CN100412194C

CN100412194C - Tomato anti insect related gene, its coding protein and application

Info

Publication number: CN100412194C
Application number: CNB2005100086094A
Authority: CN
Inventors: 李传友; 李常保; 蒋红玲; 吴晓燕
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2004-10-18
Filing date: 2005-02-23
Publication date: 2008-08-20
Anticipated expiration: 2025-02-23
Also published as: CN1824778A

Abstract

The present invention discloses an insect resisting related gene of a tomato, a coded protein thereof and an application thereof. The aim of the present invention is to provide an insect resisting related gene of a tomato, a coded protein thereof and an application for the insect resisting related gene in the process of breeding an insect resisting plant. The insect resisting related gene of a tomato has one of the following nucleotide sequences: 1) a DNA sequence of SEQ ID No. 1 in a sequence list, 2) polynucleotide for coding an SEQ ID No. 2 protein sequence in the sequence list, and 3) a nucleotide sequence which can be hybridized with the DNA sequence limited by SEQ ID No. 1 in the sequence list under the high precision condition. The tomato LeAcx 1 mutation of the present invention results in the deletion of JA synthesis and the loss of related resistance gene expression, and the present invention performs a large function in resistance breeding and quality improvement of plants for insects.

Description

Tomato anti insect related gene and proteins encoded and application

Technical field

The present invention relates to plant gene and proteins encoded thereof and application, particularly relate to a tomato anti insect related gene and proteins encoded thereof and its application in cultivating zoophobous.

Background technology

Current, worldwide, because of insect pest infestation very serious to the loss that crop yield causes.What the most generally adopt in the method for tradition control insect pest infestation is to use chemical insecticide in a large number.According to statistics, have 80% to rely on agricultural chemicals in the output of the whole world by plant protection measure increase.The use of chemical insecticide has also brought residual toxicity, environmental pollution, ecological damage, pest resistance to insecticide and serious problems such as wildness again when having obtained certain economic benefit.Therefore, thus study and utilize self resistant gene of plant effectively to control agricultural insect pest and become the target that the mankind seek assiduously.

In the evolution of long period of time process, plant is set up complicated and diversified resistance reaction mechanism for the predation of opposing insect.Generally speaking, plant is divided into two types of composing type resistance and induction type resistances to the resistance of insect.After the induction type resistance was meant that plant is subjected to insect pest infestation, synthetic rapidly a series ofly had toxic chemical substance to insect, thereby to the feed of insect, digest and assimilate, grow and the movable restraining effect that produces such as breeding, finally reach pest-resistant purpose.Compare with the composing type resistance, the induction type resistance is a kind of more active and more cost-effective resistance reactive mode, and this kind resistance has more theoretical significance and actual application value.Therefore, utilize the resistance of plant self to set up a kind of no residual hazard, the sustainable control approach of free of contamination Agricultural pests is China and even Development Trend of World Agriculture.

In research plant and agricultural pest interaction relationship, tomato is the model plant of generally acknowledging.Exist in the tomato mark-proteinase inhibitor strong and weak to the insect-resistant reaction (Proteinase Inhibitors, PIs).Proteinase inhibitor is the small molecular weight basic protein molecule that a class is rich in Serine, reaches pest-resistant purpose by the activity that suppresses digestive ferment in the insect body.After being subjected to insect pest infestation, can synthesize tomato plant the materials relevant such as PIs in a large number with resistance, and PIs does not just synthesize (being called local reaction) at injured position in a large number, at other position of plant, comprising in the unscathed blade and also can synthesize (being called system response), is a kind of resistance reaction of botanical system.

Resistance reaction for this botanical system, a kind of traditional hypothesis is interpreted as: i.e. infringement (or machinery is injured) corresponding to insect stimulates, plant can be synthesized a class signaling molecule (Signal), this class signaling molecule can long-distance transportation to each position of plant whole body, comprise unscathed blade, thus the induction of resistance Expression of Related Genes.The signaling molecule that is identified at present mainly comprise polypeptide signal molecular system element (Systemin) and derive from the plant hormone jasmonic of unsaturated fatty acids (Jasmonic acid, JA).

Systemin is the polypeptide of being made up of 18 amino-acid residues of can induced strong PIs expressing of extracting from tomato leaf, the terminal shearing of C-of the preceding systemin of forming by 200 amino acid by its precursor product-one (Prosystemin) and.Systemin plays requisite effect in tomato system resistance.External feeding can detect systemin in phloem " motion " through isotope-labeled systemin on injured blade.In view of the above, it is the signaling molecule (Ryan that carries out long-distance transportation in system's resistance that professor Ryan proposes systemin, C.A.1992.Thesearch for the proteinase inhibitor inducing factor, PIIF.Plant Mol.Biol.19,123-134.).

Injured response stimulus systemin is sheared from its precursor protein-preceding systemin.Biochemical Research shows that systemin can combine with the receptor protein SR160 specificity on being positioned at film.SR160 is one and is rich in leucic receptor protein kinase, and it contains one and is positioned at and extracellularly is rich in leucine zone, membrane spaning domain and one and is positioned at intracellular serine/threonine kinase structural domain.Systemin and SR160 in conjunction with after can cause specific phospholipase activity discharging the initial substrate linolenic acid of JA synthetic (Linolenic acid), thereby induce the biosynthesizing of JA and participate in the induction of resistance Expression of Related Genes thereupon.Systemin and JA are by the part and the system expression of a common signal transduction path regulation and control resistance related gene.

At present, relevant JA synthetic biochemical route is clear substantially, but the signal transduction path of biosynthetic regulation and control of relevant JA and JA is still known little about it.

Summary of the invention

The purpose of this invention is to provide a tomato anti insect related gene and proteins encoded thereof.

Tomato anti insect related gene provided by the present invention, name is called LeAcx1, derives from tomato and belongs to tomato (lycopersicon esculentum), has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 polynucleotide;

2) SEQ ID № in the code sequence tabulation: the DNA of 2 protein sequences;

3) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of described height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.

SEQ ID № in the sequence table: 1 by 2248 based compositions, and its encoder block is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end 108-the 2102nd bit base.

Provided by the present invention and proteins encoded LeAcx1 tomato anti insect related gene is the polypeptide with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or interpolation and the protein relevant with tomato anti insect of one to ten amino-acid residue.

SEQ ID № in the sequence table: 2 are made up of 664 amino-acid residues.

Contain that arbitrary segmental primer also belongs to protection scope of the present invention in expression vector, transgenic cell line and the host bacterium of above-mentioned and tomato anti insect related gene and amplification and the tomato anti insect related gene.

Utilize plant expression vector, tomato anti insect related gene of the present invention is imported vegetable cell or tissue, can obtain insect pest tolerance enhanced plant.

When using LeAcx1 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry LeAcx1 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, corn, wheat, also can be dicotyledonss such as tomato, Arabidopis thaliana, tobacco, cotton.

The forfeiture that tomato LeAcx1 sudden change of the present invention will cause synthetic disappearance of JA and relevant resistant gene to be expressed.This gene be positioned at JA synthetic by way of the downstream, in that (12-oxo-phytodienoic acid 12-OPDA) is converted in the process of JA and plays an important role by JA precursor substance 12-OPDA.12-OPDA itself can not be as the signaling molecule of resistance reaction, and it need be transported to peroxysome, and forming behind the JA through the three-wheel β-Yang Hua could be as signaling molecule and the induction of resistance Expression of Related Genes, thereby regulation and control are to the resistance of insect.Simultaneously because this gene be lipid acid degraded must be through rate-limiting enzyme by way of-β-Yang Hua, thereby have important value also having aspect the genetic regulation tomato fatty acid content.The content of tamato fruit lipid acid and form to its quality, local flavor, and nutritive value material impact is arranged.Utilize the transgenic line of LeAcx1 overexpression or antisense expression (the JL1 mutant is exactly the strain of antisense expression) itself, can obtain the outstanding tomato transgenic strain of quality trait.In addition, in other farm crop such as oil crops soybean, can obtain the reciprocity gene of LeAcx1 and carry out similar transgenic research.LeAcx1 will play a great role in to insect-resistant breeding and quality-improving plant.

Description of drawings

Fig. 1 is the genetic map of LeAcx1 gene map based cloning

Fig. 2 is for changeing the pest-resistant effect test result of LeAcx1 plant

Fig. 3 is the comparison that the transgenic Fructus Lycopersici esculenti plant (35S::LeAcx1) of normal tomato plant and overexpression LeAcx1 goes up the insect (tobaco hornworm) of growth

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The acquisition of embodiment 1, tomato resistance deletion mutant JL1

Selecting tomato conventional variety M82 for use is experiment material, its seed is soaked in 0.5% ethylmethane sulfonate (ethylmethyl sulfonate, EMS) carried out mutagenic treatment in 12 hours, the non-resistant that has that after cultivating its offspring's material is carried out proteinase inhibitor II detects, and the material of disappearance proteinase inhibitor II is carried out self propagated.Each all uses above-mentioned same method to detect from generation to generation, selects at last the individual plant of proteinase inhibitor resistance disappearance is carried out self propagated, through the selection of 5 generations, obtains stable tomato resistance deletion mutant material, called after JL1.

The most basic phenotypic characteristic of this mutant JL1 is the expression that injured back lacks resistance-associated protein PIs, it is connect the worm test, this mutant is lost insect-resistant as a result, the accumulating level of jasmonic (JA) only is 4% of a normal plant in the JL1 plant after further analysis revealed is injured, but the accumulation of JA synthetic mesophase product 12-OPDA is normal.

The acquisition of embodiment 2, LeAcx1

1, the method with map based cloning obtains tomato LeAcx1

By the large group that comprises 1200 individualities is carried out the fine Structure Mapping analysis; with goal gene navigate on No. 8 karyomit(e) of tomato a length be about 80KB by the BAC clone zone that 166B24 and 232L13 covered; (number of recombinant chou between digitized representation molecule marker and goal gene) as shown in Figure 1; this dna fragmentation is carried out sequential analysis; predict that this BAC clone covers 8 genes; wherein end to end two acyl-coenzyme oxydase (Acyl-CoAOxidase; ACX) gene is considered to cause the important candidate gene of JL1 resistance missing gene JL1 (will be referred to as Jl1 with the genes involved that causes JL1 proteinase inhibitor resistance disappearance); difference called after LeAcx1; LeAcx2; wherein; LeAcx1 has SEQ ID № in the sequence table: 1 polynucleotide sequence; SEQ ID № in the sequence table: 1 by 2248 based compositions; its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end 108-the 2102nd bit base.On function, the acyl-coenzyme oxydase is that the required enzyme of the vegetable fatty acid β-Yang Hua the first step also is a rate-limiting enzyme, this with the JL1 that predicts according to the phenotype of JL1 mutant in that be converted into the conclusion that plays an important role in the process of JA by 12-OPDA consistent.

2, determine the essence of JL1 according to following experiment

1) scope of utilizing genetic method further to dwindle goal gene, concrete grammar is: utilize being divided into from situation of individual above-mentioned 8 genes of checking of the reorganization that obtains in step 1 position fixing process and JL1.If certain gene is the candidate gene of JL1 gene, just should with its performance be divided into from.Concrete grammar is: above-mentioned reorganization individuality is that foundation and JL1 gene linkage obtain, for certain candidate gene, above-mentioned reorganization individuality is carried out linkage relationship PCR detects, if these reorganization individual with these gene linkages, show this gene and JL1 gene be divided into from.Only have in 8 genes as a result LeAcx1 and LeAcx2 and JL1 gene be divided into from.

2) on genomic dna and cDNA level, compare LeAcx1 and the sequence polymorphism of LeAcx2 between mutant and orthodox material, The sequencing results shows only has LeAcx1 that a sudden change by base C-T has taken place in the JL1 mutant, this sudden change causes the 139th amino acid Threonine (Threonine in the coded amino acid residue sequence of LeAcx1, Thr) sport Isoleucine (Isoleucine, Ile).SwissProt database investigation result shows this Thr ¹³⁹Be positioned at the key position in the oxidasic coenzyme flavoprotein of acyl-coenzyme active centre, and this amino acid is conservative at the same proteinoid camber of animal, microorganism and plant, in case undergo mutation, will cause the forfeiture of enzymic activity.According to this result, conclude that tentatively LeAcx1 is the JL1 gene.

3) utilize the transgenosis means to carry out function complementation experiment, cDNA sequence and the suitable restriction enzyme site design primer amplification LeAcx1 of carrier pBI121 of the LeAcx1 that obtains according to step 1, primer sequence is as follows:

Primer 1:(upstream primer) 5 '-GC TCTAGAGCGTAAGAGAGATGGAGGGT-3 ' (line part base is the XbaI recognition site)

Primer 2: (downstream primer) 5 '-C GAGCTCGAACAGTTTGCTGCAGCTCTCG-3 ' (line part base is the SacI recognition site)

The cDNA sequences Design primer amplification LeAcx1 of the LeAcx1 that obtains according to step 1, primer sequence is as follows:

Primer 3:(upstream primer) 5 '-CTGAGAGTAAGAGAGATGGAG-3 '

Primer 4:(downstream primer) 5 '-CTGGGAGGAAAAGAAGCCAAA-3 '

Extract total RNA of tomato conventional variety M82, with its reverse transcription product is template, under the guiding of primer 1 and primer 2, carry out pcr amplification, reaction finishes the back PCR product is carried out purifying, with restriction enzyme XbaI and SacI purified PCR product is carried out double digestion, then purified enzyme being cut product is connected with the carrier pBI121 that cuts through the same enzyme enzyme, connect product again and transform agrobacterium tumefaciens, after screening, select recon, under its mediation, transform tomato resistance deletion mutant JL1 then, use the method for PCR then, under the guiding of primer 3 and primer 4, the screening transfer-gen plant, transfer-gen plant is connect the worm test, and this transfer-gen plant produces resistance to insect as a result, thereby concludes that LeAcx1 is exactly the JL1 gene.

The confirmatory experiment of embodiment 3, LeAcx1 biological function

1, LeAcx1 is in the synthetic functional verification experiment that reaches in the fatty acid metabolism of JA

CDNA sequence and the suitable restriction enzyme site design primer amplification LeAcx1 of carrier pET-23b of the sudden change LeAcx1 that obtains according to step 1, primer sequence is as follows:

Primer 5:(upstream primer) 5 '-C GAGCTC(line part base is the SacI recognition site to GGTAGAGGAGATGGAGGGTGTA-3 '

Primer 6:(downstream primer) 5 '-CCG CTCGAGCGGAACAGTTTGCTGCAGCTCTCG-3 ' (line part base is the XhoI recognition site)

Extract total RNA of tomato conventional variety M82, with its reverse transcription product is template, under the guiding of primer 5 and primer 6, carry out pcr amplification, reaction finishes the back PCR product is carried out purifying, with restriction enzyme SacI and XhoI purified PCR product is carried out double digestion, then purified enzyme being cut product is connected with the carrier pET-23b that cuts through the same enzyme enzyme, to connect product transformed into escherichia coli JM109 again, after screening, select recon, under 37 ℃, make its expression, with the LeAcx1 that suddenlys change among the mutant JL1 in contrast, and make its expression with above-mentioned same method, utilize the fatty acid coa (lauric acid of different carbon chain lengths (C4-C20) then, myristic acid, palmitinic acid, linolenic acid, linolic acid or eicosanoic acid all can) determine its express spectra for substrate, the result shows that the fatty acid coa activity to containing 14 carbon atoms and 16 carbon atoms is the highest.

2, LeAcx1 is to the genetic regulation of insect-resistant

Utilize tobacco mosaic disease virus promoter (CaMV-35S, Genbank number: AY819771) make up the plant expression vector that contains LeAcx1, and with its conversion tomato, use the method for PCR then, under the guiding of primer 3 and primer 4, the screening transfer-gen plant, then transformed plant is carried out the expression analysis of resistance related gene PIs and the screening of feeding and testing in conjunction with insect, obtain the transfer-gen plant of a series of LeAcx1 overexpressions, the result as shown in Figures 2 and 3, show with the normal tomato plant of wild-type and compare that the transgenic Fructus Lycopersici esculenti plant (35S::LeAcx1) of overexpression LeAcx1 is obviously improved the resistance of insect.

Sequence table

<160>2

<210>1

<211>2248

<212>DNA

＜213〉tomato belongs to tomato (lycopersicon esculentum)

<400>1

tatcttttct?ttttttcaag?aattcaattg?ggtatttgta?aaacaaccca?agattggatt 60

ttttttcatt?gtgtaagaat?tttggttttg?atctgagagt?aagagagatg?gagggtgtag 120

attatttggc?tgatgagagg?aagaaagctg?gatttgatgt?ggatgagatg?aagattgttt 180

gggctggttc?tcgtcatgat?tttgaactta?cagatcgtat?ctctaagctt?gttgctagtg 240

atcctggctt?cagtaaggag?ggaagaacca?tgcttcctag?gaaagagcta?ttcaagaaca 300

ctctaaggaa?ggcagcatat?gcatggaaac?ggatcattga?acttcgtcta?tctcaagagg 360

aagccactat?gttaaggcgt?tacgtagatg?agcctgcttt?tactgatctt?cattggggaa 420

tgtttatacc?tgccataaaa?ggacagggca?cagataaaca?gcaggaaaag?tggctgccgc 480

tggcttacaa?gatgcaaata?attggctgtt?atgctcaaac?tgaacttggt?catgggtcca 540

atgtgcaagg?ccttgagacc?actgccacat?ttgatcctca?aacagatgaa?tttgtcattc 600

atagtccaac?attgacgtca?agcaagtggt?ggcctggtgg?attgggtaaa?gtgtctaccc 660

atgctgttgt?gtatgctcgt?cttataacag?atggtaaaga?ctatggggtt?aatggtttta 720

ttgtccaact?acggagcttg?gaggaccaca?aacctcttcc?aggtgttact?gttggagaca 780

ttggaatgaa?atttgggaac?ggagcataca?attccatgga?taatggggtg?ttaagctttg 840

atcacgtgcg?cattccaaga?gatcaaatgt?tgatgagggt?ttctcaagtt?acaaaggaag 900

gaaaatatgt?tcagtctgat?attcctcgac?aactccttta?tgggaccatg?gtatatgtac 960

ggcaatcaat?tgtagcagat?gcttcccttg?caatgtctcg?ggctgtgtgt?attgcaacca 1020

gatacagtgc?cgttcgtaga?caatttggct?ctcagaatgg?tggacaagaa?actcaggtga 1080

ttgactacaa?aactcagcaa?aacaggctct?tccccttgtt?ggcttctgca?tatgccttca 1140

gatttgttgg?tgagtggctg?aaatggttgt?acactgatgt?tacccaaaga?cttgcagcaa 1200

atgatttctc?aacattgcct?gaggcacatg?catgtaccgc?aggattgaag?tctttgacca 1260

ccagtgccac?tgctgatgga?attgaagaat?gccgaaagtt?atgcggaggt?catggttatc 1320

tttgtagcag?tgggcttcca?gaattatttg?ccgtttatgt?ccctgcctgt?acttacgaag 1380

gagataatgt?cgtgctacag?ctacaggttg?caaggtttct?tatgaagacc?atttcacaac 1440

tgggcactgg?aaagaagcca?gtaggtaccg?tatcttatat?gggaagaatc?gaacacttga 1500

tgcagtgtcg?ttctgatgtg?aaacaagccg?aggactggct?gaaacctagt?gcagtattgg 1560

aagcatttga?agcaaggtct?gccaggatgt?ctgttgcctg?cgctaagaat?cttagcaagt 1620

ttgagaatca?agaagaaggc?tttgcagaac?tagcagctga?tttagttgaa?gcagcagttg 1680

ctcattgtca?attaattgtt?gtgtccaagt?atattgagaa?gttgcaacaa?aacattcccg 1740

gtaaaggggt?taagcaacag?ctggaggtgc?tttgtggcat?ctattcactg?ttcattcttc 1800

acaaacatca?aggagatttt?ctcgggactg?gctacatcac?ctcaaagcag?gggtcgcttg 1860

ctaacgacca?gctcagagcc?ttgtattccc?agcttcgtcc?aaatgctgta?tcactggttg 1920

atgcatttaa?ctatactgat?cactaccttg?gttcaattct?tggacgttat?gatggaaacg 1980

tgtatccaaa?actgtatgag?gctgcatgga?aggatcctct?caacaaatca?gacatagcag 2040

atggcttcca?cgaatatatt?aggccactac?tcaagcagca?actacgaact?gctaagctgt 2100

gaagcgcgag?agctgcagca?aactgttgtt?ccgtgctgtg?ccaataaatt?agattttgaa 2160

atagcaacat?cctttatgac?tattaagttg?tgactgatag?tattttgttc?tgaataatat 2220

tattgaactt?ttgtaaggaa?aactgtta 2248

<210>2

<211>664

<212>Pro

＜213〉tomato belongs to tomato (lycopersicon esculentum)

<400>2

Met?Glu?Gly?Val?Asp?Tyr?Leu?Ala?Asp?Glu?Arg?Lys?Lys?Ala?Gly?Phe

1 5 10 15

Asp?Val?Asp?Glu?Met?Lys?Ile?Val?Trp?Ala?Gly?Ser?Arg?His?Asp?Phe

20 25 30

Glu?Leu?Thr?Asp?Arg?Ile?Ser?Lys?Leu?Val?Ala?Ser?Asp?Pro?Gly?Phe

35 40 45

Ser?Lys?Glu?Gly?Arg?Thr?Met?Leu?Pro?Arg?Lys?Glu?Leu?Phe?Lys?Asn

50 55 60

Thr?Leu?Arg?Lys?Ala?Ala?Tyr?Ala?Trp?Lys?Arg?Ile?Ile?Glu?Leu?Arg

65 70 75 80

Leu?Ser?Gln?Glu?Glu?Ala?Thr?Met?Leu?Arg?Arg?Tyr?Val?Asp?Glu?Pro

85 90 95

Ala?Phe?Thr?Asp?Leu?His?Trp?Gly?Met?Phe?Ile?Pro?Ala?Ile?Lys?Gly

100 105 110

Gln?Gly?Thr?Asp?Lys?Gln?Gln?Glu?Lys?Trp?Leu?Pro?Leu?Ala?Tyr?Lys

115 120 125

Met?Gln?Ile?Ile?Gly?Cys?Tyr?Ala?Gln?Thr?Glu?Leu?Gly?His?Gly?Ser

130 135 140

Asn?Val?Gln?Gly?Leu?Glu?Thr?Thr?Ala?Thr?Phe?Asp?Pro?Gln?Thr?Asp

145 150 155 160

Glu?Phe?Val?Ile?His?Ser?Pro?Thr?Leu?Thr?Ser?Ser?Lys?Trp?Trp?Pro

165 170 175

Gly?Gly?Leu?Gly?Lys?Val?Ser?Thr?His?Ala?Val?Val?Tyr?Ala?Arg?Leu

180 185 190

Ile?Thr?Asp?Gly?Lys?Asp?Tyr?Gly?Val?Asn?Gly?Phe?Ile?Val?Gln?Leu

195 200 205

Arg?Ser?Leu?Glu?Asp?His?Lys?Pro?Leu?Pro?Gly?Val?Thr?Val?Gly?Asp

210 215 220

Ile?Gly?Met?Lys?Phe?Gly?Asn?Gly?Ala?Tyr?Asn?Ser?Met?Asp?Asn?Gly

225 230 235 240

Val?Leu?Ser?Phe?Asp?His?Val?Arg?Ile?Pro?Arg?Asp?Gln?Met?Leu?Met

245 250 255

Arg?Val?Ser?Gln?Val?Thr?Lys?Glu?Gly?Lys?Tyr?Ile?Gln?Ser?Asp?Ile

260 265 270

Pro?Arg?Gln?Leu?Leu?Tyr?Gly?Thr?Met?Val?Tyr?Val?Arg?Gln?Ser?Ile

275 280 285

Val?Ala?Asp?Ala?Ser?Leu?Ala?Met?Ser?Arg?Ala?Val?Cys?Ile?Ala?Thr

290 295 300

Arg?Tyr?Ser?Ala?Val?Arg?Arg?Gln?Phe?Gly?Ser?Gln?Asn?Gly?Gly?Gln

305 310 315 320

Glu?Thr?Gln?Val?Ile?Asp?Tyr?Lys?Thr?Gln?Gln?Asn?Arg?Leu?Phe?Pro

325 330 335

Leu?Leu?Ala?Ser?Ala?Tyr?Ala?Phe?Arg?Phe?Val?Gly?Glu?Trp?Leu?Lys

340 345 350

Trp?Leu?Tyr?Thr?Asp?Val?Thr?Gln?Arg?Leu?Ala?Ala?Asn?Asp?Phe?Ser

355 360 365

Thr?Leu?Pro?Glu?Ala?His?Ala?Cys?Thr?Ala?Gly?Leu?Lys?Ser?Leu?Thr

370 375 380

Thr?Ser?Ala?Thr?Ala?Asp?Gly?Ile?Glu?Glu?Cys?Arg?Lys?Leu?Cys?Gly

385 390 395 400

Gly?His?Gly?Tyr?Leu?Cys?Ser?Ser?Gly?Leu?Pro?Glu?Leu?Phe?Ala?Val

405 410 415

Tyr?Val?Pro?Ala?Cys?Thr?Tyr?Glu?Gly?Asp?Asn?Val?Val?Leu?Gln?Leu

420 425 430

Gln?Val?Ala?Arg?Phe?Leu?Met?Lys?Thr?Ile?Ser?Gln?Leu?Gly?Thr?Gly

435 440 445

Lys?Lys?Pro?Val?Gly?Thr?Val?Ser?Tyr?Met?Gly?Arg?Ile?Glu?His?Leu

450 455 460

Met?Gln?Cys?Arg?Ser?Asp?Val?Lys?Gln?Ala?Glu?Asp?Trp?Leu?Lys?Pro

465 470 475 480

Ser?Ala?Val?Leu?Glu?Ala?Phe?Glu?Ala?Arg?Ser?Ala?Arg?Met?Ser?Val

485 490 495

Ala?Cys?Ala?Lys?Asn?Leu?Ser?Lys?Phe?Glu?Asn?Gln?Glu?Glu?Gly?Phe

500 505 510

Ala?Glu?Leu?Ala?Ala?Asp?Leu?Val?Glu?Ala?Ala?Val?Ala?His?Cys?Gln

515 520 525

Leu?Ile?Val?Val?Ser?Lys?Tyr?Ile?Glu?Lys?Leu?Gln?Gln?Asn?Ile?Pro

530 535 540

Gly?Lys?Gly?Val?Lys?Gln?Gln?Leu?Glu?Val?Leu?Cys?Gly?Ile?Tyr?Ser

545 550 555 560

Leu?Phe?Ile?Leu?His?Lys?His?Gln?Gly?Asp?Phe?Leu?Gly?Thr?Gly?Tyr

565 570 575

Ile?Thr?Ser?Lys?Gln?Gly?Ser?Leu?Ala?Asn?Asp?Gln?Leu?Arg?Ala?Leu

580 585 590

Tyr?Ser?Gln?Leu?Arg?Pro?Asn?Ala?Val?Ser?Leu?Val?Asp?Ala?Phe?Asn

595 600 605

Tyr?Thr?Asp?His?Tyr?Leu?Gly?Ser?Ile?Leu?Gly?Arg?Tyr?Asp?Gly?Asn

610 615 620

Val?Tyr?Pro?Lys?Leu?Tyr?Glu?Ala?Ala?Trp?Lys?Asp?Pro?Leu?Asn?Lys

625 630 635 640

Ser?Asp?Ile?Ala?Asp?Gly?Phe?His?Glu?Tyr?Ile?Arg?Pro?Leu?Leu?Lys

645 650 655

Gln?Gln?Leu?Arg?Thr?Ala?Lys?Leu

660

Claims

1. tomato anti insect related gene is one of following nucleotide sequence:

1) dna sequence dna shown in the SEQ ID NO:1 in the sequence table;

2) polynucleotide of protein sequence shown in the SEQ ID NO:2 in the code sequence tabulation.

2. the proteins encoded of the described tomato anti insect related gene of claim 1.

3. protein according to claim 2 is characterized in that: this proteic amino acid residue sequence is shown in SEQID NO:2.

4. the plant expression vector that contains the described tomato anti insect related gene of claim 1.

5. the transgenic plant cells system of containing the described tomato anti insect related gene of claim 1.

6. the host bacterium that contains the described tomato anti insect related gene of claim 1.

7. a cultivation is to utilize plant expression vector to the method for insect pest tolerance enhanced plant, with described gene transfered plant cell of claim 1 or tissue, obtains insect pest tolerance enhanced plant.

8. method according to claim 7 is characterized in that: described vegetable cell or tissue come from tomato, Arabidopis thaliana, tobacco, cotton, paddy rice, corn or wheat.