Asp?Tyr?Gln?Gln
245?????????????????250?????????????????255Leu?Gly?Asn?Ala?val?Gly?Thr?Gly?Ile?Gly?Met?Lys?Ala?Gly?Ile?Gln
260?????????????????265?????????????????270Ala?Leu?Asn?Asp?Ile?Gly?Thr?His?Arg?His?Ser?Ser?Thr?Arg?Ser?Phe
275?????????????????280?????????????????285Val?Asn?Lys?Gly?Asp?Arg?Ala?Met?Ala?Lys?Glu?Ile?Gly?Gln?Phe?Met
290?????????????????295?????????????????300Asp?Gln?Tyr?Pro?Glu?Val?Phe?Gly?Lys?Pro?Gln?Tyr?Gln?Lys?Gly?Pro305?????????????????310?????????????????315?????????????????320Gly?Gln?Glu?Val?Lys?Thr?Asp?Asp?Lys?Ser?Trp?Ala?Lys?Ala?Leu?Ser
325?????????????????330?????????????????335Lys?Pro?Asp?Asp?Asp?Gly?Met?Thr?Pro?Ala?Ser?Met?Glu?Gln?Phe?Asn
340?????????????????345?????????????????350Lys?Ala?Lys?Gly?Met?Ile?Lys?Arg?Pro?Met?Ala?Gly?Asp?Thr?Gly?Asn
355?????????????????360?????????????????365Gly?Asn?Leu?Gln?Ala?Arg?Gly?Ala?Gly?Gly?Ser?Ser?Leu?Gly?Ile?Asp
370 375 380Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu385,390 395 these anaphylaxis exciton polypeptide of 400Gly Ala Ala or proteic molecular weight are about 39kDa, 100 ℃ of of of of of .This anaphylaxis of pI and are about 4.3 and heat-resisting at least 10 minutes in exciton polypeptide or albumen are substantially free of halfcystine.Anaphylaxis exciton polypeptide or the albumen that must explain the starch Erwinia by oneself more fully are described in Wei, Z.-M., R.J.Laby, C.H.Zumoff, D.W.Bauer, S.-Y.He, A.Collmer and S.V.Beer, " Harpin-phytopathogen is separated the anaphylactoid exciton that the starch Erwinia produces; " Science 257:85-88 ( 1992 ) , the document is attached to herein by reference.DNASEQ.ID.No.24：AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA 60GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT 120ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG 180GGTGGCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG 240GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG 300GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CCTGGGCGAA 360GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA 420GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GGGTATTAAC 480TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC 540CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGGTGATGGG 600CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC 660GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG 720CTCCTTGGCA AACGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC 780GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG 840TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT 900ATCGGTACGC ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG 960GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC 1020CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC 1080AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC 1140ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC ACGCGGTGCC 1200GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA 1260CTTGGCAAGC TGGGCGCGGC TTAAGCTT 1288
The another kind that must explain the starch Erwinia by oneself may be disclosed in u.s. patent application serial number 09/120; 927 by suitable anaphylaxis exciton, and the document is attached to herein by reference.SEQ.ID.No.25DNA：ATGTCAATTC TTACGCTTAA CAACAATACC TCGTCCTCGC CGGGTCTGTT CCAGTCCGGG 60GGGGACAACG GGCTTGGTGG TCATAATGCA AATTCTGCGT TGGGGCAACA ACCCATCGAT 120CGGCAAACCA TTGAGCAAAT GGCTCAATTA TTGGCGGAAC TGTTAAAGTC ACTGCTATCG 180CCACAATCAG GTAATGCGGC AACCGGAGCC GGTGGCAATG ACCAGACTAC AGGAGTTGGT 240AACGCTGGCG GCCTGAACGG ACGAAAAGGC ACAGCAGGAA CCACTCCGCA GTCTGACAGT 300CAGAACATGC TGAGTGAGAT GGGCAACAAC GGGCTGGATC AGGCCATCAC GCCCGATGGC 360CAGGGCGGCG GGCAGATCGG CGATAATCCT TTACTGAAAG CCATGCTGAA GCTTATTGCA 420CGCATGATGG ACGGCCAAAG CGATCAGTTT GGCCAACCTG GTACGGGCAA CAACAGTGCC 480TCTTCCGGTA CTTCTTCATC TGGCGGTTCC CCTTTTAACG ATCTATCAGG GGGGAAGGCC 540CCTTCCGGCA ACTCCCCTTC CGGCAACTAC TCTCCCGTCA GTACCTTCTC ACCCCCATCC 600ACGCCAACGT CCCCTACCTC ACCGCTTGAT TTCCCTTCTT CTCCCACCAA AGCAGCCGGG 660GGCAGCACGC CGGTAACCGA TCATCCTGAC CCTGTTGGTA GCGCGGGCAT CGGGGCCGGA 720AATTCGGTGG CCTTCACCAG CGCCGGCGCT AATCAGACGG TGCTGCATGA CACCATTACC 780GTGAAAGCGG GTCAGGTGTT TGATGGCAAA GGACAAACCT TCACCGCCGG TTCAGAATTA 840GGCGATGGCG GCCAGTCTGA AAACCAGAAA CCGCTGTTTA TACTGGAAGA CGGTGCCAGC 900CTGAAAAACG TCACCATGGG CGACGACGGG GCGGATGGTA TTCATCTTTA CGGTGATGCC 960AAAATAGACA ATCTGCACGT CACCAACGTG GGTGAGGACG CGATTACCGT TAAGCCAAAC 1020AGCGCGGGCA AAAAATCCCA CGTTGAAATC ACTAACAGTT CCTTCGAGCA CGCCTCTGAC 1080AAGATCCTGC AGCTGAATGC CGATACTAAC CTGAGCGTTG ACAACGTGAA GGCCAAAGAC 1140TTTGGTACTT TTGTACGCAC TAACGGCGGT CAACAGGGTA ACTGGGATCT GAATCTGAGC 1200CATATCAGCG CAGAAGACGG TAAGTTCTCG TTCGTTAAAA GCGATAGCGA GCGGCTAAAC 1260GTCAATACCA GTGATATCTC ACTGGGTGAT GTTGAAAACC ACTACAAAGT GCCGATGTCC 1320GCCAACCTGA AGGTGGCTGA ATGA 1344
Referring to GenBank accession number U94513.Isolated DNA molecule coding of the present invention has the anaphylaxis exciton albumen or the polypeptide of the aminoacid sequence of following SEQ.ID.No.26: Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu1 5 10 15Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser
20??????????????????25??????????????????30Ala?Leu?Gly?Gln?Gln?Pro?Ile?Asp?Arg?Gln?Thr?Ile?Glu?Gln?Met?Ala
35??????????????????40??????????????????45Gln?Leu?Leu?Ala?Glu?Leu?Leu?Lys?Ser?Leu?Leu?Ser?Pro?Gln?Ser?Gly
50??????????????????55??????????????????60Asn?Ala?Ala?Thr?Gly?Ala?Gly?Gly?Asn?Asp?Gln?Thr?Thr?Gly?Val?Gly65??????????????????70??????????????????75??????????????????80Asn?Ala?Gly?Gly?Leu?Asn?Gly?Arg?Lys?Gly?Thr?Ala?Gly?Thr?Thr?Pro
85??????????????????90??????????????????95Gln?Ser?Asp?Ser?Gln?Asn?Met?Leu?Ser?Glu?Met?Gly?Asn?Asn?Gly?Leu
100?????????????????105?????????????????110Asp?Gln?Ala?Ile?Thr?Pro?Asp?Gly?Gln?Gly?Gly?Gly?Gln?Ile?Gly?Asp
115?????????????????120?????????????????125Asn?Pro?Leu?Leu?Lys?Ala?Met?Leu?Lys?Leu?Ile?Ala?Arg?Met?Met?Asp
130?????????????????135?????????????????140Gly?Gln?Ser?Asp?Gln?Phe?Gly?Gln?Pro?Gly?Thr?Gly?Asn?Asn?Ser?Ala145?????????????????150?????????????????155?????????????????160Ser?Ser?Gly?Thr?Ser?Ser?Ser?Gly?Gly?Ser?Pro?Phe?Asn?Asp?Leu?Ser
165?????????????????170?????????????????175Gly?Gly?Lys?Ala?Pro?Ser?Gly?Asn?Ser?Pro?Ser?Gly?Asn?Tyr?Ser?Pro
180?????????????????185?????????????????190Val?Ser?Thr?Phe?Ser?Pro?Pro?Ser?Thr?Pro?Thr?Ser?Pro?Thr?Ser?Pro
195?????????????????200?????????????????205Leu?Asp?Phe?Pro?Ser?Ser?Pro?Thr?Lys?Ala?Ala?Gly?Gly?Ser?Thr?Pro
210?????????????????215??????????????????220Val?Thr?Asp?His?Pro?Asp?Pro?Val?Gly?Ser?Ala?Gly?Ile?Gly?Ala?Gly225?????????????????230?????????????????235?????????????????235Asn?Ser?Val?Ala?Phe?Thr?Ser?Ala?Gly?Ala?Asn?Gln?Thr?Val?Leu?His
245?????????????????250?????????????????255Asp?Thr?Ile?Thr?Val?Lys?Ala?Gly?Gln?Val?Phe?Asp?Gly?Lys?Gly?Gln
260?????????????????265?????????????????270Thr?Phe?Thr?Ala?Gly?Ser?Glu?Leu?Gly?Asp?Gly?Gly?Gln?Ser?Glu?Asn
275?????????????????280?????????????????285Gln?Lys?Pro?Leu?Phe?Ile?Leu?Glu?Asp?Gly?Ala?Ser?Leu?Lys?Asn?Val
290?????????????????295?????????????????300Thr?Met?Gly?Asp?Asp?Gly?Ala?Asp?Gly?Ile?His?Leu?Tyr?Gly?Asp?Ala305?????????????????310?????????????????315?????????????????320Lys?Ile?Asp?Asn?Leu?His?Val?Thr?Asn?Val?Gly?Glu?Asp?Ala?Ile?Thr
325?????????????????330?????????????????335Val?Lys?Pro?Asn?Ser?Ala?Gly?Lys?Lys?Ser?His?Val?Glu?Ile?Thr?Asn
340?????????????????345?????????????????350Ser?Ser?Phe?Glu?His?Ala?Ser?Asp?Lys?lle?Leu?Gln?Leu?Asn?Ala?Asp
355?????????????????360?????????????????365Thr?Asn?Leu?Ser?Val?Asp?Asn?Val?Lys?Ala?Lys?Asp?Phe?Gly?Thr?Phe
370?????????????????375?????????????????380Val?Arg?Thr?Asn?Gly?Gly?Gln?Gln?Gly?Asn?Trp?Asp?Leu?Asn?Leu?Ser385?????????????????390?????????????????395?????????????????400His?Ile?Ser?Ala?Glu?Asp?Gly?Lys?Phe?Ser?Phe?Val?Lys?Ser?Asp?Ser
405?????????????????410?????????????????415Glu?Gly?Leu?Asn?Val?Asn?Thr?Ser?Asp?Ile?Ser?Leu?Gly?Asp?Val?Glu
420?????????????????425?????????????????430Asn?His?Tyr?Lys?Val?Pro?Met?Ser?Ala?Asn?Leu?Lys?Val?Ala?Glu
435 440 445 these albumen or polypeptide are tart, are rich in glycine and Serine, and the shortage halfcystine.It also is inhibition thermally-stabilised, responsive to proteolytic enzyme and that be subjected to the plant metabolism inhibitor.The molecular size of albumen of the present invention or polypeptide expection is about 4.5kDa.
The another kind that must explain the starch Erwinia by oneself may be disclosed in u.s. patent application serial number 09/120,663 by suitable anaphylaxis exciton, and the document is attached to herein by reference.This isolated DNA molecule coding of the present invention has the anaphylactoid albumen or the polypeptide of inducing plant pathogenic agent of the aminoacid sequence of following SEQ.ID.No.28: Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr1 5 10 15Ala Ala His Asn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly Ser
20??????????????????25??????????????????30Ser?Ser?Ser?Ser?Pro?Gln?Asn?Ala?Ala?Ala?Ser?Leu?Ala?Ala?Glu?Gly
35??????????????????40??????????????????45Lys?Asn?Arg?Gly?Lys?Met?Pro?Arg?Ile?His?Gln?Pro?Ser?Thr?Ala?Ala
50??????????????????55??????????????????60Asp?Gly?Ile?Ser?Ala?Ala?His?Gln?Gln?Lys?Lys?Ser?Phe?Ser?Leu?Arg65??????????????????70??????????????????75??????????????????80Gly?Cys?Leu?Gly?Thr?Lys?Lys?Phe?Ser?Arg?Ser?Ala?Pro?Gln?Gly?Gln
85??????????????????90??????????????????95Pro?Gly?Thr?Thr?His?Ser?Lys?Gly?Ala?Thr?Leu?Arg?Asp?Leu?Leu?Ala
100?????????????????105?????????????????110Arg?Asp?Asp?Gly?Glu?Thr?Gln?His?Glu?Ala?Ala?Ala?Pro?Asp?Ala?Ala
115?????????????????120?????????????????125Arg?Leu?Thr?Arg?Ser?Gly?Gly?Val?Lys?Arg?Arg?Asn?Met?Asp?Asp?Met
130?????????????????135?????????????????140Ala?Gly?Arg?Pro?Met?Val?Lys?Gly?Gly?Ser?Gly?Glu?Asp?Lys?Val?Pro145?????????????????150?????????????????155?????????????????160Thr?Gln?Gln?Lys?Arg?His?Gln?Leu?Asn?Asn?Phe?Gly?Gln?Met?Arg?Gln
165?????????????????170?????????????????175Thr?Met?Leu?Ser?Lys?Met?Ala?His?Pro?Ala?Ser?Ala?Asn?Ala?Gly?Asp
180?????????????????185?????????????????190Arg?Leu?Gln?His?Ser?Pro?Pro?His?Ile?Pro?Gly?Ser?His?His?Glu?Ile
195?????????????????200?????????????????205Lys?Glu?Glu?Pro?Val?Gly?Ser?Thr?Ser?Lys?Ala?Thr?Thr?Ala?His?Ala
210?????????????????215?????????????????220Asp?Arg?Val?Glu?Ile?Ala?Gln?Glu?Asp?Asp?Asp?Ser?Glu?Phe?Gln?Gln225?????????????????230?????????????????235?????????????????240Leu?His?Gln?Gln?Arg?Leu?Ala?Arg?Glu?Arg?Glu?Asn?Pro?Pro?Gln?Pro
245?????????????????250?????????????????255Pro?Lys?Leu?Gly?Val?Ala?Thr?Pro?Ile?Ser?Ala?Arg?Phe?Gln?Pro?Lys
260?????????????????265?????????????????270Leu?Thr?Ala?Val?Ala?Glu?Ser?Val?Leu?Glu?Gly?Thr?Asp?Thr?Thr?Gln
275?????????????????280?????????????????285Ser?Pro?Leu?Lys?Pro?Gln?Ser?Met?Leu?Lys?Gly?Ser?Gly?Ala?Gly?Val
290?????????????????295?????????????????300Thr?Pro?Leu?Ala?Val?Thr?Leu?Asp?Lys?Gly?Lye?Leu?Gln?Leu?Ala?Pro305?????????????????310?????????????????315?????????????????320Asp?Asn?Pro?Pro?Ala?Leu?Asn?Thr?Leu?Leu?Lys?Gln?Thr?Leu?Gly?Lys
325?????????????????330?????????????????335Asp?Thr?Gln?His?Tyr?Leu?Ala?His?His?Ala?Ser?Ser?Asp?Gly?Ser?Gln
340?????????????????345?????????????????350His?Leu?Leu?Leu?Asp?Asn?Lys?Gly?His?Leu?Phe?Asp?Ile?Lys?Ser?Thr
355?????????????????360?????????????????365Ala?Thr?Ser?Tyr?Ser?Val?Leu?His?Asn?Ser?His?Pro?Gly?Glu?Ile?Lys
370?????????????????375?????????????????380Gly?Lys?Leu?Ala?Gln?Ala?Gly?Thr?Gly?Ser?Val?Ser?Val?Asp?Gly?Lys385?????????????????390?????????????????395?????????????????400Ser?Gly?Lys?Ile?Sar?Leu?Gly?Ser?Gly?Thr?Gln?Ser?His?Asn?Lys?Thr
405?????????????????410?????????????????415Met?Leu?Ser?Gln?Pro?Gly?Glu?Ala?His?Arg?Ser?Leu?Leu?Thr?Gly?Ile
420?????????????????425?????????????????430Trp?Gln?His?Pro?Ala?Gly?Ala?Ala?Arg?Pro?Gln?Gly?Glu?Ser?Ile?Arg
435?????????????????440?????????????????445Leu?His?Asp?Asp?Lys?Ile?His?Ile?Leu?His?Pro?Glu?Leu?Gly?Val?Trp
450?????????????????455?????????????????460Gln?Ser?Ala?Asp?Lys?Asp?Thr?His?Ser?Gln?Leu?5er?Arg?Gln?Ala?Asp465?????????????????470?????????????????475?????????????????480Gly?Lys?Leu?Tyr?Ala?Leu?Lys?Asp?Asn?Arg?Thr?Leu?Gln?Asn?Leu?Ser
485?????????????????490?????????????????495Asp?Asn?Lys?Ser?Ser?Glu?Lys?Leu?Val?Asp?Lys?Ile?Lys?Ser?Tyr?Ser
500????????????????505??????????????????510Val?Asp?Gln?Arg?Gly?Gln?Val?Ala?Ile?Leu?Thr?Asp?Thr?Pro?Gly?Arg
515?????????????????520?????????????????525His?Lys?Net?Ser?Ile?Met?Pro?Ser?Leu?Asp?Ala?Ser?Pro?Glu?Ser?His
530?????????????????535?????????????????540Ile?Ser?Leu?Ser?Leu?His?Phe?Ala?Asp?Ala?His?Gln?Gly?Leu?Leu?His545?????????????????550?????????????????555?????????????????560Gly?Lys?Ser?Glu?Leu?Glu?Ala?Gln?Sar?Val?Ala?Ile?Ser?His?Gly?ArS
565?????????????????570?????????????????575Leu?Val?Val?Ala?Asp?Ser?Glu?Gly?Lys?Leu?Phe?Ssr?Ala?Ala?Ile?Pro
580?????????????????585?????????????????590Lys?Gln?Gly?Asp?Gly?Asn?Glu?Leu?Lys?Met?Lys?Ala?Met?Pro?Gln?His
595?????????????????600?????????????????605Ala?Leu?Asp?Glu?His?Phe?Gly?His?Asp?His?Gln?Ile?Ser?Gly?Phe?Phe
610?????????????????615?????????????????620His?Asp?Asp?His?Gly?Gln?Leu?Asn?Ale?Leu?Val?Lys?Asn?Asn?Phe?Arg625?????????????????630?????????????????635?????????????????640Gln?Gln?His?Ala?Cys?Pro?Leu?Gly?Asn?Asp?His?Gln?Phe?His?Pro?Gly
645?????????????????650?????????????????655Trp?Asn?Leu?Thr?Asp?Ala?Leu?Val?Ile?Asp?Asn?Gln?Leu?Gly?Leu?His
660?????????????????665?????????????????670His?Thr?Asn?Pro?Glu?Pro?His?Glu?Ile?Leu?Asp?Met?Gly?His?Leu?Gly
675?????????????????680?????????????????585Ser?Leu?Ala?Leu?Gln?Glu?Gly?Lys?Leu?His?Tyr?Phe?Asp?Gln?Leu?Thr
690?????????????????695?????????????????700Lys?Gly?Trp?Thr?Gly?Ala?Glu?Ser?Asp?Cys?Lys?Gln?Leu?Lys?Lys?Gly705?????????????????710?????????????????715?????????????????720Leu?Asp?Gly?Ala?Ala?Tyr?Leu?Leu?Lys?Asp?Gly?Glu?Val?Lys?Arg?Leu
725?????????????????730?????????????????735Asn?Ile?Asn?Gln?Ser?Thr?Ser?Ser?Ile?Lys?His?Gly?Thr?Glu?Asn?Val
740?????????????????745?????????????????750Phe?Ser?Leu?Pro?His?Val?Arg?Asn?Lys?Pro?Glu?Pro?Gly?Asp?Ala?Leu
755?????????????????760?????????????????765Gln?Gly?Leu?Asn?Lys?Asp?Asp?Lys?Ala?Gln?Ala?Met?Ala?Val?Ile?Gly
770?????????????????775?????????????????780Val?Asn?Lys?Tyr?Leu?Ala?Leu?Thr?Glu?Lys?Gly?Asp?Ile?Arg?Ser?Phe785?????????????????790?????????????????795?????????????????800Gln?Ile?Lys?Pro?Gly?Thr?Gln?Gln?Leu?Glu?Arg?Pro?Ala?Gln?Thr?Leu
805?????????????????810?????????????????8l5Ser?Arg?Glu?Gly?Ile?Ser?Gly?Glu?Leu?Lys?Asp?Ile?His?Val?Asp?His
820?????????????????825?????????????????830Lys?Gln?Asn?Leu?Tyr?Ala?Leu?Thr?His?Glu?Gly?Glu?Val?Phe?His?Gln
835?????????????????840?????????????????845Pro?Arg?Glu?Ala?Trp?Gln?Asn?Gly?Ala?Glu?Ser?Ser?Ser?Trp?His?Lys
850?????????????????855?????????????????860Leu?Ala?Leu?Pro?Gln?Ser?Glu?Ser?Lye?Leu?Lys?Ser?Leu?Asp?Met?Ser865?????????????????870?????????????????875?????????????????880His?Glu?His?Lys?Pro?Ile?Ala?Thr?Phe?Glu?Asp?Gly?Ser?Gln?His?Gln
885?????????????????890?????????????????895Leu?Lys?Ala?Gly?Gly?Trp?His?Ala?Tyr?Ala?Ala?Pro?Glu?Arg?Gly?Pro
900?????????????????905?????????????????910Leu?Ala?val?Gly?Thr?Ser?Gly?Ser?Gln?Thr?Val?Phe?Asn?Arg?Leu?Met
915?????????????????920?????????????????925Gln?Gly?Val?Lys?Gly?Lys?Val?Ile?Pro?Gly?Ser?Gly?Leu?Thr?Val?Lys
930?????????????????935?????????????????940Leu?Ser?Ala?Gln?Thr?Gly?Gly?Met?Thr?Gly?Ala?Glu?Gly?Arg?Lys?Val945?????????????????950?????????????????955?????????????????960Ser?Ser?Lys?Phe?Ser?Glu?Arg?Ile?Arg?Ala?Tyr?Ala?Phe?Asn?Pro?Thr
965?????????????????970?????????????????975Met?Ser?Thr?Pro?Arg?Pro?Ile?Lys?Asn?Ala?Ala?Tyr?Ala?Thr?Gln?His
980?????????????????985?????????????????990Gly?Trp?Gln?Gly?Arg?Glu?Gly?Leu?Lys?Pro?Leu?Tyr?Glu?Met?Gln?Gly
995?????????????????1000????????????????1005Ala?Leu?Ile?Lys?Gln?Leu?Asp?Ala?His?Ash?Val?Arg?His?Ash?Ala?Pro
1010????????????????1015????????????????1020Gln?Pro?Asp?Leu?Gln?Ser?Lys?Leu?Glu?Thr?Leu?Asp?Leu?Gly?Glu?His1025????????????????1030????????????????1035????????????????1040Gly?Ala?Glu?Leu?Leu?Asn?Asp?Met?Lys?Arg?Phe?Atg?Asp?Glu?Leu?Glu
1045????????????????1050????????????????1055??????????????????????????????Gln?Ser?Ala?Thr?Arg?Ser?Val?Thr?Val?Leu?Gly?Gln?His?Gln?Gly?Val
1060????????????????1065????????????????1070Leu?Lys?Ser?Asn?Gly?Glu?Ile?Asn?Ser?Glu?Phe?Lys?Pro?Ser?Pro?Gly
1075????????????????1080????????????????1085Lys?Ala?Leu?Val?Gln?Ser?Phe?Asn?Val?Asn?Arg?Ser?Gly?Gln?Asp?Leu
1090????????????????1095????????????????1100Ser?Lys?Ser?Leu?Gln?Gln?Ala?Val?His?Ala?Thr?Pro?Pro?Ser?Ala?Glu1105????????????????1110????????????????1115????????????????1120Ser?Lys?Leu?Gln?Ser?Met?Leu?Gly?His?Phe?Vel?Ser?Ala?Gly?Val?Asp
1125????????????????1130????????????????1135Met?Ser?His?Gln?Lys?Gly?Glu?Ile?Pro?Leu?Gly?Arg?Gln?Arg?Asp?Pro
1140????????????????1145????????????????1150Asn?Asp?Lys?Thr?Ala?Leu?Thr?Lys?Ser?Arg?Leu?Ile?Leu?Asp?Thr?Val
1155????????????????1160????????????????1165Thr?Ile?Gly?Glu?Leu?His?Glu?Leu?Ale?Asp?Lys?Ala?Lys?Leu?Val?Ser
1170????????????????1175????????????????1180Asp?His?Lys?Pro?Asp?Ala?Asp?Gln?Ile?Lys?Gln?Leu?Arg?Gln?Gln?Phe1185????????????????1190????????????????1195????????????????1200Asp?Thr?Leu?Arg?Glu?Lys?Arg?Tyr?Glu?Ser?Asn?Pro?Val?Lys?His?Tyr
1205????????????????1210????????????????1215Thr?Asp?Met?Gly?Phe?Thr?His?Asn?Lys?Ala?Leu?Glu?Ala?Asn?Tyr?Asp
1220????????????????1225????????????????1230Ala?Val?Lys?Ala?Phe?Ile?Ash?Ala?Phe?Lys?Lys?Glu?His?His?Gly?Val
1235????????????????1240????????????????1245Asn?Leu?Thr?Thr?Arg?Thr?Val?Leu?Glu?Ser?Gln?Gly?Ser?Ala?Glu?Leu
1250????????????????1255????????????????1260Ala?Lys?Lys?Leu?Lys?Asn?Thr?Leu?Leu?Ser?Leu?Asp?Ser?Gly?Glu?Ser1265????????????????1270????????????????1275????????????????1280Met?Ser?Phe?Ser?Arg?Ser?Tyr?Gly?Gly?Gly?Vel?Ser?Thr?Va1?Phe?Val
1285????????????????1290????????????????1295Pro?Thr?Leu?Ser?Lys?Lys?Val?Pro?Val?Pro?Val?Ile?Pro?Gly?Ala?Gly
1300????????????????1305????????????????1310Ile?Thr?Leu?Asp?Arg?Ala?Tyr?Asn?Leu?Ser?Phe?Ser?Arg?Thr?Ser?Gly
1315????????????????1320????????????????1325Gly?Leu?Asn?Val?Ser?Phe?Gly?Arg?Asp?Gly?Gly?Val?Ser?Gly?Asn?Ile
1330????????????????1335????????????????1340Met?Val?Ala?Thr?Gly?His?Asp?Val?Met?Pro?Tyr?Met?Thr?Gly?Lys?Lys1345????????????????1350????????????????1355????????????????1360Thr?Ser?Ala?Gly?Asn?Ala?Ser?Asp?Trp?Leu?Ser?Ala?Lys?His?Lys?Ile
1365????????????????1370????????????????1375Ser?Pro?Asp?Leu?Arg?Ile?Gly?Ala?Ala?Val?Ser?Gly?Thr?Leu?Gln?Gly
1380????????????????1385????????????????1390Thr?Leu?Gln?Asn?Ser?Leu?Lys?Phe?Lys?Leu?Thr?Glu?Asp?Glu?Leu?Pro
1395????????????????1400????????????????1405Gly?Phe?Ile?His?Gly?Leu?Thr?His?Gly?Thr?Leu?Thr?Pro?Ala?Glu?Leu
1410????????????????1415????????????????1420Leu?Gln?Lys?Gly?Ile?Glu?His?Gln?Met?Lys?Gln?Gly?Ser?Lys?Leu?Thr1425????????????????1430????????????????1435????????????????1440Phe?Ser?Val?Asp?Thr?Ser?Ala?Asn?Leu?Asp?Leu?Arg?Ala?Gly?Ile?Asn
1445????????????????1450????????????????1455Leu?Asn?Glu?Asp?Gly?Ser?Lys?Pro?Asn?Gly?Val?Thr?Ala?Arg?Val?Ser
1460????????????????1465????????????????1470Ala?Gly?Leu?Ser?Ala?Ser?Ala?Asn?Leu?Ala?Ala?Gly?Ser?Arg?Glu?Arg
1475????????????????1480????????????????1485Ser?Thr?Thr?Ser?Gly?Gln?Phs?Gly?Ser?Thr?Thr?Ser?Ala?Ser?Asn?Asn
1490????????????????1495????????????????1500Arg?Pro?Thr?Phe?Leu?Asn?Gly?Val?Gly?Ala?Gly?Ala?Asn?Leu?Thr?Ala1505????????????????1510????????????????1515????????????????1520Ala?Leu?Gly?Val?Ala?His?Ser?Ser?Thr?His?Glu?Gly?Lys?Pro?Val?Gly
1525????????????????1530????????????????1535Ile?Phe?Pro?Ala?Phe?Thr?Ser?Thr?Asn?Val?Ser?Ala?Ala?Leu?Ala?Leu
1540????????????????1545????????????????1550Asp?Asn?Arg?Thr?Ser?Gln?Ser?Ile?Ser?Leu?Glu?Leu?Lys?Arg?Ala?Glu
1555????????????????1560????????????????1565Pro?Val?Thr?Ser?Asn?Asp?Ile?Ser?Glu?Leu?Thr?Ser?Thr?Leu?Gly?Lys
1570????????????????1575????????????????1580His?Phe?Lys?Asp?Ser?Ala?Thr?Thr?Lys?Met?Leu?Ala?Ala?Leu?Lys?Glu1585????????????????1590????????????????1595????????????????1600Leu?Asp?Asp?Ala?Lys?Pro?Ala?Glu?Gln?Leu?His?Ile?Leu?Gln?Gln?His
1605????????????????1610????????????????1615Phe?Ser?Ala?Lys?Asp?Val?Val?Gly?Asp?Glu?Arg?Tyr?Glu?Ala?Val?Arg
1620????????????????1625????????????????1630Asn?Leu?Lys?Lys?Leu?Val?Ils?Arg?Gln?Gln?Ala?Ala?Asp?Ser?His?Ser
1635????????????????1640????????????????1645Met?Glu?Leu?Gly?Ser?Ala?Ser?His?Ser?Thr?Thr?Tyr?ASn?Asn?Leu?Ser
1650????????????????1655????????????????1660Arg?Ile?Asn?Asn?Asp?Gly?Ile?Val?Glu?Leu?Leu?His?Lys?His?Phe?Asp1665????????????????1670????????????????1675????????????????1680Ala?Ala?Leu?Pro?Ala?Ser?Ser?Ala?Lys?Arg?Leu?Gly?Glu?Met?Met?Asn
1685????????????????1690????????????????1695Asn?Asp?Pro?Ala?Leu?Lys?Asp?Ile?Ile?Lys?Gln?Leu?Gln?Ser?Thr?Pro
1700????????????????1705????????????????1710Phe?Ser?Ser?Ala?Ser?Val?Ser?Met?Glu?Leu?Lys?Asp?Gly?Leu?Arg?Glu
1715????????????????1720????????????????1725Gln?Thr?Glu?Lys?Ala?Ile?Leu?Asp?Gly?Lys?Val?Gly?Arg?Glu?Glu?Val
1730????????????????1735????????????????1740Gly?Val?Leu?Phe?Gln?Asp?Arg?Asn?Asn?Leu?Arg?Val?Lys?Ser?Val?Ser1745????????????????1750????????????????1755????????????????1760Val?Ser?Gln?Ser?Val?Ser?Lys?Ser?Glu?Gly?Phe?Asn?Thr?Pro?Ala?Leu
1765????????????????1770????????????????1775Leu?Leu?Gly?Thr?Ser?Asn?Ser?Ala?Ala?Mer?Ser?Met?Glu?Arg?Asn?Ile
1780????????????????1785????????????????1790Gly?Thr?Ile?Asn?Phe?Lys?Tyr?Gly?Gln?Asp?Gln?Asn?Thr?Pro?Arg?Arg
1795????????????????1800????????????????1805Phe?Thr?Leu?Glu?Gly?Gly?Ile?Ala?Gln?Ala?Asn?Pro?Gln?Val?Ala?Ser
1,810 1815 1820Ala Leu Thr Asp Leu Lys Lys Glu Gly Leu Glu Met Lys Ser1825 1,830 1835 these albumen or polypeptide are about 198kDa, and pI is 8.98.
The present invention relates to have the dna molecular of separation of the nucleotide sequence of following SEQ.ID.No.29: ATGACATCGT CACAGCAGCG GGTTGAAAGG TTTTTACAGT ATTTCTCCGC CGGGTGTAAA 60ACGCCCATAC ATCTGAAAGA CGGGGTGTGC GCCCTGTATA ACGAACAAGA TGAGGAGGCG 120GCGGTGCTGG AAGTACCGCA ACACAGCGAC AGCCTGTTAC TACACTGCCG AATCATTGAG 180GCTGACCCAC AAACTTCAAT AACCCTGTAT TCGATGCTAT TACAGCTGAA TTTTGAAATG 240GCGGCCATGC GCGGCTGTTG GCTGGCGCTG GATGAACTGC ACAACGTGCG TTTATGTTTT 300CAGCAGTCGC TGGAGCATCT GGATGAAGCA AGTTTTAGCG ATATCGTTAG CGGCTTCATC 360GAACATGCGG CAGAAGTGCG TGAGTATATA GCGCAATTAG ACGAGAGTAG CGCGGCATAA 420 these dna moleculars are called as the dspF gene. This isolated DNA molecule coding of the present invention has the anaphylaxis exciton albumen or the polypeptide of the aminoacid sequence of following SEQ.ID.No.30:Met Thr Ser Ser Gln Gln Arg Val Glu Arg Phe Leu Gln Tyr Phe Serl 5 10 15Ala Gly Cys Lys Thr Pro Ile His Leu Lys Asp Gly Val Cys Ala Leu
20??????????????????25??????????????????30Tyr?Asn?Glu?Gln?Asp?Glu?Glu?Ala?Ala?Val?Leu?Glu?Val?Pro?Gln?His
35??????????????????40??????????????????45Ser?Asp?Ser?Leu?Leu?Leu?His?Cys?Arg?Ile?Ile?Glu?Ala?Asp?Pro?Gln
50??????????????????55??????????????????60Thr?Ser?Ile?Thr?Leu?Tyr?Ser?Met?Leu?Leu?Gln?Leu?Asn?Phe?Glu?Met65??????????????????70??????????????????75??????????????????80Ala?Ala?Met?Arg?Gly?Cys?Trp?Leu?Ala?Leu?Asp?Glu?Leu?His?Asn?Val
85??????????????????90??????????????????95Arg?Leu?Cys?Phe?Gln?Gln?Ser?Leu?Glu?His?Leu?Asp?Glu?Ala?Ser?Phe
100?????????????????105?????????????????110Ser?Asp?Ile?Val?Ser?Gly?Phe?Ile?Glu?His?Ala?Ala?Glu?Val?Arg?Glu
115?????????????????120?????????????????125Tyr?Ile?Ala?Gln?Leu?Asp?Glu?Ser?Ser?Ala?Ala
130 135 these albumen or polypeptide are about 16kDa, and pI is 4.45.
Derive from the anaphylaxis exciton polypeptide of pseudomonas syringae or albumen and have aminoacid sequence: Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Metl 5 10 15Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser corresponding to following SEQ.ID.No.31
20??????????????????25??????????????????30Ser?Lys?Ala?Leu?Gln?Glu?Val?Val?Val?Lys?Leu?Ala?Glu?Glu?Leu?Met
35??????????????????40??????????????????45Arg?Asn?Gly?Gln?Leu?Asp?Asp?Ser?Ser?Pro?Leu?Gly?Lys?Leu?Leu?Ala
50??????????????????55??????????????????60Lys?Ser?Met?Ala?Ala?Asp?Gly?Lys?Ala?Gly?Gly?Gly?Ile?Glu?Asp?Val65??????????????????70??????????????????75??????????????????80Ile?Ala?Ala?Leu?Asp?Lys?Leu?Ile?His?Glu?Lys?Leu?Gly?Asp?Asn?Phe
85??????????????????90??????????????????95Gly?Ala?Ser?Ala?Asp?Ser?Ala?Ser?Gly?Thr?Gly?Gln?Gln?Asp?Leu?Met
100?????????????????105?????????????????110Thr?Gln?Val?Leu?Asn?Gly?Leu?Ala?Lys?Ser?Met?Leu?Asp?Asp?Leu?Leu
115?????????????????120?????????????????125Thr?Lys?Gln?Asp?Gly?Gly?Thr?Ser?Phe?Ser?Glu?Asp?Asp?Met?Pro?Met
130?????????????????135?????????????????140Leu?Asn?Lys?Ile?Ala?Gln?Phe?Met?Asp?Asp?Asn?Pro?Ala?Gln?Phe?Pro145?????????????????150?????????????????155?????????????????160Lys?Pro?Asp?Ser?Gly?Ser?Trp?Val?Asn?Glu?Leu?Lys?Glu?Asp?Asn?Phe
165?????????????????170?????????????????175LGu?Asp?Gly?Asp?Glu?Thr?Ala?Ala?Phe?Arg?Ser?Ala?Leu?Asp?Ile?Ilo
180?????????????????185?????????????????190Gly?Gln?Gln?Leu?Gly?Asn?Gln?Gln?Ser?Asp?Ala?Gly?Ser?Leu?Ala?Gly
195?????????????????200?????????????????205Thr?Gly?Gly?Gly?Leu?Gly?Thr?Pro?Ser?Ser?Phe?Ser?Asn?Asn?Ser?Ser
210?????????????????215?????????????????220Val?Met?Gly?Asp?Pro?Leu?Ile?Asp?Ala?Asn?Thr?Gly?Pro?Gly?Asp?Ser225?????????????????230?????????????????235?????????????????240Gly?Asn?Thr?Arg?Gly?Glu?Ala?Gly?Gln?Leu?Ile?Gly?Glu?Leu?Ile?Asp
245?????????????????250?????????????????255Arg?Gly?Leu?Gln?Ser?Val?Leu?Ala?Gly?Gly?Gly?Leu?Gly?Thr?Pro?Val
260?????????????????265?????????????????270Asn?Thr?Pro?Gln?Thr?Gly?Thr?Ser?Ala?Asn?Gly?Gly?Gln?Ser?Ala?Gln
275?????????????????280?????????????????285Asp?Leu?Asp?Gln?Leu?Leu?Gly?Gly?Leu?Leu?Leu?Lys?Gly?Leu?Glu?Ala
290?????????????????295?????????????????300Thr?Leu?Lys?Asp?Ala?Gly?Gln?Thr?Gly?Thr?Asp?Val?Gln?Ser?Ser?Ala305?????????????????310?????????????????315?????????????????320Ala?Gln?Ile?Ala?Thr?Leu?Leu?Val?Ser?Thr?Leu?Leu?Gln?Gly?Thr?Arg
325?????????????????330?????????????????335Asn?Gln?Ala?Ala?Ala
340 these anaphylaxis exciton polypeptide or proteic molecular weight are 34-35kDa.It is rich in glycine (about 13.5%), and lacks halfcystine and tyrosine.Further information about the anaphylaxis exciton that derives from pseudomonas syringae sees He, S.Y., H.C.Huang and A.Collmer, " the pseudomonas syringae cloves mutation Harpinpss that causes a disease:by secretion of Hrp approach and the anaphylactoid albumen of inducing plant; " Cell 73:1255-1266 ( 1993 ) , the document is attached to herein by reference.DNASEQ.ID.No.32：ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC 120GTGAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA 180AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TGAGGATGTC 240ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG 300GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC 360AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CTCCGAAGAC 420GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC ACAGTTTCCC 480AAGCCGGACT CGGGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC 540GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG 600AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC 660AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC 720GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC TTATCGACCG TGGCCTGCAA 780TCGGTATTGG CCGGTGGTGG ACTGGGCACA CCCGTAAACA CCCCGCAGAC CGGTACGTCG 840GCGAATGGCG GACAGTCCGC TCACGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG 900GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT 960GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TCAGGCTGCA 1020GCCTGA 1026
The another kind that derives from pseudomonas syringae may be disclosed in u.s. patent application serial number 09/120; 817 by suitable anaphylaxis exciton, and the document is attached to herein by reference.SEQ.ID.No.33：TCCACTTCGC TGATTTTGAA ATTGGCAGAT TCATAGAAAC GTTCAGGTGT GGAAATCAGG 60CTGAGTGCGC AGATTTCGTT GATAAGGGTG TGGTACTGGT CATTGTTGGT CATTTCAAGG 120CCTCTGAGTG CGGTGCGGAG CAATACCAGT CTTCCTGCTG GCGTGTGCAC ACTGAGTCGC 180AGGCATAGGC ATTTCAGTTC CTTGCGTTGG TTGGGCATAT AAAAAAAGGA ACTTTTAAAA 240ACAGTGCAAT GAGATGCCGG CAAAACGGGA ACCGGTCGCT GCGCTTTGCC ACTCACTTCG 300AGCAAGCTCA ACCCCAAACA TCCACATCCC TATCGAACGG ACAGCGATAC GGCCACTTGC 360TCTGGTAAAC CCTGGAGCTG GCGTCGGTCC AATTGCCCAC TTAGCGAGGT AACGCAGCAT 420GAGCATCGGC ATCACACCCC GGCCGCAACA GACCACCACG CCACTCGATT TTTCGGCGCT 480AAGCGGCAAG AGTCCTCAAC CAAACACGTT CGGCCAGCAG AACACTCAGC AAGCGATCGA 540CCCGAGTGCA CTGTTGTTCG GCAGCGACAC ACAGAAAGAC GTCAACTTCG GCACGCCCGA 600CAGCACCGTC CAGAATCCGC AGGACGCCAG CAAGCCCAAC GACAGCCAGT CCAACATCGC 660TAAATTGATC AGTGCATTGA TCATGTCGTT GCTGCAGATG CTCACCAACT CCAATAAAAA 720GCAGGACACC AATCAGGAAC AGCCTGATAG CCAGGCTCCT TTCCAGAACA ACGGCGGGCT 780CGGTACACCG TCGGCCGATA GCGGGGGCGG CGGTACACCG GATGCGACAG GTGGCGGCGG 840CGGTGATACG CCAAGCGCAA CAGGCGGTGG CGGCGGTGAT ACTCCGACCG CAACAGGCGG 900TGGCGGCAGC GGTGGCGGCG GCACACCCAC TGCAACAGGT GGCGGCAGCG GTGGCACACC 960CACTGCAACA GGCGGTGGCG AGGGTCGCGT AACACCGCAA ATCACTCCGC AGTTGGCCAA 1020CCCTAACCGT ACCTCAGGTA CTGGCTCGGT GTCGGACACC GCAGGTTCTA CCGAGCAAGC 1080CGGCAAGATC AATGTGGTGA AAGACACCAT CAAGGTCGGC GCTGGCGAAG TCTTTGACGG 1140CCACGGCGCA ACCTTCACTG CCGACAAATC TATGGGTAAC GGAGACCAGG GCGAAAATCA 1200GAAGCCCATG TTCGAGCTGG CTGAAGGCGC TACGTTGAAG AATGTGAACC TGGGTGAGAA 1260CGAGGTCGAT GGCATCCACG TGAAAGCCAA AAACGCTCAG GAAGTCACCA TTGACAACGT 1320GCATGCCCAG AACGTCGGTG AAGACCTGAT TACGGTCAAA GGCGAGGGAG GCGCAGCGGT 1380CACTAATCTG AACATCAAGA ACAGCAGTGC CAAAGGTGCA GACGACAAGG TTGTCCAGCT 1440CAACGCCAAC ACTCACTTGA AAATCGACAA CTTCAAGGCC GACGATTTCG GCACGATGGT 1500TCGCACCAAC GGTGGCAAGC AGTTTGATGA CATGAGCATC GAGCTGAACG GCATCGAAGC 1560TAACCACGGC AAGTTCGCCC TGGTGAAAAG CGACAGTGAC GATCTGAAGC TGGCAACGGG 1620CAACATCGCC ATGACCGACG TCAAACACGC CTACGATAAA ACCCAGGCAT CGACCCAACA 1680CACCGAGCTT TGAATCCAGA CAAGTAGCTT GAAAAAAGGG GGTGGACTC 1729DNAdspE。 This isolated DNA molecule coding of the present invention has the anaphylactoid albumen or the polypeptide of inducing plant pathogenic agent of the aminoacid sequence of following SEQ.ID.No.34:Met Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Thr Pro Leu1 5 10 15Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr Phe Gly
20??????????????????25??????????????????30Glu?Gln?Asn?Thr?Gln?Gln?Ala?Ile?Asp?Pro?Ser?Ala?Leu?Leu?Phe?Gly
35??????????????????40??????????????????45Ser?Asp?Thr?Gln?Lys?Asp?Val?Asn?Phe?Gly?Thr?Pro?Asp?Ser?Thr?Val
50??????????????????55??????????????????60Gln?Asn?Pro?Gln?Asp?Ala?Ser?Lys?Pro?Asn?Asp?Ser?Gln?Ser?Asn?Ile65??????????????????70??????????????????75??????????????????80Ala?Lys?Leu?Ile?Ser?Ala?Leu?Ile?Met?Ser?Leu?Leu?Gln?Met?Leu?Thr
85??????????????????90??????????????????95Asn?Ser?Asn?Lys?Lys?Gln?Asp?Thr?Asn?Gln?Glu?Gln?Pro?Asp?Ser?Gln
100?????????????????105?????????????????110Ala?Pro?Phe?Gln?Asn?Asn?Gly?Gly?Leu?Gly?Thr?Pro?Ser?Ala?Asp?Ser
115?????????????????120?????????????????125Gly?Gly?Gly?Gly?Thr?Pro?Asp?Ala?Thr?Gly?Gly?Gly?Gly?Gly?Asp?Thr
130?????????????????135?????????????????140Pro?Ser?Ala?Thr?Gly?Gly?Gly?Gly?Gly?Asp?Thr?Pro?Thr?Ala?Thr?Gly145?????????????????150?????????????????155?????????????????160Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Thr?Pro?Thr?Ala?Thr?Gly?Gly?Gly
165?????????????????170?????????????????175Ser?Gly?Gly?Thr?Pro?Thr?Ala?Thr?Gly?Gly?Gly?Glu?Gly?Gly?Val?Thr
180?????????????????185?????????????????190Pro?Gln?Ile?Thr?Pro?Gln?Lau?Ala?Asn?Pro?Asn?Arg?Thr?Ser?Gly?Thr
195?????????????????200?????????????????205Gly?Ser?Val?Ser?Asp?Thr?Ala?Gly?Ser?Thr?Glu?Gln?Ala?Gly?Lys?Ile
210?????????????????215?????????????????220Asn?Val?Val?Lys?Asp?Thr?Ile?Lys?Val?Gly?Ala?Gly?Glu?Val?Phe?Asp225?????????????????230?????????????????235?????????????????240Gly?His?Gly?Ala?Thr?Phe?Thr?Ala?Asp?Lys?Ser?Met?Gly?Asn?Gly?Asp
245?????????????????250?????????????????255Gln?Gly?Glu?Asn?Gln?Lys?Pro?Met?Phe?Glu?Leu?Ala?Glu?Gly?Ala?Thr
260?????????????????265?????????????????270Leu?Lys?Asn?Val?Asn?Leu?Gly?Glu?Asn?Glu?Vai?Asp?Gly?Ile?His?Val
275?????????????????280?????????????????285Lys?Ala?Lys?Asn?Ala?Gln?Glu?Val?Thr?Ile?Asp?Asn?Val?His?Ala?Gln
290?????????????????295?????????????????300Asn?Val?Gly?Glu?Asp?Leu?Ile?Thr?Val?Lys?Gly?Glu?Gly?Gly?Ala?Ala305?????????????????310?????????????????315?????????????????320Val?Thr?Asn?Leu?Asn?Ile?Lys?Asn?Ser?Ser?Ala?Lys?Gly?Ala?Asp?Asp
325?????????????????330?????????????????335Lys?Val?Val?Gln?Leu?Asn?Ala?Asn?Thr?His?Leu?Lys?Ile?Asp?Asn?Phe
340?????????????????345?????????????????350Lys?Ala?Asp?Asp?Phe?Gly?Thr?Met?Val?Arg?Thr?Asn?Gly?Gly?Lys?Gln
355?????????????????360?????????????????365Phe?Asp?Asp?Met?Ser?Ile?Glu?Leu?Asn?Gly?Ile?Glu?Ala?Asn?His?Gly
370?????????????????375?????????????????380Lys?Phe?Ala?Leu?Val?Lys?Ser?Asp?Ser?Asp?Asp?Leu?Lys?Leu?Ala?Thr385?????????????????390?????????????????395?????????????????400Gly?Asn?Ile?Ala?Met?Thr?Asp?Val?Lys?His?Ala?Tyr?Asp?Lys?Thr?Gln
405?????????????????410?????????????????415Ala?Ser?Thr?Gln?His?Thr?Glu?Leu
420 these albumen or polypeptide are about 42.9kDa.
Derive from the anaphylaxis exciton polypeptide of aeruginosa eggplant or albumen and have aminoacid sequence: Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln1 5 10 15Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser corresponding to following SEQ.ID.No.35
20??????????????????25??????????????????30Val?Gln?Asp?Leu?Ile?Lys?Gln?Val?Glu?Lys?Asp?Ile?Leu?Asn?Ile?Ile
35??????????????????40??????????????????45Ala?Ala?Leu?Val?Gln?Lys?Ala?Ala?Gln?Ser?Ala?Gly?Gly?Asn?Thr?Gly
50??????????????????55??????????????????60Asn?Thr?Gly?Asn?Ala?Pro?Ala?Lys?Asp?Gly?Asn?Ala?Asn?Ala?Gly?Ala65??????????????????70??????????????????75??????????????????80Asn?Asp?Pro?Ser?Lys?Asn?Asp?Pro?Ser?Lys?Ser?Gln?Ala?Pro?Gln?Ser
85??????????????????90??????????????????95Ala?Asn?Lys?Thr?Gly?Asn?Val?Asp?Asp?Ala?Asn?Asn?Gln?Asp?Pro?Met
100?????????????????105?????????????????110Gln?Ala?Leu?Met?Gln?Leu?Leu?Glu?Asp?Leu?Val?Lys?Leu?Leu?Lys?Ala
115?????????????????120?????????????????125Ala?Leu?His?Met?Gln?Gln?Pro?Gly?Gly?Asn?Asp?Lye?Gly?Asn?Gly?Val
130?????????????????135?????????????????140Gly?Gly?Ala?Asn?Gly?Ala?Lys?Gly?Ala?Gly?Gly?Gln?Gly?Gly?Leu?Ala145?????????????????150?????????????????155?????????????????160Glu?Ala?Leu?Gln?Glu?Ile?Glu?Gln?Ile?Leu?Ala?Gln?Leu?Gly?Gly?Gly
165?????????????????170?????????????????175Gly?Ala?Gly?Ala?Gly?Gly?Ala?Gly?Gly?Gly?Val?Gly?Gly?Ala?Gly?Gly
180?????????????????185?????????????????190Ala?Asp?Gly?Gly?Ser?Gly?Ala?Gly?Gly?Ala?Gly?Gly?Ala?Asn?Gly?Ala
195?????????????????200?????????????????205Asp?Gly?Gly?Asn?Gly?Val?Asn?Gly?Asn?Gln?Ala?Asn?Gly?Pro?Gln?Asn
210?????????????????215?????????????????220Ala?Gly?Asp?val?Asn?Gly?Ala?Asn?Gly?Ala?Asp?Asp?Gly?Ser?Glu?Asp225?????????????????230?????????????????235?????????????????240Gln?Gly?Gly?Leu?Thr?Gly?Val?Leu?Gln?Lys?Leu?Met?Lys?Ile?Leu?Asn
245?????????????????250?????????????????255Ala?Leu?Val?Gln?Met?Met?Gln?Gln?Gly?Gly?Leu?Gly?Gly?Gly?Asn?Gln
260?????????????????265?????????????????270Ala?Gln?Gly?Gly?Ser?Lys?Gly?Ala?Gly?Asn?Ala?Ser?Pro?Ala?Ser?Gly
275?????????????????280?????????????????285Ala?Asn?Pro?Gly?Ala?Asn?Gln?Pro?Gly?Ser?Ala?Asp?Asp?Gln?Ser?Ser
290?????????????????295?????????????????300Gly?Gln?Asn?Asn?Leu?Gln?Ser?Gln?Ila?Mat?Asp?Val?Val?Lys?Glu?Val305?????????????????310?????????????????315?????????????????320Val?Gln?Ile?Leu?Gln?Gln?Met?Leu?Ala?Ala?Gln?Asn?Gly?Gly?Ser?Gln
325?????????????????330?????????????????335Gln?Ser?Thr?Ser?Thr?Gln?Pro?Met
340SEQ.ID.No.36DNA：ATGTCAGTCG GAAACATCCA GACCCGTCG AACCTCCCGG GTCTGCAGAA CCTGAACCTC 60AACACCAACA CCAACAGCCA GCAATCGGGC CAGTCCGTGC AAGACCTGAT CAAGCAGGTC 120GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCAGA AGGCCGCACA GTCGGCGGGC 180GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC 240AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC 300GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GCTGCTGGAA 360GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CAATGACAAG 420GGCAACGGCG TGGGCGGTGC CAACGGCGCC AAGGGTGCCG GCGGCCAGGG CGGCCTGGCC 480GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC 540GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CGGTGCGGGT 600GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC 660GGCCCGCAGA ACGCAGGCGA TGTCAACGGT GCCAACGGCG CGGATGACGG CAGCGAAGAC 720CAGGGCGGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC GCTGGTGCAG 780ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC AGGGCGGCTC GAAGGGTGCC 840GGCAACGCCT CGCCGGCTTC CGGCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGGGGAT 900GATCAATCGT CCGGCCAGAA CAATCTGCAA TCCCAGATCA TGGATGTGGT GAAGGAGGTC 960GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG 1020ACGCAGCCGA TGTAA 1035Arlat； M.; F.Van Gijsegem; J.C.Huet; J.C.Pemollet and CA.Boucher; " by the Hrp approach secretion albumen that PopAl-the induced hypersensitivity sample reacts on specific petunia genotype of aeruginosa eggplant; " EMBO is (1994) J.13:543-533, the document is incorporated herein by reference.
Derive from the cause a disease anaphylaxis exciton polypeptide of mutation (Xanthomonas campestris pv.glycines) or albumen of xanthomonas campestris soybean and have aminoacid sequence corresponding to following SEQ.ID.No.37:
Thr?Leu?Ile?Glu?Leu?Met?Ile?Val?Val?Ala?Ile?Ile?Ala?Ile?Leu?Ala
1???????????????5???????????????????10??????????????????15
Ala?Ile?Ala?Leu?pro?Ala?Tyr?Gln?Asp?Tyr
20 25 these sequences are anaphylaxis exciton polypeptide or the proteic amino terminal sequence that only has 26 residues that derives from the pathogenic mutation of xanthomonas campestris soybean.It is complementary with determined fimbrial protein subunit in other xanthomonas campestris causes a disease mutation.
Derive from the xanthomonas campestris Flos Pelargonii cause a disease the anaphylaxis exciton polypeptide of mutation (Xanthomonas campestris pv.pelargonii) or albumen be thermally-stabilised, to the proteolytic enzyme sensitivity, and molecular weight is 20kDa.It comprises the aminoacid sequence corresponding to following SEQ.ID.No.38:
Ser?Ser?Gln?Gln?Ser?Pro?Ser?Ala?Gly?Ser?Glu?Gln?Gln?Leu?Asp?Gln
1???????????????5???????????????????10??????????????????15
Leu?Leu?Ala?Met
20
Cui etc., " the RsInA mutant of carrot soft rot Erwinia carrot soft rot subspecies Ecc71 bacterial strain overexpression hrpNEcc and bring out anaphylaxis sample reaction in tobacco leaf; " described the separation of carrot soft rot Erwinia anaphylaxis exciton albumen or polypeptide among MPMI9 (7): the 565-73 (1996), the document is attached to herein by reference.Ahmad etc., " Harpin is to Si Shi Erwinia pathogenic optional in corn; " 8thInt ' l.Cong.Molec.Plant-Microb.Inter.1996 14-19 in July day and Ahmad etc., " Harpin is to Si Shi Erwinia pathogenic optional in corn; " Ann Mtg.Am.Phytopath.Soc.1996 has stated anaphylaxis exciton albumen or the polypeptide of Si Shi Erwinia in 27-31 in July day, described document is attached to herein by reference.
Kaman etc., and " from the extracellular protein exciton of phytophthora: to bacterium and tool specificity (most specificity) of fungal plant pathogen and induction of resistance, " Molec.Plant-MicrobeInteract., 6 (1): 15-25 (1993); Ricci etc., " derive from pathogenic agent fungi phytophthora, in tobacco, bring out necrosis and obtain the proteic structure and the activity of resistance, " Eur.J.Biochem., 183:555-63 (1989); Ricci etc., " isolate of phytophthora parasitica difference in tobacco produces the exciton of parasiticein and downright bad and resistance, " Plant Path.41:298-307 (1992); Baillreul etc., " anaphylactoid a kind of new exciton in the tobacco: fungi glycoprotein brings out necrocytosis; defence expression of gene; salicylic generation and inducible system obtain resistance; " PlantJ.8 (4): 551-60 (1995) and Bonnet etc., " the acquisition resistance that the exciton in tobacco and other plant triggers; " Eur.J.PlantPath., 102:181-92 (1996), it is mould to describe terrible autoparasitism epidemic disease, latent ground epidemic disease is mould, the camphor tree epidemic disease is mould, Phytophthora capsici, mould and mould anaphylaxis exciton albumen or the polypeptide of oranges and tangerines brown rot epidemic disease of big male epidemic disease, these documents all are attached to herein by reference.
According to another kind of anaphylaxis exciton of the present invention is from u.s. patent application serial number 09/136, the exciton of the bad rotten subspecies of comprehensively describing in 625 of Michigan rod shape bacillus (Clavibactermichiganensis subsp.sepedonicus), the document is attached to herein by reference.
Above-mentioned exciton is typical exciton.By under the condition of the gene of expressing the coding exciton, cultivate and bring out anaphylactoid fungi or bacterium, can identify other exciton.Can derive from the suitable plant tissue of infiltration of the acellular prepared product of culture supernatant by use, test its exciton activity (being local necrosis).
The present invention includes above-mentioned anaphylaxis exciton polypeptide or proteic fragment and from the fragment of the total length exciton of other pathogenic agent.
Can produce suitable fragment by several method.In first method,, produce the subclone of the proteic gene of the known exciton of coding by the subclone gene fragment according to conventional molecular genetics operation.In external or body, these subclones are expressed in bacterial cell to produce less albumen or peptide then, can test the exciton activity of described albumen or polypeptide according to the following stated method.
As a kind of adoptable method, can by with proteolytic ferment as Quimotrase or staphylococcal protein A,SPA enzyme or tryptic digestion total length exciton albumen, produce the proteic fragment of exciton.Different proteolytic ferments may be sheared exciton albumen in different sites based on the proteic aminoacid sequence of exciton.Some fragment that produces from proteolysis can be active resistance exciton.
In another scheme, according to understanding, through adopting many group-specific primerses of round pcr and the described albumen specific part of selection representative, the fragment that can synthesize described exciton protein gene to described albumen primary structure.Then with these fragment clonings in the suitable carriers to express the peptide or the albumen of brachymemma.
Chemosynthesis also can be used to prepare suitable fragment.The known amino acid sequence of the exciton that employing is to be produced carries out such synthesizing.Perhaps, make the total length exciton stand High Temperature High Pressure and produce fragment.These fragments can be separated through conventional method (for example chromatography, SDS-PAGE) then.
Suitable segmental example comprises the fragment of separating starch Erwinia anaphylaxis exciton not bring out anaphylactoid anaphylaxis exciton.Suitable fragment comprises the interior segments of the aminoacid sequence of the N end fragment of aminoacid sequence of C end fragment, SEQ.ID.No.23 of the aminoacid sequence of SEQ.ID.No.23 or SEQ.ID.No.23.The C end fragment of the aminoacid sequence of SEQ.ID.No.23 can cover the amino acid of (span) following sequence: SEQ.ID.No.23:169 and 403,210 and 403,267 and 403 or 343 and 403.The interior segments of the aminoacid sequence of SEQ.ID.No.23 can cover the amino acid of following sequence: SEQ.ID.No.23:105 and 179,137 and 166,121 and 150 or 137 and 156.Can identify other suitable fragment according to the present invention.
Another example of the useful fragment of anaphylaxis exciton (itself does not bring out anaphylaxis described fragment) is to contain the cause a disease protein fragments of amino acid/11 90-294 of aminoacid sequence (SEQ.ID.No.31) of mutation anaphylaxis exciton of pseudomonas syringae cloves.This fragment can be used to give disease resistance and promotes plant-growth.
A useful segmental example again of anaphylaxis exciton is the peptide that has corresponding to the aminoacid sequence of SEQ.ID.No.39.This peptide derives from and brings out the mould glycoprotein of anaphylactoid big male epidemic disease, and promotes plant-growth.
For example, by disappearance or add the amino acid that influences minimum, can produce varient to characteristic, secondary structure and the water-wet behavior aspect of described polypeptide.For example, polypeptide chain can be received on signal (or leading) sequence of albumen N-end of common translation or the described protein transport of translation guiding.Also described polypeptide chain can be received on joint or other sequence so that synthetic, purifying or identify described polypeptide.
Described fragment of the present invention is preferably isolating form (promptly isolating from its host living beings), and more preferably by routine techniques, produces with the form of purifying (preferably at least about 60%, more preferably 80%, purity).Usually, produce described fragment of the present invention but be not secreted in the growth medium of recombinant host cell.Perhaps, albumen of the present invention or polypeptide are secreted in the growth medium.Under the proteic situation of non-secretion, in order to separate described protein fragments, with carry host cell (for example intestinal bacteria) breeding of recombinant plasmid, through supersound process, thermal treatment or chemical treatment cracking, then that homogenate is centrifugal to remove bacterial debris.Then with described supernatant liquor heat treated and by the described fragment of centrifugation.To contain described segmental supernatant liquor part in the dextran of suitable size or polyacrylamide post through gel-filtration to separate described fragment.In case of necessity, can be further purified described protein part through ion-exchange or HPLC.
Adopt conventional recombinant DNA technology, described anaphylaxis exciton polypeptide of coding or proteic described segmental dna molecular can be incorporated in the cell.Generally speaking, this relates to that described dna molecular is inserted into described dna molecular is in allogenic (promptly not existing usually) expression system.Sense orientation and correct frame that described allogeneic dna sequence DNA molecule is suitable are inserted in described expression system or the carrier.Described carrier contains the essential element of transcribing and translating of the albumen coded sequence of described insertion.
No. the 4th, 237,224, the United States Patent (USP) (document is attached to herein by reference) of Cohen and Boyer has been described the cutting of employing restriction enzyme and has been connected the expression system of generation recombinant plasmid form with dna ligase.Adopt multiple method for transformation that these recombinant plasmids are introduced then, and duplicate in prokaryotic organism that in comprising tissue culture, grow and the eukaryotic single cell culture thing.
Also recombination can be incorporated into virus, in vaccinia virus.Can be by plasmid transfection be produced recombinant virus in the cell with virus infection.
Suitable carriers includes but not limited to that following virus vector is such as λ carrier system gt11, gtWES.tB, Charon 4 (Charon 4) and plasmid vector are such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV40, pBluescript II SK+/-or KS+/-(referring to " Stratagene Cloning Systems " Catalog (1993) from Stratagene, LaJolla, Calif, it is attached to herein by reference), pQE, pIH821, pGEX, pET series is (referring to F.W.Studier etc., GeneExpression Technology the 185th rolls up (1990) " to adopt the gene of the direct cloning by expression of T7 RNA polymerase ", and the document is attached to herein by reference) and any derivative.Especially transduce by transforming, joint, mobilization (mobilization) or electroporation, recombinant molecule can be incorporated in the cell.Adopt as Sambrook etc., Molecular Cloning:A Laborotoruy Manual, Cold Springs Laboratory, ColdSprings Harbor, standard cloning process in the described this area of New Yodk (1989), described dna sequence dna is cloned in the described carrier, and the document is attached to herein by reference.
Can utilize various host-vector systems to express described albumen coded sequence.At first, described carrier system must be complementary with used host cell.Host-vector system includes but not limited to the following stated host-vector system: with the bacterium of phage DNA, plasmid DNA or cosmid DNA conversion; Microorganism is such as the yeast that contains yeast vector; Unite with the mammal cell line that virus (for example vaccinia virus, adenovirus etc.) infects; Insect cell system with virus (for example baculovirus) infection; With by the vegetable cell of infectation of bacteria.The Expression element of these carriers is different aspect its intensity and specificity.According to the host-vector system that is adopted, can use many suitable any in the element transcribed and translate.
Different hereditary signals and processing incident are controlled the genetic expression (for example DNA's transcribes and the translation of messenger RNA(mRNA) (mRNA)) of multiple level.
DNA transcribes and depends on the guiding RNA polymerase in conjunction with the existence that also therefore starts the promotor of mRNA synthetic section of DNA sequence.The dna sequence dna of eukaryotic promoter and the dna sequence dna of prokaryotic promoter are variant.In addition, eukaryotic promoter and the hereditary signal of following may not discerned by prokaryotic system or may be inoperative in prokaryotic system, and prokaryotic promoter is not identified with inoperative in eukaryotic cell yet.
Equally, the translation of the mRNA in the prokaryotic organism depends on the existence of the suitable prokaryotic organism signal different with Eukaryotic those signals.MRNA in the prokaryotic organism effectively translates the ribosome bind site that needs to be called on the mRNA Shine-Dalgarno (" SD ") sequence.This sequence is one section initiator codon that is positioned at the N-terminal methionine(Met) of code for said proteins, normally the mRNA nucleotide sequence of the weak point before the AUG.3 ' of SD sequence and 16S rRNA (ribosome-RNA(rRNA))-end is complementary, may be attached on the rrna by forming duplex startup mRNA with described rRNA then, so that correctly locate this rrna.About making the maximized summary of genetic expression, referring to Roberts and Lauer, Methods in Enzymology, 68:473 (1979), the document is attached to herein by reference.
Promotor is difference aspect its " intensity " (being that they start the ability of transcribing).For the gene of cloning by expression, preferably adopt strong promoter to transcribe to obtain high level, and so expressing said gene.Based on the host cell systems that is adopted, can use any in many suitable promotors.For example, when its phage of clone or plasmid in intestinal bacteria, can be with the P of promotor such as T7 phage promoter, lac promotor, trp promotor, rec promotor, ribosome-RNA(rRNA) promotor, escherichia coli RAnd P LPromotor and other promotor include but not limited to lacUV5, ompF, bla, lpp etc., are used for guiding high level to transcribe adjacent DNA section.In addition, the genetic transcription that hybrid trp-lacUV5 (tac) promotor that produces by recombinant DNA or other synthetic DNA technology or other escherichia coli promoter can be used to provide insertion.
If not bacterial host cell bacterial strain and the expression vector that can select specificity to induce then to suppress the promotor effect.In some operation, in order effectively to transcribe the DNA of insertion, the specific inductor of essential adding.For example, add lactose or IPTG (isopropylthio-) and induce the lac operon.Various other operons are to be under the different control such as trp, pro etc.
Effectively the transcribing and translating of gene in prokaryotic cell prokaryocyte also needs specific start signal.Messenger RNA(mRNA) and institute's proteinic amount of synthetic according to gene specific are measured respectively, these transcribe with translation initiation signal can be different aspect " intensity ".The DNA expression vector that contains promotor can comprise that also any various " by force " transcribe and/or the composition of translation initiation signal.For example, the effective translation in the intestinal bacteria needs the SD sequence of initiator codon (" ATG ") 5 ' about 7-9 base so that ribosome bind site to be provided.Therefore, can adopt and to make up by any SD-ATG that the host cell rrna uses.Such combination includes but not limited to from the cro gene of escherichia coli or N gene or from the SD-ATG combination of intestinal bacteria tryptophane E, D, C, B or A gene.In addition, can use and relate to any SD-ATG combination that the recombinant DNA that mixes synthetic Nucleotide or other technology produce.
Anaphylaxis exciton polypeptide or proteic segmental isolated DNA molecule are cloned in the expression system in case will encode, and just it can be incorporated in the host cell at any time.Based on described carrier/host cell systems, can carry out such mixing by above-described various forms of conversions.Proper host cell includes but not limited to bacterium, virus, yeast, mammalian cell, insect, plant etc.
The invention still further relates to conferring disease resistance in plants, promote plant-growth and/or the method that realizes preventing and treating plant insect.These methods relate to, effectively give disease resistance, promote under the condition of growth and/or pest control for described fragment, will be applied to whole strain plant or plant part or plant seed at anaphylactoid anaphylaxis exciton polypeptide of not bringing out of non-infectious form or proteic fragment.Perhaps, these fragments of anaphylaxis exciton albumen or polypeptide can be applied to plant, so as from the seed of this kind of plant self results can conferring disease resistance in plants, promote plant-growth and/or realize pest control.
As being applied to plant or plant seed, anaphylaxis exciton polypeptide or proteic fragment, can use transgenic plant or plant seed so that give plant or the plant conferring disease resistance in plants of cultivating by described plant seed, promote the alternative method of plant-growth and/or pest control.If use transgenic plant, this method relates to: provide with coding anaphylaxis exciton polypeptide or proteic segmental dna molecular transgenic plant transformed, described fragment is not brought out anaphylaxis, and cultivates described plant under the condition that effectively allows the sort of dna molecular conferring disease resistance in plants, promotion plant-growth and/or pest control.Perhaps, can provide with coding anaphylaxis exciton polypeptide or proteic segmental dna molecular transgenic plant transformed seed, described fragment is not brought out anaphylaxis; And with described planting seed in soil.Effectively allowing the sort of dna molecular conferring disease resistance in plants, promoting under the condition of plant-growth and/or pest control, then from the seminal propagation plant of sowing.
Can be in many ways, comprise: 1) application a kind of isolating fragment or 2) use the bacterium that does not cause disease and use the described segmental gene transformation of coding, enforcement is applied to anaphylaxis exciton polypeptide or albumen embodiment of the present invention of described plant or plant seed.In the embodiment of back, contain the bacterium of the dna molecular of coding described anaphylaxis exciton polypeptide or proteic fragment (described fragment is not brought out anaphylaxis) by application, described fragment can be applied to plant or plant seed.Such bacterium must secrete or export described fragment, so that described can contact plant or plant seed cell.In these embodiments, by in plant or seed or just in time before described bacterium is introduced described plant or plant seed, produce described fragment by bacterium.
Can use described method of the present invention to handle various plants or their seed to give disease resistance, to promote growth and/or pest control.Suitable plant comprises dicotyledons and monocotyledons, more particularly, useful crop can comprise: clover, paddy rice, wheat, barley, rye, cotton, Sunflower Receptacle, peanut, corn, potato, sweet potato, Kidney bean, pea, witloof, lettuce, Herba Sonchi Arvensis, wild cabbage, brussels sprouts, beet, parsnip, turnip, Cauliflower, blue and white cabbage, radish, spinach, onion, garlic, eggplant, pepper, celery, Radix Dauci Sativae, pumpkin, summer squash (pumpkin), summer squash (zucchini), cucumber, apple, pears, muskmelon, citrus, strawberry, grape, immature fruit of Juteleaf Raspberry, pineapple, soybean, tobacco, tomato, jowar and sugarcane.The example of suitable ornamental plant is: Arabidopis thaliana (Arabidopsis thaliana), African violet belong to (Saintpaulia), green winter eggplant, Flos Pelargonii, poinsettia, chrysanthemum, carnation and youth-and-old-age.
, cannot give the absolute immunity that opposing is infected, but reduce the severity of described disease, and symptom is postponed in the purposes of giving aspect the disease resistance about the fragment of anaphylaxis exciton albumen of the present invention or polypeptide.The quantity of scab number, scab size and fungal pathogens sporulation has all reduced.This method of giving disease resistance has the disease that treatment before can not treat, the disease that systemic treatment can not be treated respectively owing to the reason of expense, and has avoided using the potentiality of deleterious material on infectious factor or the environment.
The method of giving plant pathogen resistance according to the present invention can be used for giving the resistance that comprises virus, bacterium and fungi at various pathogenic agent.Obtain especially to following virus by method of the present invention: the disease resistance of tobacco mosaic virus (TMV) and Tomato mosaic virus.Also can give plant especially to the disease resistance of following bacterium according to the present invention: aeruginosa eggplant, pseudomonas syringae tobacco cause a disease mutation (Pseudomonas syringae pv.tabaci) and the pathogenic mutation of xanthomonas campestris Flos Pelargonii.By adopting method of the present invention, plant can obtain especially the disease resistance to following fungi: sharp sickle spore (Fusarium oxysporum) and phytophthora infestans (Phytophthorainfestans).
With regard to the fragment of using anaphylaxis exciton albumen of the present invention or polypeptide promotes plant-growth, can obtain various forms of plant-growths enhancings or promotion.When seed begins or afterwards the life plant, this effect all can produce as far back as plant-growth.For example, according to plant-growth of the present invention comprise higher output, the seed quality produced improves, the seed germination percentage improves, increases of plant size, higher biomass, more bigger fruits, fruit more morning painted and more precocity fruiting and plant maturation.Therefore, the present invention provides remarkable economic efficiency to the producer.For example, germination early and precocity make crop can lack the area growth that the season of growth can not be grown crop in other cases.The raising of rate of emergence causes improving crop plant density and more effective seed that utilizes.The size of higher output, increase and the biomass yield that improves make from given splat and produce higher income.
Further aspect of the present invention relates to any type of plant insect control of realization.For example, according to pest control of the present invention comprise the direct insect infringement that prevents insect contact and used the plant of anaphylaxis exciton, prevented to cause by damage that plant is ingested, make insect away from such plant, kill insect near such plant, disturb the insect of such plant that ingests larva, prevent that insect from settling down host plant, preventing that the insect of settling down from discharging plant poison etc.The present invention also prevents to infect cause plant disease is afterwards damaged by insect.
At various insect the present invention is effective.European corn borer is a kind of main insect of corn (molar shape sweet corn), but it also ingesting above on the 200 kind of plant species, comprises tender pod Kidney bean, wax bean and lima bean and human consumption soybean, pepper, potato and tomato and many weed species.The insect that ingests of the insect larva of other the various vegetable crops of infringement comprises following insect: the real noctuid of beet armyworm, cabbage looper, paddy, autumn noctuid, small cabbage moth, wild cabbage root kind fly (cabbage root maggot), onion fly, beautiful art kind fly, the wild snout moth's larva (melon worm) of melon, pepper band trypetid, the moth-eaten moth of tomato and maggot.In general, this monoid insect has been represented the worldwide most important economically insect monoid of vegetables production.
If handle whole strain or the described plant of part, comprise when leaf, stem, root, propagulum (for example transplanting a cutting) wait, can implement to comprise the method for the present invention of application anaphylaxis exciton polypeptide or proteic fragment (described fragment is not brought out anaphylaxis) by various methodologies.This method can (but needn't) relate to described anaphylaxis exciton polypeptide or proteic fragment are soaked into described plant.The suitable applications method comprises that high pressure or low pressure are sprayed, injection and use blade wear when taking place near exciton.When handling plant seed or propagulum (for example transplanting a cutting) according to application implementation scheme of the present invention, can use according to anaphylaxis exciton polypeptide of the present invention or proteic fragment by high pressure or low pressure sprinkling, coating, immersion or injection.Those skilled in the art it is contemplated that other suitable applications method, and prerequisite is that they can realize the cells contacting with described fragment and described plant or plant seed.In case after handling with the fragment of anaphylaxis exciton of the present invention, just with described planting seed in nature or artificial soil, and adopt the ordinary method cultivation with the generation plant.With plant after use the seminal propagation of handling according to the present invention to come out, can one or many use the fragment or the complete exciton of described anaphylaxis exciton albumen or polypeptide and handle described plant, with conferring disease resistance in plants, promote plant-growth and/or prevent and treat the insect of described plant.
Can be with according to anaphylaxis exciton polypeptide of the present invention or proteic fragment, be applied to plant or plant seed individually or with the mixture of other material.Perhaps, described fragment can be applied to plant individually, and use other material at different time.
Be suitable for containing anaphylaxis exciton polypeptide or proteic fragment according to the composition of application implementation scheme processing plant of the present invention or plant seed in carrier, described fragment is not brought out anaphylaxis.Suitable carriers comprises water, the aqueous solution, slurries or dry powder.In this embodiment, described composition contains the described fragment that is higher than 500nM.
Although optional, said composition can contain other additive, comprises fertilizer, sterilant, mycocide, nematocides and composition thereof.Suitable fertilizer comprises (NH 4) 2NO 3The example of suitable sterilant is the Malathion.Effectively mycocide comprises Vancide 89.
Other suitable additive comprises buffer reagent, wetting agent, coating agent and abradant.These materials can be used to help method of the present invention.In addition, can bring out anaphylactoid fragment with other conventional seed preparations with handle material (comprising clay and polysaccharide) and be applied to plant seed with described.
In relating to the alternate embodiment of the present invention of using transgenic plant and transgenic seed, do not need the fragment of anaphylaxis exciton is applied to described plant or seed usually.But, according to method well-known in the art, produce with the so segmental dna molecular transgenic plant transformed of coding.
By adopting the described recombinant DNA of micropipette mechanical transfer, can be in vegetable cell with carrier direct microinjection recited above.Crossway, Mol.Gen.Genetics, 202:179-85 (1985), the document is attached to herein by reference.Adopt polyoxyethylene glycol, also can be in described vegetable cell with transfer of genetic material.Krens etc., Nature, 296:72-74 (1982), the document is attached to herein by reference.
Another kind of scheme with the gene-transformed plant cell of giving pathogen resistance is the particle bombardment (being also referred to as biolistic transforms) of described host cell.Can be with a kind of this scheme of realization in the several method.At first relate to inert particle or bioactive particles are advanced cell.All be the United States Patent (USP) the 4th, 945,050,5,036,006 and 5,100 of Sanford etc., disclose this technology No. 792, these documents are attached to herein by reference.Generally, this method relates to, and effectively penetrating described cell outer surface and being impregnated under its inner condition, inert particle or bioactive particles is advanced described cell.If the use inert particle then can be introduced described carrier in the described cell by using the carrier bag that contains described allogeneic dna sequence DNA by described particle.Perhaps, described carrier can surround described target cell so that by the wake flow (wake) of described particle described carrier is brought in the described cell.Also bioactive particles (bacterial cell of doing that for example contains described carrier and allogeneic dna sequence DNA) can be advanced vegetable cell.
Another method of introducing is that protoplastis merges with other entity, and described other entity is or the corpusculum (fusiblelipid-surfaced bodies) on minicell, cell, lysosome or other the lipid surface that can merge.Fraley etc., Proc.Natl.Acad.Sci.USA, 79:1859-63 (1982), the document is attached to herein by reference.
Also described dna molecular can be introduced described vegetable cell by electroporation.Fromm etc., Proc.Natl.Acad.Sci.USA, 82:5824 (1985), the document is attached to herein by reference.In this technology, containing in the presence of the plasmid of expression cassette, the plant protoplast electroporation.The high field intensity electricimpulse reversibly makes saturatingization of microbial film and introduces plasmid.The plant protoplast of electroporation forms cell walls, division and regeneration again.
With the another kind of method of described dna molecular introduced plant cell is with agrobacterium tumefaciens (Agrobacterium tumefaciens) or Agrobacterium rhizogenes (A.rhizogenes) the infection plant cell of using described gene transformation in advance.Under conditions suitable known in the art, cultivate described plant transformed cell to form bud or root, further develop into plant then.Generally speaking, this method relates to bacterial suspension inoculates described plant tissue, is not containing on the antibiotic regeneration culture medium described tissue cultivating 48-72 hour in 25-28 ℃ then.
Agrobacterium (Agrobacterium) is the representative genus of of Gram-negative Rhizobiaceae.Its bacterial classification causes root knot (agrobacterium tumefaciens) and hair root (Agrobacterium rhizogenes).Vegetable cell in crown gall tumor and the root of hair is induced the amino acid derivative that is called opine with generation, and opine is only by described bacterial degradation metabolism.Cause that the bacterial gene that opine is expressed is the facility source of the controlling elements of chimeric expression box.In addition, the existence of mensuration opine can be used to identify the tissue of conversion.
With Ti-plasmids of agrobacterium tumefaciens or the Ri plasmid of Agrobacterium rhizogenes, the allogeneic heredity sequence can be introduced suitable vegetable cell.By agroinfection, Ti-plasmids or Ri plasmid are delivered to vegetable cell, and stably are incorporated in the described Plant Genome.J.Schell, Science, 237:1176-83 (1987), the document is attached to herein by reference.
After the conversion, must be with described plant transformed cell regeneration.
At Evans etc., Handbook of Plant Cell Cultures. the 1st volume: (MacMillanPublishing Co., New York, 1983) and Vasil I.R. (writing), Cell Culture andSomatic Cell Genetics ofPlants, Acad.Press, Odando, the 1st volume, 1984 and III volume (1986) in described from the protoplast regenerated plant of cultivating, described document is attached to herein by reference.
Know that in fact all plants can include but not limited to sugarcane, beet, cotton, fruit tree and fabaceous all main species from cultured cells or tissue regeneration.
The regenerated method plant species with plant between different, but generally at first provide conversion protoplastis suspension or contain the culture dish of the explant of conversion.Form callus, sprout from callus induction then, and long subsequently root.Perhaps, can in callus, induce embryogeny.These embryos are sprouted as natural embryo to form plant.Described substratum generally contains each seed amino acid and hormone such as plant hormone and phytokinin.Preferably also L-glutamic acid and proline(Pro) are joined in the described substratum, especially to the species as corn and clover.Effectively substratum, genotype and cultivation history are depended in regeneration.If controlled these three kinds of variable factors well, so regeneration normally can reproduce and repeatably.
After expression cassette stably was incorporated into transgenic plant, it can transfer to other plant by sexual hybridization.According to the species of being hybridized, can adopt any in many standard breeding techniques.
In case produce the transfer-gen plant of the type, just can be according to ordinary method with described plant self cultivation, the existence of the segmental gene of coding anaphylaxis exciton causes disease resistance, promotes plant-growth and/or prevents and treats plant insect.Perhaps, reclaim transgenic seed or propagulum (for example transplanting a cutting) from transgenic plant.Then with described planting seed in soil, and adopt the ordinary method cultivation to produce transgenic plant.Under the condition of effective conferring disease resistance in plants, promotion plant-growth and/or pest control, from the transgenic seed breeding transgene plant of sowing.Although do not wish to be limited by theory, such disease resistance, growth and/or pest control can or can produce because of the expression of described polypeptide or protein fragments by the RNA mediation.
If use transgenic plant and plant seed according to the present invention, then they can also be with handling with being used for handle using according to the segmental described Plants and Seeds identical materials of anaphylaxis exciton of the present invention.Can be by comprising the aforesaid method of high pressure or low pressure sprinkling, injection, coating and immersion, will comprise according to segmental these other the material of anaphylaxis exciton of the present invention being applied to described transgenic plant and plant seed.Equally,, described plant can be used according to segmental one or more application methodes of anaphylaxis exciton of the present invention and handle, to give disease resistance, to promote growth and/or pest control behind described transgenic plant seed breeding plant.Also available conventional plant treatment agent (for example sterilant, fertilizer etc.) is handled this kind of plant.
EXAMPLE Example 1-bacterial isolates and plasmid
In following examples used coli strain comprise respectively from Gibco BRL (GrandIsland, N.Y.) and Stratagene (La Jolla, CA) DH5 α of Gou Maiing and BL21 (DE3).(Madison WI) buys pET28 (b) carrier from Novagen.Complete hrpN gene is contained in Eco DH5 α/2139.2139 constitute the D.Bauer production of thing by Comeil university.Cut digestion with HindIII through restriction enzyme, the hrpN gene is downcut from 2139 plasmids, then the dna profiling of cloning with hrpN as the synthetic brachymemma of PCR through the sepharose purifying.Subsequently these clones are inserted into and contain Kan r(His) of gene 6Be used to select transformant among the carrier pET28 (b).Embodiment 2-DNA operation
From Boeringer Mannheim (Indianapolis, IN) or Gibco BRL obtain restriction enzyme.Obtain T4 dna ligase, calf intestinal alkaline phosphatase (CIAP) and PCR Supemix from Gibco BRL TMBuy from Qiagen (Hilden, Germany) that QIAprep rotation (spin) prepares test kit in a small amount, the Qiagen plasmid prepares test kit and QIAquick PCR purification kit in a small amount.By Lofstrand Labs company limited (Gaithersburg, MD) synthetic pcr primer thing.By Bio-SynthesisInc. (LewisVille, TX) synthetic oligopeptide.According to standard technique (Sambrook etc., Laboratory Manual, second edition, Cold Spring HarborLaboratory Press (1989)) or the scheme that provides according to manufacturers, carry out all DNA operations, cut digestion, DNA connection and PCR such as plasmid separation, restriction enzyme.The fragmentation of embodiment 3-hrpN gene
Produce the hrpN gene and the interior segments (Fig. 1) of a series of N end and the brachymemma of C end through PCR.Total length hrpN gene as dna profiling, is designed for the clone's of each brachymemma 3 ' primer and 5 ' primer (Fig. 2).3 ' primer comprises a NdeI enzyme cleavage site that contains initiator codon ATG (methionine(Met)), and 5 ' primer comprises terminator codon TAA and a HindIII enzyme cleavage site that is used for being connected to pET28 (b) carrier.At GeneAmp TMCarry out PCR in 9700 (Perkin-Elmer, Foster City CA), the 0.5ml pipe.With 45 μ lSupermix TM(Life Technology, Gaithersburg is MD) with every pair of dna primer of 20pmoles, 10ng total length harpin DNA and deionization H 2O is mixed to final volume 50 μ l.Mixture in 95 ℃ of heating after 2 minutes, is continued 1 minute, 58 ℃ in 94 ℃ and continues to continue 1.5 minutes in 1 minute and 72 ℃, carry out circulate for PCR30 time (cycles).(Novex, San Diego CA) go up this PCR product of confirmation at the 6%TBE gel.DNA QIAquick PCR purification kit purifying with amplification digested 5 hours with Nde I and Hind III in 37 ℃, and use phenol: chloroform: ethanol sedimentation is once used in primary isoamyl alcohol (25: 25: 1) extracting then.With 5 μ g pET28 (b) carrier DNAs with the Hind III of the Nde I of 15 units and 20 units in 37 ℃ of digestion 3 hours, handle to reduce background with CIAP then by incomplete single enzymic digestion was produced.With the carrier DNA of digestion QIAquick PCR purification kit purifying, be directly used in connection then.In the 15ul mixture of the pET28 (b), 30ng orientation (targeted) PCR fragment and 1 T4DNA of the unit ligase enzyme that contain the 200ng digestion of having an appointment, in the 14-16 ℃ of ligation of carrying out 5-12 hour.5-7.5 μ l is connected solution to join in the 100 μ l DH5 α competent cells in the 15ml Falcon pipe, then in cultivating 30 minutes on ice., after 45 seconds 0.9mlSOC solution or 0.45ml LB substratum are joined in every arm in 42 ℃ of heat shocks, then in 37 ℃ of incubations 1 hour.20ul, 100 μ l and 200 μ l cell transformed are added on the LB agar that contains 30 μ g/ml kantlex, are incubated overnight in 37 ℃ then.With single colony lift to 3ml LB substratum, then in 37 ℃ of overnight incubation.(QIAGEN, Hilden Germany) prepare plasmid DNA from the 2ml culture to prepare test kit in a small amount with QIAprep.The DNA that derives from described transformant is checked order to confirm described successfully conversion through restriction enzyme digestion analysis or part.Adopt aforesaid standard chemical method for transformation, the plasmid that will contain the target DNA sequence is transferred in the BL21 bacterial strain.Generation is as containing the proteic clone of total length harpin in pET28 (b) carrier of positive control, and produces the clone who only contains pET28 (b) carrier as negative control.The proteic expression of embodiment 4-anaphylaxis exciton brachymemma
E. coli bl21 (DE3) bacterial strain that will contain described hrpN clone is in the Luria broth culture that contains 30 μ g/ml kantlex (5g/L Difco yeast extract, 10g/L Difco Tryptones, 5g/L NaCl and 1mM NaOH), in 37 ℃ of grow overnight.Then in the same medium with described microbionation to 100 times volume, in 37 ℃ of OD that grow to 0.6-0.8 620Then in the same medium with described microbionation to 250 times volume, grow to about 0.3 or the OD of 0.6-0.8 in 37 ℃ 620Add 1mM IPTG then, and with culture in 19 ℃ of grow overnight (about 18 hours).Adopt this strategy all described clones successfully can not to be expressed.(12g/L bacto-tryptone, 24g/L bacterium are used yeast, 0.4% glycerine, 0.17M KH to have several clones to have to grow in Temfic meat soup 2PO 4, 0.72K 2HPO 4) in, and/or IPTG induces the back in 37 ℃ of growths, and/or early than the time results (table 1) of spending the night.
Table 1: the proteic expression of anaphylaxis exciton brachymemma
Fragment Amino acid (SEQ.ID. No.23) Growth medium Induce O.D. Express temperature Harvest time
1 (+contrast) ?1-403 ????LB About 0.3 or 0.6-0.8 19 ℃ or 25 ℃ ????16-18hr
2 (+contrasts) ????- LB and TB About 0.3 or 0.6-0.8 19 ℃ and 37 ℃ ????16-18hr
????3 ?105-403 ????LB ?0.6-0.8 ????19℃ ????16-18hr
????4 ?169-403 ????TB About 0.3 ????19℃ ????16-18hr
????5 ?210-403 LB or M9ZB ?0.6-0.8 ????19℃ ????16-18hr
????6 ?257-403 LB or M9ZB ?0.6-0.8 ????19℃ ????16-18hr
????7 ?343-403 ????LB About 0.3 ????19℃ ????5hr
????8 ?1-75 ????TB About 0.3 ????37℃ ????16-18hr
????9 ?1-104 ????TB About 0.3 ????37℃ ????16-18hr
????10 ?1-168 ????TB About 0.3 ????37℃ ????16-18hr
????11 ?1-266 ????LB About 0.3 ????37℃ ????4hr
????12 ?1-342 ????LB ?0.6-0.8 ????19℃ ????16-18hr
????13 ?76-209 ????LB About 0.3 ????37℃ ????5hr
????14 ?76-168 TB or LB About 0.3 ????37℃ 3hr or 16-18hr
????15 ?105-209 ???M9ZB About 0.3 ????37℃ ????3hr
????16 ?169-209 Do not have and express
????17 ?105-168 ????LB About 0.3 ????37℃ ????3-5hr
????18 ?99-209 ????LB About 0.3 ????37℃ ????3hr
????19 ?137-204 ????LB About 0.3 ????37℃ ????3hr
????20 ?137-180 ????LB About 0.3 ????37℃ ????16-18hr
????21 ?105-180 ????LB About 0.3 ????37℃ ????3hr
????22 ?150-209 Do not have and express
????23 ?150-180 Do not have and express
The proteic small scale purification of embodiment 5-anaphylaxis exciton brachymemma (confirming to express)
50ml hrpN clone's culture is as above grown with the albumen of the described brachymemma of abduction delivering.Behind the described culture of results, in 14, centrifugal 5 minutes of 000rpm is suspended in urea lysis buffer (8M urea, 0.1M Na once more with the described cell suspending liquid of 1.5ml 2HPO 4With 0.01M Tris-pH8.0) in, incubation is 10 minutes under room temperature, and then in 14, centrifugal 10 minutes of 000rpm preserves described supernatant liquor then.With equilibrated (His) 6-join in the described supernatant liquor in conjunction with the duplicate samples such as 50 μ l of 50% slurries of nickel agarose resin, mixed 1 hour in 4 ℃.Then the nickel agarose is used urea lavation buffer solution (8M urea, 0.1M Na 2HPO 4With 0.01M Tris--pH6.3) washing three times, the washing between in 5, centrifugal 5 minutes of 000rpm.With 50 μ l urea elution buffer (8M urea, 0.1M Na 2HPO 4, 0.01M Tris and 0.1M EDTA--pH6.3), described albumen is eluted from this resin.According to the proteic size of described brachymemma, with described eluate 4-20%, 16% or 10-20%Tris-glycine precast gel on electrophoresis, to confirm described expression.Embodiment 6-tobacco HR induces
Growth is used for the duplicate samples such as 1.5ml of the proteic 50ml culture of the described brachymemma of small scale purification, and in 14, centrifugal 4 minutes of 000rpm is suspended in the potassium phosphate buffer of isopyknic 5mM, pH6.8 once more.With about 30 seconds of described cell suspending liquid supersound process, use potassium phosphate buffer then with 1: 2 and dilution in 1: 10.By hole of mill in individual blade, two kinds of dilutions are added the 4th to the 9th leaf that clean cell lysate soaks into the tobacco plant of 10-15 sheet leaf, and adopt the syringe of needle-less that described bacterial lysate is soaked in the blade intercellular space.Soaked into the back 24-48 hour, record HR reaction.Envrionment temperature, the periodicity of illumination of tobacco (Nicotiana tabacum v.Xanthi) seedling in 20-25 ℃ is 12 hours illumination/12 hour dark and grows for about 40%RH time.Because on a small scale urea purification produces considerably less because the albumen of the sex change that purge process causes is measured (with the albumen of the active brachymemma of screening HR) so cell lysate is used for initial HR.Embodiment 7-is used for anaphylaxis exciton brachymemma proteic of comprehensive organism determination of activity
Extensive natural purifying
As discussed previously, cultivate the albumen of 6 bottles of 500ml hrpN clones' culture with the described brachymemma of abduction delivering.Behind the described culture of results, with described cell in 7, centrifugal 5 minutes of 000rpm, being suspended in imidazoles lysis buffer (5mM imidazoles, 0.5MNaCl and 20mMTris) once more adds in 0.05%Triton X-100 and the 0.1mg/ml N,O-Diacetylmuramidase, in 30 ℃ of incubations 10 minutes, supersound process 2 minutes was once more in 15, centrifugal 20 minutes of 000rpm preserves described supernatant liquor then.With equilibrated (His) 6-join in the described supernatant liquor in conjunction with the duplicate samples such as 4ml of 50% liquid of nickel agarose resin slurry, mixed about 4 hours in 4 ℃.Then the nickel agarose is washed three times with imidazoles lavation buffer solution (20mM imidazoles, 0.5M NaCl and 20mM Tris), in 5, centrifugal 5 minutes of 000rpm places disposable chromatography post then between washing.With described chromatography column in centrifugal 1 minute of 1100rpm to remove the lavation buffer solution of any remnants, then through described post and elution buffer one being arised under the room temperature incubation 10 minutes, then in 1100rpm with centrifugal 1 minute of described post, with 4ml imidazoles elution buffer (1M imidazoles, 0.5M NaCl and 20mM Tris), described albumen is eluted from this resin.According to the proteic size of described brachymemma, with described eluate 4-20%, 16% or 10-20%Tris-glycine precast gel on electrophoresis, to confirm described expression.Through described protein band is compared with the standard protein band in Mark 12 molecular weight markers, determine described proteic concentration.Embodiment 8-is used for anaphylaxis exciton brachymemma proteic of comprehensive organism determination of activity
Extensive urea purification
Present method is identical with the method for above-mentioned extensive natural purifying, only is to use urea lysate, urea lavation buffer solution and urea elution buffer, and described cell supersound process unlike above-mentioned natural purifying.Behind the purifying, to the more and more urea dialysis of lower concentration, warp is to 10mM Tris/20mM NaCl dialysed overnight, with described protein renaturation then in 8 hours in elder generation.Described renaturation process causes N end protein precipitation.Described sedimentary 1-168 albumen through adding 100mM Tris-HCl, pH10.4 dissolving, is heated described albumen about 1 hour in 30 ℃ then.Through described protein band is compared with the standard protein band in Mark 12 molecular weight markers, determine described proteic concentration.Adopt this strategy, successfully do not dissolve 1-75 and 1-104 protein fragments, so their supersound process in 100mM Tris-HCl, pH10.4 and are exposed described proteic avtive spot and are used for biological activity determination with the many described albumen of dissolving as much as possible.Embodiment 9-growth-promoting effect (GE) induces
With 60 tomatoes (Lycopersicon spp.cv.Marglobe) seed soaked overnight in the albumen of 10 μ g/ml that use 5mM potassium phosphate buffer, pH6.8 dilution and the described brachymemma of 20 μ g/ml.The next morning, described 60 planting seeds in 3 basins, after 12-15 days and after 18-20 days, are measured the height of high tomato plant of every basin 10 strains, and compare with the height of the adjoining tree of only handling with phosphate buffered saline buffer.Aspect height, analyze the height of the plant that contrasts with the height of the plant determining to handle and described damping fluid and compare whether there is significant difference, thereby determine whether described protein induced growth-promoting effect with the albumen of described brachymemma.Embodiment 10-system obtains inducing of resistance (SAR)
3 strain tobaccos (the Nicotiana tabacum cv.Xanthi) plant (about 75 days plant) that will have 8-12 sheet leaf are used for described mensuration.Wherein a slice leaf of described tobacco plant is covered, and remaining blade sprays with the protein solution of the described brachymemma of 20 μ g/ml of the 5mM potassium phosphate buffer dilution of about 50ml.After 5-7 days, 2 leaves (not blade of Pen Saing and the just sprinkling blade on the blade that sprays in contrast) are inoculated with 20 μ l solution and the pugil diatomite of 1.8 μ g/ml TMV, promptly inoculated by rubbing along the upper surface of described blade with described mixture.TMV enters in this plant by the atomic little damage that diatomite forms.TMV inoculates back about 3-4 days, the scab number on the processing blade of scab number on these two leaves and the slow middle liquid of negative control is compared the number of counting TMV scab.By comparing, analyze to determine to reduce the usefulness of TMV scab number by this protein fragments with described damping fluid contrast.The percentage that calculates usefulness is: the TMV scab reduces (% usefulness)=100 * (mean number of scab on the mean number of scab on the 1-processing blade/damping fluid contrast blade).The proteic expression of embodiment 11-anaphylaxis exciton brachymemma
Carrying out proteic small-scale expression of described fragment and purifying expresses and HR activity (table 2) with screening.
Table 2
The proteic expression of anaphylaxis exciton brachymemma and HR activity (screening on a small scale)
Fragment # Amino acid (SEQ.ID. No.23) Express The HR activity
1 (+contrast) ????1-403 ????+ ????+
2 (contrasts) ??????- The albumen of only having powerful connections ????-
????3 ????105-403 ????+ ????+
????4 ????169-403 ????+ ????-
????5 ????210-403 ????+ ????-
????6 ????267-403 ????+ ????-
????7 ????343-403 ???+/- ????-
????8 ????1-75 ????+ ????-
????9 ????1-104 ????+ ???+/-
????10 ????1-168 ????+ ????+
????11 ????1-266 ????+ ????+
????12 ????1-342 ????+ ????+
????13 ????76-209 ????+ ????+
????14 ????76-168 ????+ ????-
????15 ????105-209 ????+ ????+
????16 ????169-209 ????- ????-
????17 ????105-168 ????+ ????-
????18 ????99-209 ????+ ????+
????19 ????137-204 ????+ ????+
????20 ????137-180 ????+ ????+
????21 ????105-180 ????+ ????+
????22 ????150-209 ????- ????-
????23 ????150-180 ????- ????-
All described clones' fragment albumen is the horizontal expression to change all, just except three small segments (amino acid/11 69-209,150-209 and 150-180).Fragment 210-403 and 267-403 express very goodly, produce the albumen of high density from small scale purification, occur protein band clearly on sds gel electrophoresis.Other fragment (such as amino acid/11-168 and 1-104) produces the albumen of much less, occurs fuzzy protein band on electrophoresis.Be difficult to determine whether to express fragment 343-403, i.e. Zui Xiao C end protein is because having several tangible background albumen on the described gel except that the 343-403 albumen of suspecting., test comprises described total length anaphylaxis exciton albumen respectively and only comprises the proteic positive control albumen of background and proteic expression of negative control and HR activity.
Carry out proteic extensive expression of described fragment and purifying to determine expression level and active tire (table 3) of HR.
Table 3
The proteic expression level and the HR of anaphylaxis exciton brachymemma tires (pure on a large scale
Change)
Fragment # Amino acid (SEQ ID.No.23) Express HR tires
1 (+contrast) ????1-403 ???3.7mg/ml ????5-7μg/ml
2 (contrasts) ?????- ??????- Dilution in 1: 2
????4 ????169-403 ???2mg/ml ???????-
????5 ????210-403 ???5mg/ml ???????-
????6 ????267-403 ???4mg/ml ???????-
????7 ????343-402 ???200μg/ml ???????-
????8 ????1-75 ???50μg/ml ???????-
????9 ????1-104 ???50μg/ml 3 μ g/ml (dilution in 1: 16)
????10 ????1-168 ???1mg/ml ???1μg/ml
????13 ????76-209 ???2.5mg/ml ???5μg/ml
????14 ????76-168 ???2mg/ml ???????-
????15 ????105-209 ???5mg/ml ???5-10μg/ml
????17 ????105-168 ???250μg/ml ???????-
????19 ????137-204 ???3.6mg/ml ???3.5μg/ml
????20 ????137-180 ???250μg/ml ???16μg/ml
Be chosen in the evaluation anaphylaxis exciton and think that the albumen of most important described brachymemma is used for extensive expression.Positive control (total length anaphylaxis exciton) is with the relative high level expression of 3.7mg/ml.All described C end proteins are with the relative high level expression of 2-5mg/ml, are except the fragment 343-403 as discussed previously.Described N end fragment is also expressed very goodly; Yet, during purge process, described albumen precipitation and dissolving seldom again.Described concentration in the table 3 has only reflected described dissolved albumen.Described interior segments is expressed in the scope of 2-3.6mg/ml.The utmost point be difficult to determine fragment 105-168 concentration (described according to estimates concentration may than shown in much higher) because the protein band on the sds gel is big fragment, but dyeing is very shallow.Described negative control comprises several background albumen as expection, but does not induce advantage albumen significantly.Embodiment 12-tobacco HR induces
Described total length positive control albumen just brings out HR to 5-7 μ g/ml only less.Only comprise that the proteic described negative control of background (pET28) imidazoles purifying " albumen " is few to bring out the HR reaction to 1: 2 extent of dilution, the susceptibility of this mensuration is reduced to 1: 1 and 1: 2 extent of dilution, can not use this albumen.This false HR may cause owing to thereby used imidazoles in the purge process is not exclusively given in conjunction with the proteic affinity of one or more backgrounds.The imidazoles of about 60mM concentration just brings out false HR reaction.
Comprise that from amino acid/11 37-180 (SEQ.ID.No.23) only a decisive structural domain (definitive domain) of the little interior region of 44 amino acid proteins is accredited as minimum HR structural domain.Other potential structural domain is thought to be positioned at from amino acid/11-104 (possible amino acid/11-75) this proteic N end (SEQ.ID.No.23).Because the difficulty that runs in these fragment albumen of purifying causes, is difficult to confirmation or dwindles N end HR structural domain.Because be not recovered to albumen when adopting natural purge process, described N end fragment albumen has to use urea purification.Therefore, these albumen precipitate during renaturation process, are difficult to or may be dissolved in solution more hardly, thereby make this albumen be difficult to measure by HR, because have only soluble albumen can bring out HR.The difficulty of dwindling N end HR structural domain only is due to the fact that formation: negative control brings out false HR at ordinary times at low dilution water, thereby has reduced the susceptibility of described mensuration.
Unexpected is, if amino acid/11 68 and 169 (fragment 76-168 and 105-168) (SEQ.ID.No.23) between, with described inner HR structural domain cutting, then described fragment is lost its HR activity.This shows, the HR activity of fragment 1-168 (SEQ.ID.No.23) should be owing to described inner HR structural domain, but owing to some other structural domain, causes imagination to find second HR structural domain at described proteic N end regions.Yet, as discussed previously, be difficult to confirm this imagination.
Anaphylaxis exciton C end (amino acid 210-403 (SEQ.ID.No.23)) does not contain the HR structural domain.Adopt present HR to measure, but it does not bring out HR on detection level.Even the big C end fragment that derives from amino acid/11 69-403 (SEQ.ID.No.23) do not bring out HR yet, although it comprises partial interior HR structural domain.As mentioned above, scinderin causes the active forfeiture of HR between amino acid/11 68 and 169 (SEQ.ID.No.23).
Do not express because some has 61 amino acid or little clone's still less albumen, synthetic have 30 amino acid whose several oligopeptides to dwindle the functional area of inner HR structural domain.Synthetic described oligopeptides in amino acid/11 21-179 (SEQ.ID.No.23) scope.Yet these oligopeptides do not bring out HR.Expection oligopeptides 137-166,121-150 and 137-156 (SEQ.ID.No.23) may bring out HR, because these fragments do not comprise requisite amino acid/11 68 and 169 (SEQ.ID.No.23).Expection oligopeptides 150-179 (SEQ.ID.No.23) may bring out HR.May be 30 amino acid for described albumen and Yan Taixiao so that can not bring out any activity, in default of folding and therefore lack combination; Perhaps may be during synthetic described peptide, lose important amino acid (perhaps in described process or only because select which 30 amino acid to synthesize), therefore, described fragment can not be brought out HR.Embodiment 13-plant-growth promoter action (PGE) induces
Described C end fragment strengthens 9% to 21% with tomato growth.Described N end fragment strengthens 4% to 13% with tomato growth.Described interior segments will be grown and be strengthened 9% to 20%.The 76-209 fragment will be grown under concentration 60 μ g/ml and be strengthened 18%, but not promote growth under common 20 μ g/ml.This is owing to the inaccuracy (table 4) of described quantitative process.
Table 4
Fragment # Amino acid PGE hr＞Huan Chongye @10 μ g/ml PGE hr＞Huan Chongye @20 μ g/ml
1 (+contrast) ?1-403 ?????12％ ???11％
2 (contrasts) ????- ?????-3％ ???-2％
????4 ?169-403 ?????9％ ???12％
????5 ?210-403 ?????13％ ???14％ 16％@40μg/ml
????6 ?267-403 ?????2l％ ????21％ 23％@40μg/ml
????7 ?343-403 ?????7％ ????7％
????9 ?1-104 ?????4％ ????8％
????10 ?1-168 ?????13％ ????5％
????13 ?76-209 ?????7％ ????4％ 18％@40μg/ml
????14 ?76-168 ?????18％ ????20％
????15 ?105-209 ?????14％ ????19％
????17 ?105-168 ?????19％ ????16％
????19 ?137-204 ?????11％ ????13％
????20 ?137-180 ??????- ?????9％
* be higher than the plant height of described damping fluid contrast 10% or more and must measure the enhancing from 7.4% to 17.3% (table 5) of will growing of described oligopeptides by PGE.
Table 5
Fragment Amino acid Express HR tires TMV usefulness PGE ht＞damping fluid
Oligopeptides ????150-179 ????NA ??- ????72.9％ ????10.1％
Oligopeptides ????137-166 ????NA ??- ????61.2％ ????12.0％
Oligopeptides ????121-150 ????NA ??- ????60.0％ ????17.3％
Oligopeptides ????137-156 ????NA ??- ???-87.7％ ????7.4％
Described data show have the PGE structural domain that surpasses to exist, and preponderate than described N end structure territory although described C end structure territory and internal structure territory be it seems, because described N end fragment minimum quantity promotes growth.Embodiment 14-system obtains inducing of resistance (SAR)
It seems that the described anaphylaxis exciton of all of being tested fragment all have 60% usefulness or higher so far, just except the oligopeptides 137-156 (table 5 and 6).
Table 6
Fragment # Amino acid The usefulness of TMV control
1 (+contrast) ????1-403 84% and 72%
2 (contrasts) ??????- 40% and 31%
????4 ????169-403 64% and 79%
????5 ????210-403 77% and 78%
????6 ????267-403 70% and 72%
????9 ?????1-104 ????????82％
????10 ?????1-168 ????????69％
????13 ????76-209 44% and 84%
????14 ????76-168 83% and 87%
????15 ????105-209 57% and 67%
????17 ????105-168 ????????89％
????19 ????137-204 89% and 77%
????20 ????137-180 64% and 58%
These data show a plurality of SAR structural domains in described albumen.Relation between embodiment 15-HR, PGE and the SAR
Obviously, described anaphylaxis activity can promote activity to separate with described plant-growth.Described C end fragment just promotes tomato growth about 20% when concentration has only 20 μ g/ml significantly, but these same clip even can not bring out tobacco HR being higher than 200 μ g/ml concentration.The SAR activity also be it seems and can be separated with the HR activity.This discovery is very important to future in the work of the transgenosis application facet of anaphylaxis exciton technology.The described fragment of inducing PGE and/or SAR but not bringing out HR is absolutely necessary to this technology, even because the constitutive expression of low-level HR exciton also may kill and wound plant.Embodiment 16-derive from the pseudomonas syringae cloves cause a disease mutation the anaphylaxis exciton not
The fragment of bringing out HR is induced in tobacco the disease resistance of TMV and short
Advance the growth of tomato
Derive from order to test that HrpZ-causing a disease the fragment of not bringing out HR of anaphylaxis exciton of mutation from the pseudomonas syringae cloves whether can inducing anti-disease, prepare several fragments constitute the HR of fragment albumen in tobacco that things and test express bring out induce with disease resistance and tomato in growth-promoting effect.
Adopt Pfu Turbo (Stratagene), through pcr amplification hrpZ-coding from the gene of the pathogenic mutation anaphylaxis exciton of pseudomonas syringae cloves with lower curtate: the zone of coded amino acid 152-190, amino acid/11 52-294, amino acid/11 90-294, amino acid 301-341 and total length HrpZ (amino acid/11-341).Described dna fragmentation is cloned among the pCAL-n (Stratagene) to produce the C end fusion rotein of calmodulin binding peptide.Select pCAL-n because described fusion rotein can be easily and on the calmodulin resin gentle purifying.Described DNA is transformed in the bacillus coli DH 5 alpha, and identifies correct clone.Then described clone is transferred among the intestinal bacteria BLR DE3 and be used for protein expression.With described bacterial growth OD to 0.8-1.0 in Terrific meat soup 620Express with the IPTG inducible protein then, and described bacterium was cultivated 3 hours again.All HrpZ fragments can both in this way be expressed.
Selecting amino acid fragment 152-294 and 190-294 is used for further analyzing and identifying.Expection fragment 152-294 comprises a structural domain that brings out HR, and fragment 190-294 does not contain the structural domain that brings out HR.Described culture is centrifugal, then with described bacterium resuspending in 40ml, 10mM Tris pH8.0.Add 20 μ l defoamers and 40 μ l, 200mM PMSF, and with described bacterium supersound process with smudge cells.Through centrifugal removal bacterial debris, then described supernatant liquor was placed in boiling water bath 10 minutes.Through the described precipitation of centrifugal removal, and keep described supernatant liquor-a kind of crude protein preparation be used for the test.
Every kind of supernatant liquor of 15 μ l electrophoresis on gel also dyes to determine whether described albumen exists.The 152-294 fragment is Duoed 5 times approximately than the amount that the 190-294 fragment exists according to estimates.The blade that several diluents of every kind of preparation is soaked into two strain tobacco plants is used for HR test (table 7).As shown in table 7, the 152-294 fragment is brought out HR, and the 190-294 fragment is not brought out HR.
The extent of dilution of the segmental HR test result of table 7HrpZ HrDZ fragment fragment preparation a
1: 21: 51: 25 1: 125152-294+,+ b+ ,++ ,+-,-190-294-,--,--,--,- aDilute described preparation with MilliQ water. bThe result represents every strain of two strain plants.+, HR;-, no HR.
Then TMV disease resistance and growth-promoting effect are induced in described fragment preparation test.Since the difference of HrpZ fragment concentrations aspect, with 40 times of 152-294 preparation dilutions, and with 8 times of 190-294 preparation dilutions.The result shows, compare with the damping fluid contrast, the 190-294 amino acid fragment with the reduced number of TMV scab 85% (table 8).By contrast, the 152-294 amino acid fragment has only reduced 55% with the number of TMV scab.Also as shown in table 8, Duo 4.64% with the plant that the 152-294 amino acid fragment is handled than the plant-growth of handling with damping fluid, and Duo 2.62% than the plant-growth of handling with damping fluid with the plant that the 190-294 amino acid fragment is handled.
Table 8
HR test, TMV and PGE test-results HrpZ fragment HR fall out effect aTMV (% usefulness) bPGE (%＞damping fluid ht) c152-294+54.64 4.64190-294-85.25 2.62 a+, bring out HR in the tobacco leaf;-, there is not HR in the tobacco leaf. bThe % that reduces of the TMV scab in the tobacco leaf of Pen Saing not. cThe % that the plant height that sprays than damping fluid is Duoed.
These test-results show that it seems that amino acid/11 52-294 participate in the HR fall out effect, because the ability of bringing out HR has been eliminated in their removal.These two kinds of fragment preparations are realized disease control and growth-promoting effect.Therefore, the ability of bringing out HR is not that TMV infects the deciding factor that weakens with growth-promoting effect.Embodiment 17-uses to such an extent that 13 mould amino acid whose peptides of arrogant male epidemic disease stimulate the tomato seedling growth
The parsley blade comprises supersensitivity necrocytosis, the activation of defense minister's correlation gene and the plain generation of plant defendance at a kind of typical disease resistance response of the mould generation of the big male epidemic disease of soybean pathogenic agent.Before several years, 42kDa glycoprotein is excited son purifying (Parker etc. from the mould fungal cultures filtrate of big male epidemic disease, " the outer glycoprotein of born of the same parents of arrogant male epidemic disease tempeh microspecies (Phytophthora megasperma f.sp.glycinea) to bring out the parsley cell and the defendance of the plant in the protoplastis of cultivation plain synthetic; " Mol.Plant Microbe Interact.4:19-27 (1991), the document is attached to herein by reference).Then, 13 amino acid whose oligopeptides are accredited as and are included in the 42kDa glycoprotein.It seems that these 13 amino acid whose peptides have and the similar biological activity of total length glycoprotein (42kDa).It enough brings out complicated defense response in the parsley cell, comprise that H+/Ca2+ flows into, K+/Cl-flows out, active oxygen produces, SAR is gene induced and plant is safeguarded plain compound accumulation (Nurnberger etc., " fungi oligopeptides exciton to the high-affinity of parsley plasma membrane in conjunction with triggering multiple defense response; " Cell 78:449-460 (1994), the document is attached to herein by reference).
To derive from proteic 13 the amino acid whose peptides of 42kDa and whether also promote plant-growth in order to test, by the described oligopeptides of the Synthetic 2 0mg of Biosynthesis company.This synthetic peptide sequence is NH2-Val-Trp-Asn-Gln-Pro-Val-Arg-Gly-Phe-Lys-Val-Tyr-Glu-COOH (SEQ.ID.No.39).Institute's synthetic peptide is suspended in 10ml, the 5mM potassium phosphate buffer again, is diluted to 1ng/ml and 100ng/ml with identical damping fluid then.With 100 tomato seeds (kind, Marglobe) soaked overnight in the 20ml peptide solution.With the planting seed that soaked in 8 inches basins that artificial soil is housed.To be immersed in seed in the damping fluid that does not have described peptide with comparing.After coming up and at first two true leaves extend fully, the height of record tomato seedling.In the plant of tobacco and other test, described peptide can not bring out HR.Yet it has a significant impact plant-growth promoter action tool.Table 9 shows that the tomato seedling of handling with described peptide is increasing by 12.6% aspect the height, shows that the fungal peptide that derives from 42kDa glycoprotein can promote the tomato seedling growth.Studies show that further described peptide comprises also having similar growth effect in the tobacco other crop.Spray plant with described peptide solution and obtain similar growth-promoting effect.
Table 9 processing seedling height (cm) average (cm) variation % damping fluid 6.0 6.0 6.0 5.5 5.5 5.55-
5.5 5.5 5.0 5.0 5.5 peptide solutions (100ng/ml) 6.5 6.0 6.5 6.5 6.5 6.25 12.6
6.0??6.0??6.0??6.0??6.5
Although described the present invention in detail in order to illustrate, much less this details is only for this purpose, and under situation without departing from the spirit and scope of the present invention, those skilled in the art can change therein, and the spirit and scope of the present invention are limited by following claims.
＜110〉Eden Bioscienoe Corporation＜120〉＜130〉21829/34＜140〉＜141〉＜150〉60/103,050＜151〉1998-10-05＜160〉39＜170〉PatentIn Ver.2.1＜210〉1＜211〉31＜212〉DNA＜213〉＜400〉1gggaattcat atgagtctga atacaagtgg g 31＜210〉2＜211〉31＜212〉DNA＜213〉＜400〉2gggaattcat atgggcggtg gcttaggcggt 31＜210〉3＜211〉29＜212〉DNA＜213〉＜400〉3ggcatatgtc gaacgcgctg aacgatatg 29＜210〉4＜211〉31＜212〉DNA＜213〉＜400〉4gggaattcat atgttaggcg gttcgctgaa c 31＜210〉5＜211〉29＜212〉DNA＜213〉＜400〉5ggcatatgct gaacacgctg ggctcgaaa 29＜210〉6＜211〉29＜212〉DNA＜213〉＜400〉6ggcatatgtc aacgtcccaa aacgacgat 29＜210〉7＜211〉27＜212〉DNA＜213〉＜400〉7ggcatatgtc cacctcagac tccagcg 27＜210〉8＜211〉34＜212〉DNA＜213〉＜400〉8gggaattcat atgcaaagcc tgtttggtga tggg 34＜210〉9＜211〉31＜212〉DNA＜213〉＜400〉9gggaattcat atgggtaatg gtctgagcaa g 31＜210〉10＜211〉31＜212〉DNA＜213〉＜400〉10gggaattcat atgaaagcgg gcattcaggc g 31＜210〉11＜211〉34＜212〉DNA＜213〉＜400〉11gggaattcat atgacaccag ccagtatgga gcag 34＜210〉12＜211〉31＜212〉DNA＜213〉＜400〉12gcaagcttaa cagcccacca ccgcccatca t 31＜210〉13＜211〉31＜212〉DNA＜213〉＜400〉13gcaagcttaa atcgttcagc gcgttcgaca g 31＜210〉14＜211〉34＜212〉DNA＜213〉＜400〉14gcaagcttaa tatctcgctg aacatcttca gcag 34＜210〉15＜211〉30＜212〉DNA＜213〉＜400〉15gcaagcttaa ggtgccatct tgcccatcac 30＜210〉16＜211〉34＜212〉DNA＜213〉＜400〉16gcaagcttaa atcagtgact ccttttttat aggc 34＜210〉17＜211〉31＜212〉DNA＜213〉＜400〉17gcaagcttaa caggcccgac agcgcatcag t 31＜210〉18＜211〉31＜212〉DNA＜213〉＜400〉18gcaagcttaa accgataccg gtacccacgg c 31＜210〉19＜211〉34＜212〉DNA＜213〉＜400〉19gcaagcttaa tccgtcgtca tctggcttgc tcag 34＜210〉20＜211〉25＜212〉DNA＜213〉＜400〉20gcaagcttaa gccgcgccca gcttg 25＜210〉21＜211〉338＜212〉PRT＜213〉＜400〉21Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser1 5 10 15Gly Leu Gly Ala Gin Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser
20??????????????????25??????????????????30Leu?Gly?Ser?Ser?Val?Asp?Lys?Leu?Ser?Ser?Thr?Ile?Asp?Lys?Leu?Thr
35???????????????????40???????????????????45Ser?Ala?Leu?Thr?Ser?Met?Met?Phe?Gly?Gly?Ala?Leu?Ala?Gln?Gly?Leu
50??????????????????55??????????????????60Gly?Ala?Ser?Ser?Lys?Gly?Leu?Gly?Met?Ser?Asn?Gln?Leu?Gly?Gln?Ser?65??????????????????70??????????????????75??????????????????80Phe?Gly?Asn?Gly?Ala?Gln?Gly?Ala?Ser?Asn?Leu?Leu?Ser?Val?Pro?Lys
85??????????????????90??????????????????95Ser?Gly?Gly?Asp?Ala?Leu?Ser?Lys?Met?Phe?Asp?Lys?Ala?Leu?Asp?Asp
100?????????????????105?????????????????110Leu?Leu?Gly?His?Asp?Thr?Val?Thr?Lys?Leu?Thr?Asn?Gln?Ser?Asn?Gln
115?????????????????120?????????????????125Leu?Ala?Asn?Ser?Met?Leu?Asn?Ala?Ser?Gln?Met?Thr?Gln?Gly?Asn?Met
130?????????????????135??????????????????140Asn?Ala?Phe?Gly?Ser?Gly?Val?Asn?Asn?Ala?Leu?Ser?Ser?Ile?Leu?Gly145?????????????????150?????????????????155?????????????????160Asn?Gly?Leu?Gly?Gln?Ser?Met?Ser?Gly?Phe?Ser?Gln?Pro?Ser?Leu?Gly
165?????????????????170?????????????????175Ala?Gly?Gly?Leu?Gln?Gly?Leu?Ser?Gly?Ala?Gly?Ala?Phe?Asn?Gln?Leu
180?????????????????185?????????????????190Gly?Asn?Ala?Ile?Gly?Met?Gly?Val?Gly?Gln?Asn?Ala?Ala?Leu?Ser?Ala
195?????????????????200?????????????????205Leu?Ser?Asn?Val?Ser?Thr?His?Val?Asp?Gly?Asn?Asn?Arg?His?Phe?Val
210?????????????????215?????????????????220Asp?Lys?Glu?Asp?Arg?Gly?Met?Ala?Lys?Glu?Ile?Gly?Gln?Phe?Met?Asp225?????????????????230?????????????????235?????????????????240Gln?Tyr?Pro?Glu?Ile?Phe?Gly?Lys?Pro?Glu?Tyr?Gln?Lys?Asp?Gly?Trp
245?????????????????250?????????????????255Ser?Ser?Pro?Lys?Thr?Asp?Asp?Lys?Ser?Trp?Ala?Lys?Ala?Leu?Ser?Lys
260?????????????????265?????????????????270Pro?Asp?Asp?Asp?Gly?Met?Thr?Gly?Ala?Ser?Met?Asp?Lys?Phe?Arg?Gln
275?????????????????280?????????????????285Ala?Met?Gly?Met?Ile?Lys?Ser?Ala?Val?Ala?Gly?Asp?Thr?Gly?Asn?Thr
290?????????????????295?????????????????300Asn?Leu?Asn?Leu?Arg?Gly?Ala?Gly?Gly?Ala?Ser?Leu?Gly?Ile?Asp?Ala305?????????????????310?????????????????315?????????????????320Ala?Val?Val?Gly?Asp?Lys?Ile?Ala?Asn?Met?Ser?Leu?Gly?Lys?Leu?Ala
325 330 335Asn Ala＜210〉22＜21l〉2l 4l＜212〉DNA＜213〉＜400〉22cgattttacc cgggtgaacg tgctatgacc gacagcatca cggtattcga caccgttacg 60gcgtttatgg ccgcgatgaa ccggcatcag gcggcgcgct ggtcgccgca atccggcgtc 120gatctggtat ttcagtttgg ggacaccggg cgtgaactca tgatgcagat tcagccgggg 180cagcaatatc ccggcatgtt gcgcacgctg ctcgctcgtc gttatcagca ggcggcagag 240tgcgatggct gccatctgtg cctgaacggc agcgatgtat tgatcctctg gtggccgctg 300ccgtcggatc ccggcagtta tccgcaggtg atcgaacgtt tgtttgaact ggcgggaatg 360acgttgccgt cgctatccat agcaccgacg gcgcgtccgc agacagggaa cggacgcgcc 420cgatcattaa gataaaggcg gcttttttta ttgcaaaacg gtaacggtga ggaaccgttt 480caccgtcggc gtcactcagt aacaagtatc catcatgatg cctacatcgg gatcggcgtg 540ggcatccgtt gcagatactt ttgcgaacac ctgacatgaa tgaggaaacg aaattatgca 600aattacgatc aaagcgcaca tcggcggtga tttgggcgtc tccggtctgg ggctgggtgc 660tcagggactg aaaggactga attccgcggc ttcatcgctg ggttccagcg tggataaact 720gagcagcacc atcgataagt tgacctccgc gctgacttcg atgatgtttg gcggcgcgct 780ggcgcagggg ctgggcgcca gctcgaaggg gctggggatg agcaatcaac tgggccagtc 840tttcggcaat ggcgcgcagg gtgcgagcaa cctgctatcc gtaccgaaat ccggcggcga 900tgcgttgtca aaaatgtttg ataaagcgct ggacgatctg ctgggtcatg acaccgtgac 960caagctgact aaccagagca accaactggc taattcaatg ctgaacgcca gccagatgac 1020ccagggtaat atgaatgcgt tcggcagcgg tgtgaacaac gcactgtcgt ccattctcgg 1080caacggtctc ggccagtcga tgagtggctt ctctcagcct tctctggggg caggcggctt 1140gcagggcctg agcggcgcgg gtgcattcaa ccagttgggt aatgccatcg gcatgggcgt 1200ggggcagaat gctgcgctga gtgcgttgag taacgtcagc acccacgtag acggtaacaa 1260ccgccacttt gtagataaag aagatcgcgg catggcgaaa gagatcggcc agtttatgga 1320tcagtatccg gaaatattcg gtaaaccgga ataccagaaa gatggctgga gttcgccgaa 1380gacggacgac aaatcctggg ctaaagcgct gagtaaaccg gatgatgacg gtatgaccgg 1440cgccagcatg gacaaattcc gtcaggcgat gggtatgatc aaaagcgcgg tggcgggtga 1500taccggcaat accaacctga acctgcgtgg cgcgggcggt gcatcgctgg gtatcgatgc 1560ggctgtcgtc ggcgataaaa tagccaacat gtcgctgggt aagctggcca acgcctgata 1620atctgtgctg gcctgataaa gcggaaacga aaaaagagac ggggaagcct gtctcttttc 1680ttattatgcg gtttatgcgg ttacctggac cggttaatca tcgtcatcga tctggtacaa 1740acgcacattt tcccgttcat tcgcgtcgtt acgcgccaca atcgcgatgg catcttcctc 1800gtcgctcaga ttgcgcggct gatggggaac gccgggtgga atatagagaa actcgccggc 1860cagatggaga cacgtctgcg ataaatctgt gccgtaacgt gtttctatcc gcccctttag 1920cagatagatt gcggtttcgt aatcaacatg gtaatgcggt tccgcctgtg cgccggccgg 1980gatcaccaca atattcatag aaagctgtct tgcacctacc gtatcgcggg agataccgac 2040aaaatagggc agtttttgcg tggtatccgt ggggtgttcc ggcctgacaa tcttgagttg 2100gttcgtcatc atctttctcc atctgggcga cctgatcggt t 2141＜210〉23＜21l〉403＜212〉PRT＜213〉＜400〉23Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser 1 5 10 15Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln
20??????????????????25??????????????????30Asn?Ala?Gly?Leu?Gly?Gly?Asn?Ser?Ala?Leu?Gly?Leu?Gly?Gly?Gly?Asn
35??????????????????40??????????????????45Gln?Asn?Asp?Thr?Val?Asn?Gln?Leu?Ala?Gly?Leu?Leu?Thr?Gly?Met?Met
50??????????????????55??????????????????60Met?Met?Met?Ser?Met?Met?Gly?Gly?Gly?Gly?Leu?Met?Gly?Gly?Gly?Leu?65??????????????????70??????????????????75??????????????????80Gly?Gly?Gly?Leu?Gly?Asn?Gly?Leu?Gly?Gly?Ser?Gly?Gly?Leu?Gly?Glu
85??????????????????90??????????????????95Gly?Leu?Ser?Asn?Ala?Leu?Asn?Asp?Met?Leu?Gly?Gly?Ser?Leu?Asn?Thr
100?????????????????105?????????????????110Leu?Gly?Ser?Lys?Gly?Gly?Asn?Asn?Thr?Thr?Ser?Thr?Thr?Asn?Ser?Pro
115?????????????????120?????????????????125Leu?Asp?Gln?Ala?Leu?Gly?Ile?Asn?Ser?Thr?Ser?Gln?Asn?Asp?Asp?Ser
130?????????????????135?????????????????140Thr?Ser?Gly?Thr?Asp?Ser?Thr?Ser?Asp?Ser?Ser?Asp?Pro?Met?Gln?Gln145?????????????????150?????????????????155?????????????????160Leu?Leu?Lys?Met?Phe?Ser?Glu?Ile?Met?Gln?Ser?Leu?Phe?Gly?Asp?Gly
165?????????????????170?????????????????175Gln?Asp?Gly?Thr?Gln?Gly?Ser?Ser?Ser?Gly?Gly?Lys?Gln?Pro?Thr?Glu
180?????????????????185?????????????????190Gly?Glu?Gln?Asn?Ala?Tyr?Lys?Lys?Gly?Val?Thr?Asp?Ala?Leu?Ser?Gly
195?????????????????200?????????????????205Leu?Met?Gly?Asn?Gly?Leu?Ser?Gln?Leu?Leu?Gly?Asn?Gly?Gly?Leu?Gly
210?????????????????215?????????????????220Gly?Gly?Gln?Gly?Gly?Asn?Ala?Gly?Thr?Gly?Leu?Asp?Gly?Ser?Ser?Leu225?????????????????230?????????????????235?????????????????240Gly?Gly?Lys?Gly?Leu?Gln?Asn?Leu?Ser?Gly?Pro?Val?Asp?Tyr?Gln?Gln
245?????????????????250?????????????????255Leu?Gly?Asn?Ala?Val?Gly?Thr?Gly?Ile?Gly?Met?Lys?Ala?Gly?Ile?Gln
260?????????????????265?????????????????270Ala?Leu?Asn?Asp?Ile?Gly?Thr?His?Arg?His?Ser?Ser?Thr?Arg?Ser?Phe
275?????????????????280?????????????????285Val?Asn?Lys?Gly?Asp?Arg?Ala?Met?Ala?Lys?Glu?Ile?Gly?Gln?Phe?Met
290?????????????????295?????????????????300Asp?Gln?Tyr?Pro?Glu?Val?Phe?Gly?Lys?Pro?Gln?Tyr?Gln?Lys?Gly?Pro305?????????????????310?????????????????315?????????????????320Gly?Gln?Glu?Val?Lys?Thr?Asp?Asp?Lys?Ser?Trp?Ala?Lys?Ala?Leu?Ser
325?????????????????330?????????????????335Lys?Pro?Asp?Asp?Asp?Gly?Met?Thr?Pro?Ala?Ser?Met?Glu?Gln?Phe?Asn
340?????????????????345?????????????????350Lys?Ala?Lys?Gly?Met?Ile?Lys?Arg?Pro?Met?Ala?Gly?Asp?Thr?Gly?Asn
355?????????????????360?????????????????365Gly?Asn?Leu?Gin?Ala?Arg?Gly?Ala?Gly?Gly?Ser?Ser?Leu?Gly?Ile?Asp
370 375 380Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu385 390 395 400Gly Ala Ala＜210〉24＜211〉1288＜212〉DNA＜213〉＜400〉24aagcttcggc atggcacgtt tgaccgttgg gtcggcaggg tacgtttgaa ttattcataa 60gaggaatacg ttatgagtct gaatacaagt gggctgggag cgtcaacgat gcaaatttct 120atcggcggtg cgggcggaaa taacgggttg ctgggtacca gtcgccagaa tgctgggttg 180ggtggcaatt ctgcactggg gctgggcggc ggtaatcaaa atgataccgt caatcagctg 240gctggcttac tcaccggcat gatgatgatg atgagcatga tgggcggtgg tgggctgatg 300ggcggtggct taggcggtgg cttaggtaat ggcttgggtg gctcaggtgg cctgggcgaa 360ggactgtcga acgcgctgaa cgatatgtta ggcggttcgc tgaacacgct gggctcgaaa 420ggcggcaaca ataccacttc aacaacaaat tccccgctgg accaggcgct gggtattaac 480tcaacgtccc aaaacgacga ttccacctcc ggcacagatt ccacctcaga ctccagcgac 540ccgatgcagc agctgctgaa gatgttcagc gagataatgc aaagcctgtt tggtgatggg 600caagatggca cccagggcag ttcctctggg ggcaagcagc cgaccgaagg cgagcagaac 660gcctataaaa aaggagtcac tgatgcgctg tcgggcctga tgggtaatgg tctgagccag 720ctccttggca acgggggact gggaggtggt cagggcggta atgctggcac gggtcttgac 780ggttcgtcgc tgggcggcaa agggctgcaa aacctgagcg ggccggtgga ctaccagcag 840ttaggtaacg ccgtgggtac cggtatcggt atgaaagcgg gcattcaggc gctgaatgat 900atcggtacgc acaggcacag ttcaacccgt tctttcgtca ataaaggcga tcgggcgatg 960gcgaaggaaa tcggtcagtt catggaccag tatcctgagg tgtttggcaa gccgcagtac 1020cagaaaggcc cgggtcagga ggtgaaaacc gatgacaaat catgggcaaa agcactgagc 1080aagccagatg acgacggaat gacaccagcc agtatggagc agttcaacaa agccaagggc 1140atgatcaaaa ggcccatggc gggtgatacc ggcaacggca acctgcaggc acgcggtgcc 1200ggtggttctt cgctgggtat tgatgccatg atggccggtg atgccattaa caatatggca 1260cttggcaagc tgggcgcggc ttaagctt 1288＜210〉25＜21l〉1344＜212〉DNA＜213〉＜400〉25atgtcaattc ttacgcttaa caacaatacc tcgtcctcgc cgggtctgtt ccagtccggg 60ggggacaacg ggcttggtgg tcataatgca aattctgcgt tggggcaaca acccatcgat 120cggcaaacca ttgagcaaat ggctcaatta ttggcggaac tgttaaagtc actgctatcg 180ccacaatcag gtaatgcggc aaccggagcc ggtggcaatg accagactac aggagttggt 240aacgctggcg gcctgaacgg acgaaaaggc acagcaggaa ccactccgca gtctgacagt 300cagaacatgc tgagtgagat gggcaacaac gggctggatc aggccatcac gcccgatggc 360cagggcggcg ggcagatcgg cgataatcct ttactgaaag ccatgctgaa gcttattgca 420cgcatgatgg acggccaaag cgatcagttt ggccaacctg gtacgggcaa caacagtgcc 480tcttccggta cttcttcatc tggcggttcc ccttttaacg atctatcagg ggggaaggcc 540ccttccggca actccccttc cggcaactac tctcccgtca gtaccttctc acccccatcc 600acgccaacgt cccctacctc accgcttgat ttcccttctt ctcccaccaa agcagcoggg 660ggcagcacgc cggtaaccga tcatcctgac cctgttggta gcgcgggcat cggggccgga 720aattcggtgg ccttcaccag cgccggcgct aatcagacgg tgctgcatga caccattacc 780gtgaaagcgg gtcaggtgtt tgatggcaaa ggacaaacct tcaccgccgg ttcagaatta 840ggcgatggcg gccagtctga aaaccagaaa ccgctgttta tactggaaga cggtgccagc 900ctgaaaaacg tcaccatggg cgacgacggg gcggatggta ttcatcttta cggtgatgcc 960aaaatagaca atctgcacgt caccaacgtg ggtgaggacg cgattaccgt taagccaaac 1020agcgcgggca aaaaatccca cgttgaaatc actaacagtt ccttcgagca cgcctctgac 1080aagatcctgc agctgaatgc cgatactaac ctgagcgttg acaacgtgaa ggccaaagac 1140tttggtactt ttgtacgcac taacggcggt caacagggta actgggatct gaatctgagc 1200catatcagcg cagaagacgg taagttctcg ttcgttaaaa gcgatagcga ggggctaaac 1260gtcaatacca gtgatatctc actgggtgat gttgaaaacc actacaaagt gccgatgtcc 1320gccaacctga aggtggctga atga 1344＜210〉26＜211〉447＜212〉PRT＜213〉＜400〉26Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu 1 5 10 15Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser
20??????????????????25??????????????????30Ala?Leu?Gly?Gln?Gln?Pro?Ile?Asp?Arg?Gln?Thr?Ile?Glu?Gln?Met?Ala
35??????????????????40??????????????????45Gln?Leu?Leu?Ala?Glu?Leu?Leu?Lys?Ser?Leu?Leu?Ser?Pro?Gln?Ser?Gly
50??????????????????55??????????????????60Asn?Ala?Ala?Thr?Gly?Ala?Gly?Gly?Asn?Asp?Gln?Thr?Thr?Gly?Val?Gly?65??????????????????70??????????????????75??????????????????80Asn?Ala?Gly?Gly?Leu?Asn?Gly?Arg?Lys?Gly?Thr?Ala?Gly?Thr?Thr?Pro
85???????????????????90?????????????????95Gln?Ser?Asp?Ser?Gln?Asn?Met?Leu?Ser?Glu?Met?Gly?Asn?Asn?Gly?Leu
100?????????????????105?????????????????110Asp?Gln?Ala?Ile?Thr?Pro?Asp?Gly?Gln?Gly?Gly?Gly?Gln?Ile?Gly?Asp
115?????????????????120?????????????????125Asn?Pro?Leu?Leu?Lys?Ala?Met?Leu?Lys?Leu?Ile?Ala?Arg?Met?Met?Asp
130?????????????????135?????????????????140Gly?Gln?Ser?Asp?Gln?Phe?Gly?Gln?Pro?Gly?Thr?Gly?Asn?Asn?Ser?Ala145?????????????????150?????????????????155?????????????????160Ser?Ser?Gly?Thr?Ser?Ser?Ser?Gly?Gly?Ser?Pro?Phe?Asn?Asp?Leu?Ser
165?????????????????170?????????????????175Gly?Gly?Lys?Ala?Pro?Ser?Gly?Asn?Ser?Pro?Ser?Gly?Asn?Tyr?Ser?Pro
180?????????????????185?????????????????190Val?Ser?Thr?Phe?Ser?Pro?Pro?Ser?Thr?Pro?Thr?Ser?Pro?Thr?Ser?Pro
195?????????????????200?????????????????205Leu?Asp?Phe?Pro?Ser?Ser?Pro?Thr?Lys?Ala?Ala?Gly?Gly?Ser?Thr?Pro
210?????????????????215?????????????????220Val?Thr?Asp?His?Pro?Asp?Pro?Val?Gly?Ser?Ala?Gly?Ile?Gly?Ala?Gly225?????????????????230?????????????????235?????????????????240Asn?Ser?Val?Ala?Phe?Thr?Ser?Ala?Gly?Ala?Asn?Gln?Thr?Val?Leu?His
245?????????????????250?????????????????255Asp?Thr?Ile?Thr?Val?Lys?Ala?Gly?Gln?Val?Phe?Asp?Gly?Lys?Gly?Gln
260?????????????????265?????????????????270Thr?Phe?Thr?Ala?Gly?Ser?Glu?Leu?Gly?Asp?Gly?Gly?Gln?Ser?Glu?Asn
275?????????????????280?????????????????285Gln?Lys?Pro?Leu?Phe?Ile?Leu?Glu?Asp?Gly?Ala?Ser?Leu?Lys?Asn?Val
290?????????????????295?????????????????300Thr?Met?Gly?Asp?Asp?Gly?Ala?Asp?Gly?Ile?His?Leu?Tyr?Gly?Asp?Ala305?????????????????310?????????????????315?????????????????320Lys?Ile?Asp?Asn?Leu?His?Val?Thr?Asn?Val?Gly?Glu?Asp?Ala?Ile?Thr
325?????????????????330?????????????????335Val?Lys?Pro?Asn?Ser?Ala?Gly?Lys?Lys?Ser?His?Val?Glu?Ile?Thr?Asn
340?????????????????345?????????????????350Ser?Ser?Phe?Glu?His?Ala?Ser?Asp?Lys?Ile?Leu?Gln?Leu?Asn?Ala?Asp
355?????????????????360?????????????????365Thr?Asn?Leu?Ser?Val?Asp?Asn?Val?Lys?Ala?Lys?Asp?Phe?Gly?Thr?Phe
370?????????????????375?????????????????380Val?Arg?Thr?Asn?Gly?Gly?Gln?Gln?Gly?Asn?Trp?Asp?Leu?Asn?Leu?Ser385?????????????????390?????????????????395?????????????????400His?Ile?Ser?Ala?Glu?Asp?Gly?Lys?Phe?Ser?Phe?Val?Lys?Ser?Asp?Ser
405?????????????????410?????????????????415Glu?Gly?Leu?Asn?Val?Asn?Thr?Ser?Asp?Ile?Ser?Leu?Gly?Asp?Val?Glu
420?????????????????425?????????????????430Asn?His?Tyr?Lys?Val?Pro?Met?Ser?Ala?Asn?Leu?Lys?Val?Ala?Glu
20??????????????????25??????????????????30Ser?Ser?Ser?Ser?Pro?Gln?Asn?Ala?Ala?Ala?Ser?Leu?Ala?Ala?Glu?Gly
35??????????????????40??????????????????45Lys?Asn?Arg?Gly?Lys?Met?Pro?Arg?Ile?His?Gln?Pro?Ser?Thr?Ala?Ala
50??????????????????55??????????????????60Asp?Gly?Ile?Ser?Ala?Ala?His?Gln?Gln?Lys?Lys?Ser?Phe?Ser?Leu?Arg?65??????????????????70??????????????????75??????????????????80Gly?Cys?Leu?Gly?Thr?Lys?Lys?Phe?Ser?Arg?Ser?Ala?Pro?Gln?Gly?Gln
85???????????????????90??????????????????95Pro?Gly?Thr?Thr?His?Ser?Lys?Gly?Ala?Thr?Leu?Arg?Asp?Leu?Leu?Ala
100?????????????????105?????????????????110Arg?Asp?Asp?Gly?Glu?Thr?Gln?His?Glu?Ala?Ala?Ala?Pro?Asp?Ala?Ala
115?????????????????120?????????????????125Arg?Leu?Thr?Arg?Ser?Gly?Gly?Val?Lys?Arg?Arg?Asn?Met?Asp?Asp?Met
130?????????????????135?????????????????140Ala?Gly?Arg?Pro?Met?Val?Lys?Gly?Gly?Ser?Gly?Glu?Asp?Lys?Val?Pro145?????????????????150?????????????????155?????????????????160Thr?Gln?Gln?Lys?Arg?His?Gln?Leu?Asn?Asn?Phe?Gly?Gln?Met?Arg?Gln
165?????????????????170?????????????????175Thr?Met?Leu?Ser?Lys?Met?Ala?His?Pro?Ala?Ser?Ala?Asn?Ala?Gly?Asp
180?????????????????185?????????????????190Arg?Leu?Gln?His?Ser?Pro?Pro?His?Ile?Pro?Gly?Ser?His?His?Glu?Ile
195?????????????????200?????????????????205Lys?Glu?Glu?Pro?Val?Gly?Ser?Thr?Ser?Lys?Ala?Thr?Thr?Ala?His?Ala
210?????????????????215?????????????????220Asp?Arg?Val?Glu?Ile?Ala?Gln?Glu?Asp?Asp?Asp?Ser?Glu?Phe?Gln?Gln225?????????????????230?????????????????235?????????????????240Leu?His?Gln?Gln?Arg?Leu?Ala?Arg?Glu?Arg?Glu?Asn?Pro?Pro?Gln?Pro
245?????????????????250?????????????????255Pro?Lys?Leu?Gly?Val?Ala?Thr?Pro?Ile?Ser?Ala?Arg?Phe?Gln?Pro?Lys
260?????????????????265?????????????????270Leu?Thr?Ala?Val?Ala?Glu?Ser?Val?Leu?Glu?Gly?Thr?Asp?Thr?Thr?Gln
275?????????????????280?????????????????285Ser?Pro?Leu?Lys?Pro?Gln?Ser?Met?Leu?Lys?Gly?Ser?Gly?Ala?Gly?Val
290?????????????????295?????????????????300Thr?Pro?Leu?Ala?Val?Thr?Leu?Asp?Lys?Gly?Lys?Leu?Gln?Leu?Ala?Pro305?????????????????310?????????????????315?????????????????320Asp?Asn?Pro?Pro?Ala?Leu?Asn?Thr?Leu?Leu?Lys?Gln?Thr?Leu?Gly?Lys
325?????????????????330?????????????????335Asp?Thr?Gln?His?Tyr?Leu?Ala?His?His?Ala?Ser?Ser?Asp?Gly?Ser?Gln
340???????????????????345????????????????350His?Leu?Leu?Leu?Asp?Asn?Lys?Gly?His?Leu?Phe?Asp?Ile?Lys?Ser?Thr
355?????????????????360?????????????????365Ala?Thr?Ser?Tyr?Ser?Val?Leu?His?Asn?Ser?His?Pro?Gly?Glu?Ile?Lys
370?????????????????375?????????????????380Gly?Lys?Leu?Ala?Gln?Ala?Gly?Thr?Gly?Ser?Val?Ser?Val?Asp?Gly?Lys385?????????????????390?????????????????395?????????????????400Ser?Gly?Lys?Ile?Ser?Leu?Gly?Ser?Gly?Thr?Gln?Ser?His?Asn?Lys?Thr
405?????????????????4l0?????????????????415Met?Leu?Ser?Gln?Pro?Gly?Glu?Ala?His?Arg?Ser?Leu?Leu?Thr?Gly?Ile
420?????????????????425?????????????????430Trp?Gln?His?Pro?Ala?Gly?Ala?Ala?Arg?Pro?Gln?Gly?Glu?Ser?Ile?Arg
435?????????????????440?????????????????445Leu?His?Asp?Asp?Lys?Ile?His?Ile?Leu?His?Pro?Glu?Leu?Gly?Val?Trp
450?????????????????455?????????????????460Gln?Ser?Ala?Asp?Lys?Asp?Thr?His?Ser?Gln?Leu?Ser?Arg?Gln?Ala?Asp465?????????????????470?????????????????475?????????????????480Gly?Lys?Leu?Tyr?Ala?Leu?Lys?Asp?Asn?Arg?Thr?Leu?Gln?Ash?Leu?Ser
485?????????????????490?????????????????495Asp?Asn?Lys?Ser?Ser?Glu?Lys?Leu?Val?Asp?Lys?Ile?Lys?Ser?Tyr?Ser
500?????????????????505?????????????????510Val?Asp?Gln?Arg?Gly?Gln?Val?Ala?Ile?Leu?Thr?Asp?Thr?Pro?Gly?Arg
515?????????????????520?????????????????525His?Lys?Met?Ser?Ile?Met?Pro?Ser?Leu?Asp?Ala?Ser?Pro?Glu?Ser?His
530?????????????????535?????????????????540Ile?Ser?Leu?Ser?Leu?His?Phe?Ala?Asp?Ala?His?Gln?Gly?Leu?Leu?His545?????????????????550?????????????????555?????????????????560Gly?Lys?Ser?Glu?Leu?Glu?Ala?Gln?Ser?Val?Ala?Ile?Ser?His?Gly?Arg
565?????????????????570?????????????????575Leu?Val?Val?Ala?Asp?Ser?Glu?Gly?Lys?Leu?Phe?Ser?Ala?Ala?Ile?Pro
580?????????????????585?????????????????590Lys?Gln?Gly?Asp?Gly?Asn?Glu?Leu?Lys?Met?Lys?Ala?Met?Pro?Gln?His
595?????????????????600?????????????????605Ala?Leu?Asp?Glu?His?Phe?Gly?His?Asp?His?Gln?Ile?Ser?Gly?Phe?Phe
610?????????????????615?????????????????620His?Asp?Asp?His?Gly?Gln?Leu?Asn?Ala?Leu?Val?Lys?Asn?Asn?Phe?Arg625?????????????????630?????????????????635?????????????????640Gln?Gln?His?Ala?Cys?Pro?Leu?Gly?Asn?Asp?His?Gln?Phe?His?Pro?Gly
645?????????????????650?????????????????655Trp?Asn?Leu?Thr?Asp?Ala?Leu?Val?Ile?Asp?Asn?Gln?Leu?Gly?Leu?His
660?????????????????665?????????????????670His?Thr?Asn?Pro?Glu?Pro?His?Glu?Ile?Leu?Asp?Met?Gly?His?Leu?Gly
675?????????????????680?????????????????685Ser?Leu?Ala?Leu?Gln?Glu?Gly?Lys?Leu?His?Tyr?Phe?Asp?Gln?Leu?Thr
690?????????????????695?????????????????700Lys?Gly?Trp?Thr?Gly?Ala?Glu?Ser?Asp?Cys?Lys?Gln?Leu?Lys?Lys?Gly705?????????????????710?????????????????715?????????????????720Leu?Asp?Gly?Ala?Ala?Tyr?Leu?Leu?Lys?Asp?Gly?Glu?Val?Lys?Arg?Leu
725?????????????????730?????????????????735Asn?Ile?Asn?Gln?Ser?Thr?Ser?Ser?Ile?Lys?His?Gly?Thr?Glu?Asn?Val
740?????????????????745?????????????????750Phe?Ser?Leu?Pro?His?Val?Arg?Asn?Lys?Pro?Glu?Pro?Gly?Asp?Ala?Leu
755?????????????????760?????????????????765Gln?Gly?Leu?Asn?Lys?Asp?Asp?Lys?Ala?Gln?Ala?Met?Ala?Val?Ile?Gly
770?????????????????775?????????????????780Val?Asn?Lys?Tyr?Leu?Ala?Leu?Thr?Glu?Lys?Gly?Asp?Ile?Arg?Ser?Phe785?????????????????790?????????????????795?????????????????800Gln?Ile?Lys?Pro?Gly?Thr?Gln?Gln?Leu?Glu?Arg?Pro?Ala?Gln?Thr?Leu
805?????????????????810?????????????????815Ser?Arg?Glu?Gly?Ile?Ser?Gly?Glu?Leu?Lys?Asp?Ile?His?Val?Asp?His
820?????????????????825?????????????????830Lys?Gln?Asn?Leu?Tyr?Ala?Leu?Thr?His?Glu?Gly?Glu?Val?Phe?His?Gln
835?????????????????840?????????????????845Pro?Arg?Glu?Ala?Trp?Gln?Asn?Gly?Ala?Glu?Ser?Ser?Ser?Trp?His?Lys
850?????????????????855?????????????????860Leu?Ala?Leu?Pro?Gln?Ser?Glu?Ser?Lys?Leu?Lys?Ser?Leu?Asp?Met?Ser865?????????????????870?????????????????875?????????????????880His?Glu?His?Lys?Pro?Ile?Ala?Thr?Phe?Glu?Asp?Gly?Ser?Gln?His?Gln
885?????????????????890?????????????????895Leu?Lys?Ala?Gly?Gly?Trp?His?Ala?Tyr?Ala?Ala?Pro?Glu?Arg?Gly?Pro
900?????????????????905?????????????????910Leu?Ala?Val?Gly?Thr?Ser?Gly?Ser?Gln?Thr?Val?Phe?Asn?Arg?Leu?Met
915?????????????????920?????????????????925Gln?Gly?Val?Lys?Gly?Lys?Val?Ile?Pro?Gly?Ser?Gly?Leu?Thr?Val?Lys
930?????????????????935?????????????????940Leu?Ser?Ala?Gln?Thr?Gly?Gly?Met?Thr?Gly?Ala?Glu?Gly?Arg?Lys?Val945?????????????????950?????????????????955?????????????????960Ser?Ser?Lys?Phe?Ser?Glu?Arg?Ile?Arg?Ala?Tyr?Ala?Phe?Asn?Pro?Thr
965?????????????????970?????????????????975Met?Ser?Thr?Pro?Arg?Pro?Ile?Lys?Asn?Ala?Ala?Tyr?Ala?Thr?Gln?His
980?????????????????985?????????????????990Gly?Trp?Gln?Gly?Arg?Glu?Gly?Leu?Lys?Pro?Leu?Tyr?Glu?Met?Gln?Gly
995?????????????????1000????????????????1005Ala?Leu?Ile?Lys?Gln?Leu?Asp?Ala?His?Asn?Val?Arg?His?Asn?Ala?Pro
1010????????????????1015????????????????1020Gln?Pro?Asp?Leu?Gln?Ser?Lys?Leu?Glu?Thr?Leu?Asp?Leu?Gly?Glu?His1025????????????????1030????????????????1035????????????????1040Gly?Ala?Glu?Leu?Leu?Asn?Asp?Met?Lys?Arg?Phe?Arg?Asp?Glu?Leu?Glu
1045????????????????1050????????????????1055Gln?Ser?Ala?Thr?Arg?Ser?Val?Thr?Val?Leu?Gly?Gln?His?Gln?Gly?Val
1060????????????????1065????????????????1070Leu?Lys?Ser?Asn?Gly?Glu?Ile?Asn?Ser?Glu?Phe?Lys?Pro?Ser?Pro?Gly
1075????????????????1080????????????????1085Lys?Ala?Leu?Val?Gln?Ser?Phe?Asn?Val?Asn?Arg?Ser?Gly?Gln?Asp?Leu
1090????????????????1095????????????????1100Ser?Lys?Ser?Leu?Gln?Gln?Ala?Val?His?Ala?Thr?Pro?Pro?Ser?Ala?Glu1105???????????????1110????????????????1115????????????????1120Ser?Lys?Leu?Gln?Ser?Met?Leu?Gly?His?Phe?Val?Ser?Ala?Gly?Val?Asp
1125????????????????1130????????????????1135Met?Ser?His?Gln?Lys?Gly?Glu?Ile?Pro?Leu?Gly?Arg?Gln?Arg?Asp?Pro
1140????????????????1145????????????????1150Asn?Asp?Lys?Thr?Ala?Leu?Thr?Lys?Ser?Arg?Leu?Ile?Leu?Asp?Thr?Val
1155????????????????1160????????????????1165Thr?Ile?Gly?Glu?Leu?His?Glu?Leu?Ala?Asp?Lys?Ala?Lys?Leu?Val?Ser???1170????????????????1175????????????????1180Asp?His?Lys?Pro?Asp?Ala?Asp?Gln?Ile?Lys?Gln?Leu?Arg?Gln?Gln?Phe1185???????????????1190????????????????1195????????????????1200Asp?Thr?Leu?Arg?Glu?Lys?Arg?Tyr?G1u?Ser?Asn?Pro?Val?Lys?His?Tyr
1205????????????????1210????????????????1215Thr?Asp?Met?Gly?Phe?Thr?His?Asn?Lys?Ala?Leu?Glu?Ala?Asn?Tyr?Asp
1220????????????????1225????????????????1230Ala?Val?Lys?Ala?Phe?Ile?Asn?Ala?Phe?Lys?Lys?Glu?His?His?Gly?Val
1235????????????????1240????????????????1245Asn?Leu?Thr?Thr?Arg?Thr?Val?Leu?Glu?Ser?Gln?Gly?Ser?Ala?Glu?Leu???1250????????????????1255????????????????1260Ala?Lys?Lys?Leu?Lys?Asn?Thr?Leu?Leu?Ser?Leu?Asp?Ser?Gly?Glu?Ser1265???????????????1270????????????????1275????????????????1280Met?Ser?Phe?Ser?Arg?Ser?Tyr?Gly?Gly?Gly?Val?Ser?Thr?Val?Phe?Val
1285????????????????1290????????????????1295Pro?Thr?Leu?Ser?Lys?Lys?Val?Pro?Val?Pro?Val?Ile?Pro?Gly?Ala?Gly
1300????????????????1305????????????????1310Ile?Thr?Leu?Asp?Arg?Ala?Tyr?Asn?Leu?Ser?Phe?Ser?Arg?Thr?Ser?Gly
1315????????????????1320????????????????1325Gly?Leu?Asn?Val?Ser?Phe?Gly?Arg?Asp?Gly?Gly?Val?Ser?Gly?Asn?Ile???1330????????????????1335????????????????1340Met?Val?Ala?Thr?Gly?His?Asp?Val?Met?Pro?Tyr?Met?Thr?Gly?Lys?Lys1345???????????????1350????????????????1355????????????????1360Thr?Ser?Ala?Gly?Asn?Ala?Ser?Asp?Trp?Leu?Ser?Ala?Lys?His?Lys?Ile
1365????????????????1370????????????????1375Ser?Pro?Asp?Leu?Arg?Ile?Gly?Ala?Ala?Val?Ser?Gly?Thr?Leu?Gln?Gly
1380????????????????1385????????????????1390Thr?Leu?Gln?Asn?Ser?Leu?Lys?Phe?Lys?Leu?Thr?Glu?Asp?Glu?Leu?Pro
1395????????????????1400????????????????1405Gly?Phe?Ile?His?Gly?Leu?Thr?His?Gly?Thr?Leu?Thr?Pro?Ala?Glu?Leu???1410????????????????1415????????????????1420Leu?Gln?Lys?Gly?Ile?Glu?His?Gln?Met?Lys?Gln?Gly?Ser?Lys?Leu?Thr1425???????????????1430????????????????1435????????????????1440Phe?Ser?Val?Asp?Thr?Ser?Ala?Asn?Leu?Asp?Leu?Arg?Ala?Gly?Ile?Asn
1445????????????????1450????????????????1455Leu?Asn?Glu?Asp?Gly?Ser?Lys?Pro?Asn?Gly?Val?Thr?Ala?Arg?Val?Ser
1460????????????????1465????????????????1470Ala?Gly?Leu?Ser?Ala?Ser?Ala?Asn?Leu?Ala?Ala?Gly?Ser?Arg?Glu?Arg
1475????????????????1480????????????????1485Ser?Thr?Thr?Ser?Gly?Gln?Phe?Gly?Ser?Thr?Thr?Ser?Ala?Ser?Asn?Asn???1490????????????????1495????????????????1500Arg?Pro?Thr?Phe?Leu?Asn?Gly?Val?Gly?Ala?Gly?Ala?Asn?Leu?Thr?Ala1505???????????????1510????????????????1515????????????????1520Ala?Leu?Gly?Val?Ala?His?Ser?Ser?Thr?His?Glu?Gly?Lys?Pro?Val?Gly
1525????????????????1530????????????????1535Ile?Phe?Pro?Ala?Phe?Thr?Ser?Thr?Asn?Val?Ser?Ala?Ala?Leu?Ala?Leu
1540????????????????1545????????????????1550Asp?Asn?Arg?Thr?Ser?Gln?Ser?Ile?Ser?Leu?Glu?Leu?Lys?Arg?Ala?Glu
1555????????????????1560????????????????1565Pro?Val?Thr?Ser?Asn?Asp?Ile?Ser?Glu?Leu?Thr?Ser?Thr?Leu?Gly?Lys???1570????????????????1575????????????????1580His?Phe?Lys?Asp?Ser?Ala?Thr?Thr?Lys?Met?Leu?Ala?Ala?Leu?Lys?Glu1585???????????????1590????????????????1595????????????????1600Leu?Asp?Asp?Ala?Lys?Pro?Ala?Glu?Gln?Leu?His?Ile?Leu?Gln?Gln?His
1605????????????????1610????????????????1615Phe?Ser?Ala?Lys?Asp?Val?Val?Gly?Asp?Glu?Arg?Tyr?Glu?Ala?Val?Arg
1620????????????????1625????????????????1630Asn?Leu?Lys?Lys?Leu?Val?Ile?Arg?Gln?Gln?Ala?Ala?Asp?Ser?His?Ser
1635????????????????1640????????????????1645Met?Glu?Leu?Gly?Ser?Ala?Ser?His?Ser?Thr?Thr?Tyr?Asn?Asn?Leu?Ser???1650????????????????1655????????????????1660Arg?Ile?Asn?Asn?Asp?Gly?Ile?Val?Glu?Leu?Leu?His?Lys?His?Phe?Asp1665???????????????1670????????????????1675????????????????1680Ala?Ala?Leu?Pro?Ala?Ser?Ser?Ala?Lys?Arg?Leu?Gly?Glu?Met?Met?Asn
1685????????????????1690????????????????1695Asn?Asp?Pro?Ala?Leu?Lys?Asp?Ile?Ile?Lys?Gln?Leu?Gln?Ser?Thr?Pro
1700????????????????1705????????????????1710Phe?Ser?Ser?Ala?Ser?Val?Ser?Met?Glu?Leu?Lys?Asp?Gly?Leu?Arg?Glu
1715????????????????1720????????????????1725Gln?Thr?Glu?Lys?Ala?Ile?Leu?Asp?Gly?Lys?Val?Gly?Arg?Glu?Glu?Val???1730????????????????1735????????????????1740Gly?Val?Leu?Phe?Gln?Asp?Arg?Asn?Asn?Leu?Arg?Val?Lys?Ser?Val?Ser1745???????????????1750????????????????1755????????????????1760Val?Ser?Gln?Ser?Val?Ser?Lys?Ser?Glu?Gly?Phe?Asn?Thr?Pro?Ala?Leu
1765????????????????1770????????????????1775Leu?Leu?Gly?Thr?Ser?Asn?Ser?Ala?Ala?Met?Ser?Met?Glu?Arg?Asn?Ile
1780????????????????1785????????????????1790Gly?Thr?Ile?Asn?Phe?Lys?Tyr?Gly?Gln?Asp?Gln?Asn?Thr?Pro?Arg?Arg
1795 1800 1805Phe Thr Leu Glu Gly Gly Ile Ala Gln Ala Asn Pro Gln Val Ala Ser 1810 1815 1820Ala Leu Thr Asp Leu Lys Lys Glu Gly Leu Glu Met Lys Ser1825 1830 1835＜210〉29＜211〉420＜212〉DNA＜213〉＜400〉29atgacatcgt cacagcagcg ggttgaaagg tttttacagt atttctccgc cgggtgtaaa 60acgcccatac atctgaaaga cggggtgtgc gccctgtata acgaacaaga tgaggaggcg 120gcggtgctgg aagtaccgca acacagcgac agcctgttao tacactgccg aatcattgag 180gctgacccac aaacttcaat aaccctgtat tcgatgctat tacagotgaa ttttgaaatg 240gcggccatgc goggctgttg gctggcgctg gatgaactgc acaacgtgcg tttatgtttt 300cagcagtcgc tggagcatct ggatgaagca agttttagcg atatcgttag cggcttcatc 360gaacatgcgg cagaagtgcg tgagtatata gcgcaattag acgagagtag cgcggcataa 420＜210〉30＜211〉139＜212〉PRT＜213〉＜400〉30Met Thr Ser Ser Gln Gln Arg Val Glu Arg Phe Leu Gln Tyr Phe Ser 1 5 10 15Ala Gly Cys Lys Thr Pro Ile His Leu Lys Asp Gly Val Cys Ala Leu
20??????????????????25??????????????????30Tyr?Asn?Glu?Gln?Asp?Glu?Glu?Ala?Ala?Val?Leu?Glu?Val?Pro?Gln?His
35??????????????????40??????????????????45Ser?Asp?Ser?Leu?Leu?Leu?His?Cys?Arg?Ile?Ile?Glu?Ala?Asp?Pro?Gln
50??????????????????55??????????????????60Thr?Ser?Ile?Thr?Leu?Tyr?Ser?Met?Leu?Leu?Gln?Leu?Asn?Phe?Glu?Met?65??????????????????70??????????????????75??????????????????80Ala?Ala?Met?Arg?Gly?Cys?Trp?Leu?Ala?Leu?Asp?Glu?Leu?His?Asn?Val
85??????????????????90??????????????????95Arg?Leu?Cys?Phe?Gln?Gln?Ser?Leu?Glu?His?Leu?Asp?Glu?Ala?Ser?Phe
100?????????????????105?????????????????110Ser?Asp?Ile?Val?Ser?Gly?Phe?Ile?Glu?His?Ala?Ala?Glu?Val?Arg?Glu
115?????????????????120?????????????????125Tyr?Ile?Ala?Gln?Leu?Asp?Glu?Ser?Ser?Ala?Ala
130 135＜210〉3l＜211〉341＜212〉PRT＜213〉pseudomonas syringae＜400〉31Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met, 15 10 15Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser
20??????????????????25??????????????????30Ser?Lys?Ala?Leu?Gln?Glu?Val?Val?Val?Lys?Leu?Ala?Glu?Glu?Leu?Met
35??????????????????40??????????????????45Arg?Asn?Gly?Gln?Leu?Asp?Asp?Ser?Ser?Pro?Leu?Gly?Lys?Leu?Leu?Ala
50??????????????????55??????????????????60Lys?Ser?Met?Ala?Ala?Asp?Gly?Lys?Ala?Gly?Gly?Gly?Ile?Glu?Asp?Val?65??????????????????70??????????????????75??????????????????80Ile?Ala?Ala?Leu?Asp?Lys?Leu?Ile?His?Glu?Lys?Leu?Gly?Asp?Asn?Phe
85??????????????????90??????????????????95Gly?Ala?Ser?Ala?Asp?Ser?Ala?Ser?Gly?Thr?Gly?Gln?Gln?Asp?Leu?Met
100?????????????????105?????????????????110Thr?Gln?Val?Leu?Asn?Gly?Leu?Ala?Lys?Ser?Met?Leu?Asp?Asp?Leu?Leu
115?????????????????120?????????????????125Thr?Lys?Gln?Asp?Gly?Gly?Thr?Ser?Phe?Ser?Glu?Asp?Asp?Met?Pro?Met
130?????????????????135?????????????????140Leu?Asn?Lys?Ile?Ala?Gln?Phe?Met?Asp?Asp?Asn?Pro?Ala?Gln?Phe?Pro145?????????????????150?????????????????155?????????????????160Lys?Pro?Asp?Ser?Gly?Ser?Trp?Val?Asn?Glu?Leu?Lys?Glu?Asp?Asn?Phe
165?????????????????170?????????????????175Leu?Asp?Gly?Asp?Glu?Thr?Ala?Ala?Phe?Arg?Ser?Ala?Leu?Asp?Ile?Ile
180?????????????????185?????????????????190Gly?Gln?Gln?Leu?Gly?Asn?Gln?Gln?Ser?Asp?Ala?Gly?Ser?Leu?Ala?Gly
195?????????????????200?????????????????205Thr?Gly?Gly?Gly?Leu?Gly?Thr?Pro?Ser?Ser?Phe?Ser?Asn?Asn?Ser?Ser
210?????????????????215?????????????????220Val?Met?Gly?Asp?Pro?Leu?Ile?Asp?Ala?Asn?Thr?Gly?Pro?Gly?Asp?Ser225?????????????????230?????????????????235?????????????????240Gly?Asn?Thr?Arg?Gly?Glu?Ala?Gly?Gln?Leu?Ile?Gly?Glu?Leu?Ile?Asp
245?????????????????250?????????????????255Arg?Gly?Leu?Gln?Ser?Va1?Leu?Ala?Gly?Gly?Gly?Leu?Gly?Thr?Pro?Val
260?????????????????265?????????????????270Asn?Thr?Pro?Gln?Thr?Gly?Thr?Ser?Ala?Asn?Gly?Gly?Gln?Ser?Ala?Gln
275?????????????????280?????????????????285Asp?Leu?Asp?Gln?Leu?Leu?Gly?Gly?Leu?Leu?Leu?Lys?Gly?Leu?Glu?Ala
290?????????????????295?????????????????300Thr?Leu?Lys?Asp?Ala?Gly?Gln?Thr?Gly?Thr?Asp?Val?Gln?Ser?Ser?Ala305?????????????????310?????????????????315?????????????????320Ala?Gln?Ile?Ala?Thr?Leu?Leu?Val?Ser?Thr?Leu?Leu?Gln?Gly?Thr?Arg
325?????????????????330?????????????????335Asn?Gln?Ala?Ala?Ala
340
<210>32
<211>1026
<212>DNA
＜213〉pseudomonas syringae
＜400〉32atgcagagtc tcagtcttaa cagcagctcg ctgcaaaccc cggcaatggc ccttgtcctg 60gtacgtcctg aagccgagac gactggcagt acgtcgagca aggcgcttca ggaagttgtc 120gtgaagctgg ccgaggaact gatgcgcaat ggtcaactcg acgacagctc gccattggga 180aaactgttgg ccaagtcgat ggccgcagat ggcaaggcgg gcggcggtat tgaggatgtc 240atcgctgcgc tggacaagct gatccatgaa aagctcggtg acaacttcgg cgcgtctgcg 300gacagcgcct cgggtaccgg acagcaggac ctgatgactc aggtgctcaa tggcctggcc 360aagtcgatgc tcgatgatct tctgaccaag caggatggcg ggacaagctt ctccgaagac 420gatatgccga tgctgaacaa gatcgcgcag ttcatggatg acaatcccgc acagtttccc 480aagccggact cgggctcctg ggtgaacgaa ctcaaggaag acaacttcct tgatggcgac 540gaaacggctg cgttccgttc ggcactcgac atcattggcc agcaactggg taatcagcag 600agtgacgctg gcagtctggc agggacgggt ggaggtctgg gcactccgag cagtttttcc 660aacaactcgt ccgtgatggg tgatccgctg atcgacgcca ataccggtcc cggtgacagc 720ggcaataccc gtggtgaagc ggggcaactg atcggcgagc ttatcgaccg tggcctgcaa 780tcggtattgg ccggtggtgg actgggcaca cccgtaaaca ccccgcagac cggtacgtcg 840gcgaatggcg gacagtccgc tcaggatctt gatcagttgc tgggcggctt gctgctcaag 900ggcctggagg caacgctcaa ggatgccggg caaacaggca ccgacgtgca gtcgagcgct 960gcgcaaatcg ccaccttgct ggtcagtacg ctgctgcaag gcacccgcaa tcaggctgca 1020gcctga 1026＜210〉33＜211〉1729＜212〉DNA＜213〉＜400〉33tccacttcgc tgattttgaa attggcagat tcatagaaac gttcaggtgt ggaaatcagg 60ctgagtgcgc agatttcgtt gataagggtg tggtactggt cattgttggt catttcaagg 120cctetgagtg cggtgcggag caataccagt cttcctgctg gcgtgtgcac actgagtcgc 180aggcataggc atttcagttc cttgcgttgg ttgggcatat aaaaaaagga acttttaaaa 240acagtgcaat gagatgecgg caaaacggga accggtcgct gcgctttgcc actcacttcg 300agcaagctca accccaaaca tccacatccc tatcgaacgg acagcgatac ggccacttgc 360tctggtaaac cctggagctg gcgtcggtcc aattgcccac ttagcgaggt aacgcagcat 420gagcatcggc atcacacccc ggccgcaaca gaccaccacg ccactcgatt tttcggcgct 480aagcggcaag agtcctcaac caaacacgtt cggcgagcag aacactcagc aagcgatcga 540cccgagtgca ctgttgttcg gcagcgacac acagaaagac gtcaacttcg gcacgcccga 600cageaccgtc cagaatccgc aggacgccag caagcccaac gacagccagt ccaacatcgc 660taaattgatc agtgcattga tcatgtcgtt gctgcagatg ctcaccaact ccaataaaaa 720gcaggacacc aatcaggaac agcctgatag ccaggctcct ttccagaaca acggcgggct 780cggtacaccg tcggccgata gcgggggcgg cggtacaccg gatgcgacag gtggcggcgg 840cggtgatacg ccaagcgcaa caggcggtgg cggcggtgat actccgaccg caacaggcgg 900tggcggcagc ggtggcggcg gcacacccac tgcaacaggt ggcggcagcg gtggcacacc 960cactgcaaca ggcggtggcg agggtggcgt aacaccgcaa atcactccgc agttggccaa 1020ccctaaccgt acctcaggta ctggctcggt gtcggacacc gcaggttcta ccgagcaagc 1080cggcaagatc aatgtggtga aagacaccat caaggtcggc gctggcgaag tctttgacgg 1140ccacggcgca accttcactg ccgacaaatc tatgggtaac ggagaccagg gcgaaaatca 1200gaagcccatg ttcgagctgg ctgaaggcgc tacgttgaag aatgtgaacc tgggtgagaa 1260cgaggtcgat ggcatccacg tgaaagccaa aaacgctcag gaagtcacca ttgacaacgt 1320gcatgcccag aacgtcggtg aagacctgat tacggtcaaa ggcgagggag gcgcagcggt 1380cactaatctg aacatcaaga acagcagtge caaaggtgca gacgacaagg ttgtccagct 1440caacgccaac actcacttga aaatcgacaa cttcaaggcc gacgatttcg gcacgatggt 1500tcgcaccaac ggtggcaagc agtttgatga catgagcatc gagctgaacg gcatcgaagc 1560taaccacggc aagttcgccc tggtgaaaag cgacagtgac gatctgaagc tggcaacggg 1620caacatcgcc atgaccgacg tcaaacacgc ctacgataaa acccaggcat cgacccaaca 1680caccgagctt tgaatccaga caagtagctt gaaaaaaggg ggtggactc 1729＜210〉34＜211〉424＜212〉PRT＜213〉＜400〉34Met Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Thr Pro Leu 1 5 10 15Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr Phe Gly
20??????????????????25??????????????????30Glu?Gln?Asn?Thr?Gln?Gln?Ala?Ile?Asp?Pro?Ser?Ala?Leu?Leu?Phe?Gly
35??????????????????40??????????????????45Ser?Asp?Thr?Gln?Lys?Asp?Val?Asn?Phe?Gly?Thr?Pro?Asp?Ser?Thr?Val
50??????????????????55??????????????????60Gln?Asn?Pro?Gln?Asp?Ala?Ser?Lys?Pro?Asn?Asp?Ser?Gln?Ser?Asn?Ile?65??????????????????70??????????????????75??????????????????80Ala?Lys?Leu?Ile?Ser?Ala?Leu?Ile?Met?Ser?Leu?Leu?Gln?Met?Leu?Thr
85??????????????????90??????????????????95Asn?Ser?Asn?Lys?Lys?Gln?Asp?Thr?Asn?Gln?Glu?Gln?Pro?Asp?Ser?Gln
100?????????????????105?????????????????110Ala?Pro?Phe?Gln?Asn?Asn?Gly?Gly?Leu?Gly?Thr?Pro?Ser?Ala?Asp?Ser
115?????????????????120?????????????????125Gly?Gly?Gly?Gly?Thr?Pro?Asp?Ala?Thr?Gly?Gly?Gly?Gly?Gly?Asp?Thr
130?????????????????135?????????????????140Pro?Ser?Ala?Thr?Gly?Gly?Gly?Gly?Gly?Asp?Thr?Pro?Thr?Ala?Thr?Gly145?????????????????150?????????????????155?????????????????160Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Thr?Pro?Thr?Ala?Thr?Gly?Gly?Gly
165?????????????????170?????????????????175Ser?Gly?Gly?Thr?Pro?Thr?Ala?Thr?Gly?Gly?Gly?Glu?Gly?Gly?Val?Thr
180?????????????????185?????????????????190Pro?Gln?Ile?Thr?Pro?Gln?Leu?Ala?Ash?Pro?Asn?Arg?Thr?Ser?Gly?Thr
195?????????????????200?????????????????205Gly?Ser?Val?Ser?Asp?Thr?Ala?Gly?Ser?Thr?Glu?Gln?Ala?Gly?Lys?Ile
210?????????????????215?????????????????220Asn?Val?Val?Lys?Asp?Thr?Ile?Lys?Val?Gly?Ala?Gly?Glu?Val?Phe?Asp?225????????????????230?????????????????235?????????????????240Gly?His?Gly?Ala?Thr?Phe?Thr?Ala?Asp?Lys?Ser?Met?Gly?Asn?Gly?Asp
245?????????????????250?????????????????255Gln?Gly?Glu?Asn?Gln?Lys?Pro?Met?Phe?Glu?Leu?Ala?Glu?Gly?Ala?Thr
260?????????????????265?????????????????270Leu?Lys?Asn?Val?Asn?Leu?Gly?Glu?Asn?Glu?Val?Asp?Gly?Ile?His?Val
275?????????????????280?????????????????285Lys?Ala?Lys?Asn?Ala?Gln?Glu?Val?Thr?Ile?Asp?Asn?Val?His?Ala?Gln
290?????????????????295?????????????????300Asn?Val?Gly?Glu?Asp?Leu?Ile?Thr?Val?Lys?Gly?Glu?Gly?Gly?Ala?Ala305?????????????????310?????????????????315?????????????????320Val?Thr?Asn?Leu?Asn?Ile?Lys?Asn?Ser?Ser?Ala?Lys?Gly?Ala?Asp?Asp
325?????????????????330?????????????????335Lys?Val?Val?Gln?Leu?Asn?Ala?Asn?Thr?His?Leu?Lys?Ile?Asp?Asn?Phe
340?????????????????345?????????????????350Lys?Ala?Asp?Asp?Phe?Gly?Thr?Met?Val?Arg?Thr?Asn?Gly?Gly?Lys?Gln
355?????????????????360?????????????????365Phe?Asp?Asp?Met?Ser?Ile?Glu?Leu?Asn?Gly?Ile?Glu?Ala?Asn?His?Gly
370?????????????????375?????????????????380Lys?Phe?Ala?Leu?Val?Lys?Ser?Asp?Ser?Asp?Asp?Leu?Lys?Leu?Ala?Thr385?????????????????390?????????????????395?????????????????400Gly?Asn?Ile?Ala?Met?Thr?Asp?Val?Lys?His?Ala?Tyr?Asp?Lys?Thr?Gln
405?????????????????410?????????????????415Ala?Ser?Thr?Gln?His?Thr?Glu?Leu
420＜210〉35＜211〉344＜212〉PRT＜213〉aeruginosa eggplant＜220〉＜and the description of 223〉unknown bodies: aeruginosa eggplant＜400〉35Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln 15 10 15Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser
20??????????????????25??????????????????30Val?Gln?Asp?Leu?Ile?Lys?Gln?Val?Glu?Lys?Asp?Ile?Leu?Asn?Ile?Ile
35??????????????????40??????????????????45Ala?Ala?Leu?Val?Gln?Lys?Ala?Ala?Gln?Ser?Ala?Gly?Gly?Asn?Thr?Gly
50??????????????????55??????????????????60Asn?Thr?Gly?Asn?Ala?Pro?Ala?Lys?Asp?Gly?Asn?Ala?Asn?Ala?Gly?Ala?65??????????????????70??????????????????75??????????????????80Asn?Asp?Pro?Ser?Lys?Asn?Asp?Pro?Ser?Lys?Ser?Gln?Ala?Pro?Gln?Ser
85??????????????????90??????????????????95Ala?Asn?Lys?Thr?Gly?Asn?Val?Asp?Asp?Ala?Asn?Asn?Gln?Asp?Pro?Met
100?????????????????105?????????????????110Gln?Ala?Leu?Met?Gln?Leu?Leu?Glu?Asp?Leu?Val?Lys?Leu?Leu?Lys?Ala
115?????????????????120?????????????????125Ala?Leu?His?Met?Gln?Gln?Pro?Gly?Gly?Asn?Asp?Lys?Gly?Asn?Gly?Val
130?????????????????135?????????????????140Gly?Gly?Ala?Asn?Gly?Ala?Lys?Gly?Ala?Gly?Gly?Gln?Gly?Gly?Leu?Ala145?????????????????150?????????????????155?????????????????160Glu?Ala?Leu?Gln?Glu?Ile?Glu?Gln?Ile?Leu?Ala?Gln?Leu?Gly?Gly?Gly
165?????????????????170?????????????????175Gly?Ala?Gly?Ala?Gly?Gly?Ala?Gly?Gly?Gly?Val?Gly?Gly?Ala?Gly?Gly
180?????????????????185?????????????????190Ala?Asp?Gly?Gly?Ser?Gly?Ala?Gly?Gly?Ala?Gly?Gly?Ala?Asn?Gly?Ala
195?????????????????200?????????????????205Asp?Gly?Gly?Asn?Gly?Val?Asn?Gly?Asn?Gln?Ala?Asn?Gly?Pro?Gln?Asn
210?????????????????215?????????????????220Ala?Gly?Asp?Val?Asn?Gly?Ala?Asn?Gly?Ala?Asp?Asp?Gly?Ser?Glu?Asp225?????????????????230?????????????????235?????????????????240Gln?Gly?Gly?Leu?Thr?Gly?Val?Leu?Gln?Lys?Leu?Met?Lys?Ile?Leu?Asn
245?????????????????250?????????????????255Ala?Leu?Val?Gln?Met?Met?Gln?Gln?Gly?Gly?Leu?Gly?Gly?Gly?Asn?Gln
260?????????????????265?????????????????270Ala?Gln?Gly?Gly?Ser?Lys?Gly?Ala?Gly?Asn?Ala?Ser?Pro?Ala?Ser?Gly
275?????????????????280?????????????????285Ala?Asn?Pro?Gly?Ala?Asn?Gln?Pro?Gly?Ser?Ala?Asp?Asp?Gln?Ser?Ser
290?????????????????295?????????????????300Gly?Gln?Asn?Asn?Leu?Gln?Ser?Gln?Ile?Met?Asp?Val?Val?Lys?Glu?Val205?????????????????310?????????????????315?????????????????320Val?Gln?Ile?Leu?Gln?Gln?Met?Leu?Ala?Ala?Gln?Asn?Gly?Gly?Ser?Gln
325?????????????????330?????????????????335Gln?Ser?Thr?Ser?Thr?Gln?Pro?Met
340＜210〉36＜211〉1035＜212〉DNA＜213〉＜400〉36atgtcagtcg gaaacatcca gagcccgtcg aacctcccgg gtctgcagaa cctgaacctc 60aacaccaaca ccaacagcca gcaatcgggc cagtccgtgc aagacctgat caagcaggtc 120gagaaggaca tcctcaacat catcgcagcc ctcgtgcaga aggccgcaca gtcggcgggc 180ggcaacaccg gtaacaccgg caacgcgccg gcgaaggacg gcaatgccaa cgcgggcgcc 240aacgacccga gcaagaacga cccgagcaag agccaggctc cgcagtcggc caacaagacc 300ggcaacgtcg acgacgccaa caaccaggat ccgatgcaag cgctgatgca gctgctggaa 360gacctggtga agctgctgaa ggoggccctg cacatgcagc agcccggcgg caatgacaag 420ggcaacggcg tgggcggtgc caacggcgcc aagggtgccg gcggccaggg cggcctggcc 480gaagcgctgc aggagatcga gcagatcctc gcccagctcg gcggcggcgg tgctggcgcc 540ggcggcgcgg gtggcggtgt cggcggtgct ggtggcgcgg atggcggctc cggtgcgggt 600ggcgcaggcg gtgcgaacgg cgccgacggc ggcaatggcg tgaacggcaa ccaggcgaac 660ggcccgcaga acgcaggcga tgtcaacggt gccaacggcg cggatgacgg cagcgaagac 720cagggcggcc tcaccggcgt gctgcaaaag ctgatgaaga tcctgaacgc gctggtgcag 780atgatgcagc aaggcggcct cggcggcggc aaccaggcgc agggcggctc gaagggtgcc 840ggcaacgcct cgccggcttc cggcgcgaac ccgggcgcga accagcccgg ttcggcggat 900gatcaatcgt ccggccagaa caatctgcaa tcccagatca tggatgtggt gaaggaggtc 960gtccagatcc tgcagcagat gctggcggcg cagaacggcg gcagccagca gtccacctcg 1020acgcagccga tgtaa 1035＜210〉37＜211〉26＜212〉PRT＜213〉＜400〉37Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala Ile Leu Ala 1 5 10 15Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr
20 25＜210〉38＜211〉the pathogenic mutation of 20＜212〉PRT＜213〉xanthomonas campestris Flos Pelargoniis＜400〉38Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln l, 5 10 15Leu Leu Ala Met
20＜210〉39＜211〉13＜212〉PRT＜213〉big male epidemic disease mould＜400〉39Val Trp Asn Gln Pro Val Arg Gly Phe Lys Val Tyr Glu l 5 10

Claims (46)

1. the isolating fragment of anaphylaxis exciton albumen or polypeptide, wherein said fragment is not brought out anaphylaxis in plant, but has other activity.
2. according to the isolating fragment of claim 1, wherein said anaphylaxis exciton albumen or polypeptide derive from erwinia, Rhodopseudomonas, xanthomonas or phytophthora.
3. according to the isolating fragment of claim 2, wherein said anaphylaxis exciton albumen or polypeptide must be explained the starch Erwinia by oneself.
4. according to the isolating fragment of claim 3, wherein said fragment is selected from the interior segments of the aminoacid sequence of the N end fragment of aminoacid sequence of C end fragment, SEQ.ID.No.23 of the aminoacid sequence of SEQ.ID.No.23 and SEQ.ID.No.23.
5. according to the isolating fragment of claim 4, wherein said fragment is the following amino acid whose C end fragment that covers SEQ.ID.No.23 in the aminoacid sequence of SEQ.ID.No.23: 169 and 403,210 and 403,267 and 403 or 343 and 403.
6. according to the isolating fragment of claim 4, wherein said fragment is the N end fragment of the aminoacid sequence of SEQ.ID.No.23.
7. according to the isolating fragment of claim 4, wherein said fragment is the following amino acid whose interior segments that covers SEQ.ID.No.23 in the aminoacid sequence of SEQ.ID.No.23: 105 and 179,137 and 166,121 and 150 or 137 and 156.
8. according to the isolating fragment of claim 2, wherein said anaphylaxis exciton derives from pseudomonas syringae.
9. according to the isolating fragment of claim 8, wherein said fragment comprises the amino acid/11 90-294 of SEQ.ID.No.31.
10. isolated DNA molecule is encoded according to the fragment of claim 1.
11. according to the isolated DNA molecule of claim 10, wherein said anaphylaxis exciton albumen or polypeptide derive from erwinia, Rhodopseudomonas, xanthomonas or phytophthora.
12. according to the isolated DNA molecule of claim 11, wherein said anaphylaxis exciton albumen or polypeptide must be explained the starch Erwinia by oneself.
13. according to the isolated DNA molecule of claim 12, wherein said fragment is selected from the interior segments of the aminoacid sequence of the N end fragment of aminoacid sequence of C end fragment, SEQ.ID.No.23 of the aminoacid sequence of SEQ.ID.No.23 and SEQ.ID.No.23.
14. according to the isolated DNA molecule of claim 12, wherein said fragment is the following amino acid whose C end fragment that covers SEQ.ID.No.23 in the aminoacid sequence of SEQ.ID.No.23: 169 and 403,210 and 403,267 and 403 or 343 and 403.
15. according to the isolated DNA molecule of claim 12, wherein said fragment is the N end fragment of the aminoacid sequence of SEQ.ID.No.23.
16. according to the isolated DNA molecule of claim 12, wherein said fragment is the following amino acid whose interior segments that covers SEQ.ID.No.23 in the aminoacid sequence of SEQ.ID.No.23: 105 and 179,137 and 166,121 and 150 or 137 and 156.
17. according to the isolated DNA molecule of claim 11, wherein said anaphylaxis exciton derives from pseudomonas syringae.
18. according to the isolated DNA molecule of claim 18, wherein said fragment comprises the amino acid/11 90-294 of SEQ.ID.No.31.
19. use expression system according to the dna molecular conversion of claim 10.
20. according to the expression system of claim 19, wherein said dna molecular is with suitable sense orientation with and in correct frame.
21. use dna molecular transformed host cells according to claim 10.
22. according to the host cell of claim 21, wherein said host cell is selected from vegetable cell and bacterial cell.
23. according to the host cell of claim 21, wherein said dna molecular transforms with expression system.
24. dna molecular transgenic plant transformed with claim 10.
25. according to the transgenic plant of claim 24, wherein said plant is selected from: clover, paddy rice, wheat, barley, rye, cotton, Sunflower Receptacle, peanut, corn, potato, sweet potato, Kidney bean, pea, witloof, lettuce, Herba Sonchi Arvensis, wild cabbage, brussels sprouts, beet, parsnip, turnip, Cauliflower, blue and white cabbage, radish, spinach, onion, garlic, eggplant, pepper, celery, Radix Dauci Sativae, pumpkin, summer squash (pumpkin), summer squash (zucchini), cucumber, apple, pears, muskmelon, citrus, strawberry, grape, immature fruit of Juteleaf Raspberry, pineapple, soybean, tobacco, tomato, jowar and sugarcane.
26. according to the transgenic plant of claim 24, wherein said plant is selected from: Arabidopis thaliana, African violet belong to, green winter eggplant, Flos Pelargonii, poinsettia, chrysanthemum, carnation and youth-and-old-age.
27. dna molecular transgenic plant transformed seed with claim 10.
28. according to the transgenic plant seed of claim 27, wherein said plant seed is selected from: clover, paddy rice, wheat, barley, rye, cotton, Sunflower Receptacle, peanut, corn, potato, sweet potato, Kidney bean, pea, witloof, lettuce, Herba Sonchi Arvensis, wild cabbage, brussels sprouts, beet, parsnip, turnip, Cauliflower, blue and white cabbage, radish, spinach, onion, garlic, eggplant, pepper, celery, Radix Dauci Sativae, pumpkin, summer squash (pumpkin), summer squash (zucchini), cucumber, apple, pears, muskmelon, citrus, strawberry, grape, immature fruit of Juteleaf Raspberry, pineapple, soybean, tobacco, tomato, jowar and sugarcane.
29. according to the transgenic plant seed of claim 27, wherein said plant seed is selected from: Arabidopis thaliana, African violet belong to, green winter eggplant, Flos Pelargonii, poinsettia, chrysanthemum, carnation and youth-and-old-age.
30. the method for conferring disease resistance in plants, described method comprises:
Under the condition of effectively giving disease resistance, with the form of non-infection the fragment of anaphylaxis exciton albumen or polypeptide is applied to plant or plant seed, described fragment is not brought out anaphylaxis.
31. according to the method for claim 30, wherein during described application with plant treatment.
32. according to the method for claim 30, wherein during described application plant seed is handled, described method also comprises:
In natural soils or artificial soil, sowing with the seed of the fragment of described anaphylaxis exciton processing and
From the seminal propagation plant of described soil, sowing.
33. promote the method for plant-growth, described method comprises:
Effectively promoting under the condition of plant-growth that with the form of non-infection the fragment of anaphylaxis exciton albumen or polypeptide is applied to plant or plant seed, described fragment is not brought out anaphylaxis.
34. according to the method for claim 33, wherein during described application with plant treatment.
35. according to the method for claim 33, wherein during described application plant seed is handled, described method also comprises:
In natural soils or artificial soil, sowing with the seed of the fragment of described anaphylaxis exciton processing and
From the seminal propagation plant of described soil, sowing.
36. the method for plant insect control, described method comprises:
Under the condition of effective pest control, with the form of non-infection the fragment of anaphylaxis exciton albumen or polypeptide is applied to plant or plant seed, described fragment is not brought out anaphylaxis.
37. according to the method for claim 36, wherein during described application with plant treatment.
38. according to the method for claim 36, wherein during described application plant seed is handled, described method also comprises:
In natural soils or artificial soil, sowing with the seed of the fragment of described anaphylaxis exciton processing and
From the seminal propagation plant of described soil, sowing.
39. the method for conferring disease resistance in plants, described method comprises:
Segmental dna molecular transgenic plant transformed or plant seed with coding anaphylaxis exciton albumen or polypeptide are provided, and described fragment is not brought out anaphylaxis, then
Under the condition of effectively giving disease resistance, cultivate described transgenic plant or by transgenic plant that described transgenic plant seed produced.
40., wherein provide transgenic plant according to the method for claim 39.
41., wherein provide transgenic plant seed according to the method for claim 39.
42. promote the method for plant-growth, described method comprises:
Segmental dna molecular transgenic plant transformed or plant seed with coding anaphylaxis exciton albumen or polypeptide are provided, and described fragment is not brought out anaphylaxis, then
Effectively promoting under the condition of plant-growth, cultivating described transgenic plant or by transgenic plant that described transgenic plant seed produced.
43., wherein provide transgenic plant according to the method for claim 42.
44., wherein provide transgenic plant seed according to the method for claim 42.
45. the method for plant insect control, described method comprises:
Segmental dna molecular transgenic plant transformed or plant seed with coding anaphylaxis exciton albumen or polypeptide are provided, and described fragment is not brought out anaphylaxis, then
Under the condition of effective pest control, cultivate described transgenic plant or by transgenic plant that described transgenic plant seed produced.
46., wherein provide transgenic plant according to the method for claim 45.
47., wherein provide transgenic plant seed according to the method for claim 45.
CN99814028A 1998-10-05 1999-10-05 Hypersensitive response elicitor fragments which are active but do not elicit hypersensitive response Pending CN1329619A (en)

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