CN106399328A

CN106399328A - Pathogenicity correlated Botrytis cinerea gene BcSEP6 and application thereof

Info

Publication number: CN106399328A
Application number: CN201610973325.7A
Authority: CN
Inventors: 秦庆明; 冯会强; 李桂华; 杨松; 张明哲
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2016-11-04
Filing date: 2016-11-04
Publication date: 2017-02-15
Anticipated expiration: 2036-11-04
Also published as: CN106399328B

Abstract

A pathogenicity correlated Botrytis cinerea gene BcSEP6 and an application thereof belong to the technical field of microbial gene engineering. The DNA sequence of the gene BcSEP6 derived from Botrytis cinerea and used for controlling the growth and the pathogenicity of an invasion structure is composed of 1345 nucleotides and is represented by SEQ ID No:1. The amino acid sequence of a protein encoded by the BcSEP6 gene is composed of 362 amino acids and is represented by SEQ ID No:2. The BcSEP6 gene can be applied in the plant anti-gray mold gene engineering field; and the protein encoded by the gene BcSEP6 derived from Botrytis cinerea and used for controlling the growth and the pathogenicity of the invasion structure can be applied in design and screening of anti-Botrytis cinerea reagents as a target.

Description

A kind of ash arrhizus bacteria gene BcSEP6 related to pathogenicity and its application

Technical field

The invention belongs to technical field of microbial genetic engineering is and in particular to control fungal infection structure in plant protection art Formed and the gene of pathogenicity and its application of coded protein.

Background technology

Ash arrhizus bacteria (Botrytis cinerea) is generally also referred to as Botrytis cinerea, belongs to Ascomycota (Ascomycota) Fungi, is the pathogen of gray mold, can infect 400 various plants, including almost all of vegetables and fruit tree crop.Host is from seedling Phase all can fall ill to period, and, each position of plant all can be infected by ash arrhizus bacteria, and the classical symptom of leaf portion morbidity shows as " V " shape scab, flower portion is mainly shown as that rotten and tune withers, and fruit is mainly shown as and rots and come off.The generation of disease and spreading There is close relationship with the humidity of environment, temperature, at 20 DEG C -23 DEG C, occur serious during relative humidity more than 90%.Therefore, Gray mold belongs to low temperature and high relative humidity type disease, easily occurs in rainy season or Protected production, the warp leading to because of this disease every year Ji loss is up to hundred million dollars of 100-1000.Due to host range extensively, production endanger seriously, along with correlation molecule studies skill Art is ripe, and botrytis cinerea has become one of most important model plant disease fungus, by widely studied.

Ash arrhizus bacteria is typical necrotrophic disease fungus, can generate multiple virulence factors and participate in causing a disease, main bag Include cell wall degradation enzyme, cutinase, toxin, plant hormone, the enzyme of opposing defense enzymes, tiny RNA and small-molecule substance etc., These factors cooperate enables ash arrhizus bacteria to kill host cell and to decompose the host tissue of death as nutrition.Natural bar Under part, botrytis cinerea is many to infect source using conidium as the First aggression infecting host and again.Ash arrhizus bacteria often with mycelium, point Raw spore or sclerotium are attached on plant invalid body or survive the winter in soil and more summer, and the First aggression becoming next Growing season comes Source.When condition is suitable, sclerotial germination aerial mycelium and conidiophore, and produce substantial amounts of conidium.Ripe divides Raw spore can operate etc. by wind, rainwater, irrigation water and farming and be propagated.Under the conditions of low temperature and high relative humidity, conidium sprouts Send out formation germ tube, germ tube end expand slightly develop into appresorium or further formed infect the Infection structures such as pad, mainly from Floral organ, wound and the slough decayed invade.

Appresorium, infect the growth of the Infection structures such as pad, host is infected for ash arrhizus bacteria it is critical that, if invaded Dye structural development is affected, and ash arrhizus bacteria will be difficult to invade host, and its extent of injury can be seriously undermined therewith.Gray mold The combined regulating that the growth of bacterium Infection structure is subject to the external source condition such as nutrition, interface and develops signal, correlation molecule mechanism is still not Clear, this field is carried out further investigate not only facilitating and disclose the molecule that the necrotrophic disease funguses such as ash arrhizus bacteria cause a disease Mechanism, and for research and development preventing and treating, the medicament of the plant pathogenic fungi including ash arrhizus bacteria has significant application value.

From conidia germination to the Infection structure developing complete function, it is a fine regulation process, identification should The important component of regulation process, and verify the pathogenic function of related gene it is possible to therefrom find to make as fungicide With the protein of target, prevent and treat gray mold for exploitation and theory and technology basis established by the efficient medicament of other similar disease.

Sep6 is a kind of gtp binding protein matter, is widely present in Bao Kuo the eucaryote (except plant) including fungi, should Protein by aggregating into hetero-oligomer protein complex or can form fibril further, participates in the multiple key of regulating cell Life process.By the analysis to ash arrhizus bacteria Sep6 encoding gene, evaluate the work in ash arrhizus bacteria pathogenic course for this gene With being conducive to identifying potential preventing and treating target, for screening new antifungal medicament.

Content of the invention

The purpose of the present invention aims to provide a kind of control ash arrhizus bacteria Infection structure and develops and pathogenic gene and its coding Protein.

Control Infection structure provided by the present invention is developed and pathogenic gene derives from ash arrhizus bacteria, entitled BcSEP6, its DNA sequence dna such as SEQ ID No:Shown in 1.This DNA sequence dna is BcSEP6 gene open reading frame, by 1345 cores Thuja acid forms, and wherein comprises 4 extrons, is located at SEQ ID No respectively:Between 1 the 1st to 105 nucleotides, the 169th Position, to 331 nucleotides, between the 397th to 635 nucleotides and the 764th to 1345 nucleotides between, forms Coding section length add up to 1089 nucleotides.

The invention provides controlling coded by Infection structure growth and pathogenic gene BcSEP6 from ash arrhizus bacteria Protein, its amino acid sequence such as SEQ ID No:Shown in 2, this sequence is made up of 362 amino acid.

Develop from the control Infection structure of ash arrhizus bacteria and pathogenic gene BcSEP6 can be applicable to plant botrytis resistant Genetic engineering field.

The protein coded by Infection structure growth and pathogenic gene BcSEP6 that controls from ash arrhizus bacteria can conduct Target is applied to design and the screening of botrytis resistant bacterium medicament.

Present invention demonstrates that the disappearance of BcSEP6 gene or mutation, lead to ash arrhizus bacteria Infection structure formation rate and pathogenicity Significantly reduce, illustrate that BcSEP6 gene is that ash arrhizus bacteria causes gene necessary to crops gray mold.Therefore, screening can hinder Stop the compound of this gene expression and its protein expression, modification and positioning, can with the generation of effective control gray mold, thus Contribute to developing new type bactericide.I.e. an important use of BcSEP6 gene provided by the present invention is：The expression of this gene The protein encoding with it, can be used as important candidate targets site, the design for botrytis resistant bacterium medicament and screening.

Ash arrhizus bacteria bacterial strain B05.10 used in the present invention, buys from U.S.'s genetic of fungi material collection (Fungal Genetics Stock Center, FGSC), other staff, if desired for this bacterial strain, can buy from this collection and obtain , relevant information is as follows：Collection address：Fungal Genetics Stock Center, Department ofPlant Pathology, Kansas State University, 4024Throckmorton Plant Sciences Center, Manhattan,KS 66506USA；Network address：http://www.fgsc.net/scripts/ StrainSearchReturnPage.asp？OrgID=23812；Strain number：FGSC 10317.

Brief description

Fig. 1 is gene structure collection of illustrative plates in ash arrhizus bacteria genome for the BcSEP6

Wherein BcSEP6 gene is numbered is BC1G_02868, predicts that this gene has a GTPase Structure and function domain

Fig. 2 is knockout strategy (carrying out gene replacement by the homologous recombination) schematic diagram of ash arrhizus bacteria BcSEP6 gene

Wherein：B05.10 is wild-type strain, and pSEP6 is knockout carrier, and Δ Bcsep6 is BcSEP6 deletion mutant Body.A, b, c, d, e, f, HpTa, HpTb are checking primer and its direction.

Fig. 3 is that the PCR of BcSEP6 deletion mutant body verifies electrophoretogram

Wherein：A, b, c, d, e, f, HpTa, HpTb are checking primer, and Fig. 2 is seen in relevant position, on a+b primer checking gene Whether trip there is Homo~logous exchange, and whether c+d primer checking downstream of gene occurs Homo~logous exchange；E+f primer verifying purpose gene is No it is knocked.Whether HpTa+HpTb primer checking resistance screening gene inserts.Δ Bcsep6-1 and Δ Bcsep6-2 is two The independent BcSEP6 deletion mutant body obtaining of strain.RT is radom insertion transformant

Fig. 4 is that the cultural characteristic of knockout mutations body Δ Bcsep6 and wild-type strain B05.10 contrasts photo

Wherein：Minimal medium used is PDA, and B05.10 is wild type, and Δ Bcsep6-1 and Δ Bcsep6-2 is two plants The independent BcSEP6 deletion mutant obtaining, RT-1 and RT-2 is radom insertion transformant.

Fig. 5 is the sporulation quantity statistical analysis with wild-type strain for the deletion mutant of BcSEP6 gene

Wherein：B05.10 is wild type, and Δ Bcsep6-1 and Δ Bcsep6-2 is two plants of independent BcSEP6 disappearances obtaining Mutant, RT-1 is radom insertion transformant, and * * * represents has significant difference.

Fig. 6 is the conidium form comparison in difference with wild-type strain for the deletion mutant of BcSEP6 gene

Wherein：B05.10 is wild type, and Δ Bcsep6-1 and Δ Bcsep6-2 is two plants of independent BcSEP6 disappearances obtaining Mutant, RT-1 is radom insertion transformant, and scale is 25 μm.

Fig. 7 is that the deletion mutant of BcSEP6 gene develops comparative analysis with the pad that infects of wild-type strain

Wherein：B05.10 is wild type, and Δ Bcsep6-1 and Δ Bcsep6-2 is two plants of independent BcSEP6 disappearances obtaining Mutant, RT-1 is radom insertion transformant, and scale is 50 μm.

Fig. 8 is deletion mutant and the wild-type strain pathogenicity comparative analysis of BcSEP6 gene

Wherein：Selected host is Phaseolus Leaves, and using Isolated leaf inoculation method, left side is inoculation mycelia block, right side For inoculating spores suspension, evaluated after 3 days.B05.10 is wild type, Δ Bcsep6-1 and Δ Bcsep6-2 and Δ Bcsep6-3 is three plants of independent BcSEP6 deletion mutants obtaining, and RT-1, RT-2 are radom insertion transformant；Dpi represents and connects Number of days after kind.

Specific embodiment

In order to preferably describe the present invention, it is further described below by specific embodiment, following embodiments In method, if no special instructions, be conventional method.

The correlation analysis of embodiment 1 BcSEP6 gene

The ORFs of ash arrhizus bacteria BcSEP6 gene is made up of 1345 nucleotides, comprises 4 extrons, code area CDNA total length is 1089 nucleotides, and the protein of coding is made up of 362 amino acid.To BcSEP6 gene in gray mold Bacterium genome website (https://www.broadinstitute.org/fungal-genome-initiative/ botrytis-cinerea- genome-project) in be predicted analyze, it is BC1G_02868 that BcSEP6 gene is numbered, should Gene has a conservative GTPase Structure and function domain.

The knockout of embodiment 2 BcSEP6 gene and genetic complement

1) structure of knockout carrier

Design primer using Primer 5.0, primer synthesis is carried out by Suzhou Jin Weizhi Bioisystech Co., Ltd, adopt Primer Sep6-UP-F (5'-GATCTTCACTAGTGGGAATTCTCGATGTGCGAATGGAAGTG-3') and Sep6-UP-R (5'- TTGGGTACCGAGCTCGAATTCGCTGTCGGTGGGTTTGTGGT-3'), with the genomic DNA of ash arrhizus bacteria bacterial strain B05.10 For template amplification BcSEP6 upstream region of gene 600bp fragment；Using primer Sep6-DN-F (5'- TGGGGATCCTCTAGAGTCGACCCACGATGTAATTCATGCTCCT-3') with Sep6-DN-R (5'- CTTGCATGCCTGCAGGTCGACATCGGTGAACTACTACGAAACTCC-3') expand ash arrhizus bacteria BcSEP6 downstream of gene 809bp fragment, reaction system (25 μ L) is：10mmol/L dNTP Mixture, 0.5 μ L；10 × PCR buffer, 2.5 μ L； The each 1 μ L of upstream and downstream primer (10 μm of ol/mL)；Template DNA, 1 μ L；Ex-Taq, 0.2 μ L (5U)；ddH₂O, 18.8 μ L；Amplification program For：94 DEG C of denaturations 3 minutes, then (1) 94 DEG C, denaturation 30 seconds；(2) 58 DEG C, anneal 30 seconds；(3) 72 DEG C, extend 60 seconds；(4) By 1-3 step circulation 30 times；(5) 72 DEG C extend 10 minutes.Above-mentioned DNA cloning product is carried out purifying to be reclaimed after quantitation, using NEB The ClonExpress kit of biotech firm, by fragment upstream and segments downstream respectively directed cloning to pXEH carrier EcoR I site and Sal I site, are built into knockout carrier pSEP6 (as shown in Figure 2), and carry out sequence verification.

2) conversion of ash arrhizus bacteria

A. the culture of Agrobacterium tumefaciems

Picking contains the Agrobacterium tumefaciens strain Agl-1 single bacterium colony of binary vector pSEP6, be seeded to containing 50 μ g/ml cards that Mycin, MM fluid nutrient medium (dipotassium hydrogen phosphate 0.205%, potassium dihydrogen phosphate 0.145%, the sodium chloride of 10 μ g/ml rifampins 0.015%, epsom salt 0.05%, calcium chloride hexahydrate 0.01%, ferrous sulfate heptahydrate 0.00025%, ammonium sulfate 0.05%, Glucose 0.2%) in, 250rpm, 28 DEG C of shaken cultivation 48h；4000rpm, is centrifuged 5 minutes, abandons supernatant, IM fluid nutrient medium (dipotassium hydrogen phosphate 0.205%, potassium dihydrogen phosphate 0.145%, sodium chloride 0.015%, epsom salt 0.05%, six water chlorinations Calcium 0.01%, ferrous sulfate heptahydrate 0.00025%, ammonium sulfate 0.05%, glucose 0.2%, 200 μM of AS, MES 0.854%, Glycerine 0.5%) resuspended, 4000rpm is centrifuged 5 minutes, abandons supernatant；IM culture medium is resuspended, 28 DEG C, and 250rpm shaken cultivation 6h is entered Row pre-induced.

B. the product spore culture of ash arrhizus bacteria

From B05.10 bacterial strain, a small amount of spore is taken to coat PDA culture medium (the well-done filtration of potato 20%, glucose 2%, agar 1.5%), put 28 DEG C of culture 8h and make spore fast-germination, be then transferred to 20 DEG C and cultivate 3-5 days, treat phage surface After being covered by grey spore, with IM fluid nutrient medium scraping, collect spore, micro- sem observation, adjusted using haemocytometer Spore concentration is 1 × 10⁶/mL.

C. Agrobacterium tumefaciems is co-cultured and transformant screening with ash arrhizus bacteria conidium

Agrobacterium bacterium solution and the mixing of ash arrhizus bacteria spore liquid equal-volume of 6h will be induced in advance in IM fluid nutrient medium, plus Enter AS, make final concentration reach 500 μM, mix, then press 250～350 μ L/ wares, uniform application is cultivated to the IM being covered with glassine paper On base, 22 DEG C of dark culturing 48h；After co-cultivation finishes, glassine paper is transferred to the PDA culture medium containing 100 μ g/mL hygromycin On, continue culture under the same terms.After 4～7 days, the bacterium colony of picking extension is on the screening and culturing medium containing same antibiotic.

3) checking of deletion mutant

From four, primer is screened to transformant by PCR amplification.Amplification meets being defined as of following result BcSEP6 deletion mutant body：Hygromycin gene internal primer HpTa (5'-TGCGCCCAAGCTGCATCAT-3') and HpTb (5'-TGAACTCACCGCGACGTCTGT-3') can expand 800bp fragment (wild-type strain no amplified band)；On Primer a (5'-TCATTCGTCCAACTCCCACAG-3') on genome outside trip homology arm is drawn with hygromycin gene Thing b (5'-ACAGACGTCGCGGTGAGTTCA-3') pairing can expand the recombinant fragment of expection (1.2kb)；Downstream simultaneously Primer d (5'-CAACAAACTCAGACTCCGCTG-3') on genome outside the homology arm and primer c of hygromycin gene (5'-ATGATGCAGCTTGGGCGCA-3') pairing can expand the recombinant fragment of expection (1.2kb)；And code area primer e (5'-GAATCTGGACTTGGAAAGAC-3') with f (5'-CTTCCTAGGTCTAGCTTCAC-3') no amplified band (wild type Bacterial strain amplifiable to 0.94kb fragment).As a result, 2 plants of independent BcSEP6 deletion mutant bodies are screened from transformant：Δ Bcsep6-1 and Δ Bcsep6-2 (as shown in Figure 3), for follow-up function analysis.

Effect in ash arrhizus bacteria growth and development process for the embodiment 3 BcSEP6 gene

Using plating method, evaluate the variation situation of the Relevant phenotype such as grow of BcSEP6 mutant.Will be to be measured Bacterial strain is beaten and takes bacteria cake to be inoculated into the PDA culture medium containing 100mg/L hygromycin and the PDA culture medium not containing hygromycin respectively On, 20 DEG C of dark culturing are taken pictures after 3 to 5 days observation.It was found that the growth colony diameter of BcSEP6 mutant and wild type B05.10 and radom insertion transformant RT-1, RT-2 compare seriously delayed, and BcSEP6 mutant colonies (mycelia) form is sent out Raw significant change (see Fig. 4), this explanation BcSEP6 gene plays critical function in the normal vegetative growth phase of ash arrhizus bacteria, adjusts Control growing of ash arrhizus bacteria.

Effect in terms of ash arrhizus bacteria produces spore and conidium form for the embodiment 4 BcSEP6 gene

Using plating method, evaluate the variation situation of the product spore ability of BcSEP6 mutant.Test strains are inoculated into Do not contain in the PDA culture medium of hygromycin, 20 DEG C of dark culturing, after 7 to 10 days, cover with after mycelia and start after flat board to produce spore, use one Quantitative sterilized water scrapes flat panel collector spore suspension, and carries out quantitative analysis (see Fig. 5), finds the product of BcSEP6 mutant Spore amount is remarkably decreased compared with wild type；Take simultaneously 10ul about spore suspension drip on slide, make interim slide, show Microcosmic examines the conidium form (see Fig. 6) of BcSEP6 mutant, find the conidium form of BcSEP6 mutant with wild Type is compared and also be there occurs variation, and sporinite deformation is little, becomes more circular by ellipse；In sum, BcSEP6 gene is in ash Mould germ produces spore and conidium form is built up aspect and also played a significant role.

Embodiment 5 BcSEP6 gene infects the effect in pad growth course in ash arrhizus bacteria Infection structure

Using slide culture, observe BcSEP6 mutant and develop into the ability infecting pad.By PDB spore suspension (5 × 10⁴mL-¹) drop on slide, 36h is cultivated in room temperature moisturizing.Microexamination finds, wild-type strain B05.10 and radom insertion turn The mycelium of beggar RT can form a large amount of multi-branched mat-like structure and infect pad, and mutant then almost loses and develops into The ability of this complexity Infection structure；It is only capable of forming the less deformity of some fragmentary branches and infect pad (see Fig. 7).This research Result shows, BcSEP6 gene plays very important effect in infecting pad growth course, and this gene is that ash arrhizus bacteria infects Necessary to structural development.

Embodiment 6 BcSEP6 gene is in the effect of the pathogenic aspect of ash arrhizus bacteria

Using Isolated leaf inoculation method, evaluate the pathogenicity situation of change of BcSEP6 mutant.Host from hot-house culture On plant, collection has the blade of certain leaf age, and level is put to moisture-keeping container, beaten using card punch and take test strains bacteria cake, just Face down button to blade, 20 DEG C of moisturizing dark culturing, the pathogenicity (left see Fig. 8) of 3 days post-evaluation test strains.In addition, will After test strains carry out producing spore culture, collect conidium with PDB cushioning liquid, and spore concentration is adjusted to 2 × 10⁶/ mL, takes 5ul spore suspension is inoculated on blade, 20 DEG C of moisturizing dark culturing, and the pathogenicity of 3 days post-evaluation test strains is (see Fig. 8 Right).Test result indicate that, no matter being inoculated by hypha block or spore suspension inoculation, BcSEP6 mutant pathogenicity is all notable Decline, radom insertion transformant pathogenicity is similar to wild type, to sum up research shows, BcSEP6 is a crucial pathogenic base Cause, plays a significant role during ash arrhizus bacteria infects host.BcSEP6 passes through to regulate and control the growth of ash arrhizus bacteria Infection structure, And then affect its pathogenic function, if this gene or its coded protein loss of activity, ash arrhizus bacteria infects host and causes disease Ability by degradation.

Sequence table

SEQ ID No:1 sequence

（i）Sequence signature：（A）Length：1345 bp；（B）Type：Nucleotides；（C）Chain：Single-stranded

（ii）Molecule type：DNA

（iii）Sequence description：SEQ ID No:1

1 ATGGCACCAC CCACCGCCGA AAGCGCTTCC CCCATCGGAA TTGCGAATTT

51 GCCCAACCAA AGACACAAGA TTGTCGCAAA GAGAGGTGCT GCCTTCACTA

101 TTATGGTATG TGGTTATCTA TGCTGCTTTC AAATCTCTCA GCAACTTTAT

151 ACTAATCACC TCTTGTAGGT CGCTGGCGAA TCTGGACTTG GAAAGACAAC

201 CTTCATTAAT ACCCTCTTCT CTACCACAAT CAAAAACTAT GCCGATCACA

251 AACGCAGACA CGCAAAGCAA GTGGATAAAA CTGTTGAGAT TGAAATCACA

301 AAGGCTGAAT TAGAAGAGAA ATTCTTCAAG GGTATGGAAT CCAACACATT

351 CAGTTCACTT GAGATAATTA ATTGGCCATT GTTGACACTG TCTTAGTTCG

401 TTTGACTGTT ATCGATACAC CTGGTTTTGG AGATTATGTC AACAATCGCG

451 ACTCCTGGAT GCCAATCATC GAGTTTTTGG ATGATCAACA TGAGTCCTAT

501 ATGTTGCAAG AGCAACAGCC TCGCCGTGTT GATAAAATCG ATTTACGTGT

551 ACACGCTTGC TTGTACTTCA TCCGACCTAC TGGCCACACA CTGAAACCCC

601 TCGATATTGA GGTTATGAAG AGACTCAGTT CAAGGGTAAA CTTGATTCCA

651 GTAGTTGCTA AGGCTGATAC CTTAAGCCCG GCTGATTTGG CCCGTTATAA

701 GCAAAAGGTA TAGTGTATTT GAGTAAACCA TTATCGTTGT CGAAGTTAAC

751 GTATTTTCTA CAGATCCAAG CTGTCATTGA AGCACAAGGT ATCAAGATCT

801 ACACACCACC TATCGAGGAA GATGACGAGG CTGCTGCTCA ACATGCCCGA

851 AGTTTGATGG CTGCTATGCC ATTTGCAGTC ATTGGCTCAG AGAAGGATGT

901 CAAGACTAGT GATGGACGTA TCGTTAAGGG TCGTCAATAC GCATGGGGTG

951 TTGCTGAAGT CGAGAACGAA GACCACTGCG ATTTCAAGAA GTTGAGATCC

1001 ATTCTTATCC GAACACACAT GCTCGATCTC ATTCACACTA CCGAAGAAGC

1051 ACACTACGAG GCTTACCGCG CCCAACAAAT GGAGACTCGC AAATTCGGTG

1101 AAGCTAGACC TAGGAAGCTT GATAACCCTA AATTCAAGGA AGAGGAAGAG

1151 AGCTTAAGAA AGCGTTTCAC CGAGCAGGTT AAGATTGAGG AGCACCGTTT

1201 CAGACAGTGG GAGCAGAAGC TCATCAGTGA GCGTGATCGT TTGAACAAGG

1251 ATTTGGAGAG TACACATGCT GCCATCAAGT CCTTAGAACA AGAACTTGAG

1301 CAAATGCAAG GCGGTGCCGC AACTCGCAGT CACGGCCGTC GTTAA

SEQ ID No:2 sequence

（i）Sequence signature：（A）Length：362 amino acid；(B) type：Amino acid；（C）Chain：Single-stranded.

（ii）Molecule type：Polypeptide

（iii）Sequence description：SEQ ID No:2

1 MAPPTAESAS PIGIANLPNQ RHKIVAKRGA AFTIMVAGES GLGKTTFINT

51 LFSTTIKNYA DHKRRHAKQV DKTVEIEITK AELEEKFFKV RLTVIDTPGF

101 GDYVNNRDSW MPIIEFLDDQ HESYMLQEQQ PRRVDKIDLR VHACLYFIRP

151 TGHTLKPLDI EVMKRLSSRI QAVIEAQGIK IYTPPIEEDD EAAAQHARSL

201 MAAMPFAVIG SEKDVKTSDG RIVKGRQYAW GVAEVENEDH CDFKKLRSIL

251 IRTHMLDLIH TTEEAHYEAY RAQQMETRKF GEARPRKLDN PKFKEEEESL

301 RKRFTEQVKI EEHRFRQWEQ KLISERDRLN KDLESTHAAI KSLEQELEQM

351 QGGAATRSHG RR

Claims

1. a kind of control Infection structure from ash arrhizus bacteria (Botrytis cinerea) is developed and pathogenic gene BcSEP6, It is characterized in that its DNA sequence dna such as SEQ ID No:Shown in 1.

2. a kind of control Infection structure from ash arrhizus bacteria according to claim 1 is developed and pathogenic gene BcSEP6 Coded protein is it is characterised in that its amino acid sequence such as SEQ ID No:Shown in 2.

3. the control Infection structure from ash arrhizus bacteria described in claim 1 is developed and pathogenic gene BcSEP6 is in Genes For Plant Tolerance Application in gray mold genetic engineering field.

4. the controlling coded by Infection structure growth and pathogenic gene BcSEP6 from ash arrhizus bacteria described in claim 2 Protein is designing and is screening the application in botrytis resistant bacterium medicament as target.