CN109652431A - It is a kind of improve disease resistance of plant gene GmAP1 and its application - Google Patents

Abstract

The present invention provide it is a kind of improve disease resistance of plant gene GmAP1 and its application, belong to molecular biology of plants and genetically engineered plant field, the present invention relates to a kind of plant source disease-resistant gene GmAP1 and its recombinant expression carrier and applications.The gene source has nucleotide sequence shown in SEQ ID NO.1 in soybean, or has 70% or more homology with sequence SEQ ID NO.1.The present invention also provides a kind of gene silencing and recombinant expression carriers.It is overexpressed the gene conspicuousness to promote soybean to the disease resistance of soyabean phytophthora and promote tobacco to the disease resistance of phytophthora blight of pepper, is a kind of gene of ideal enhancing disease resistance of plant.The gene is set to express the resistance capacity for obtaining it to a variety of pathogens in soybean or tobacco by the method for genetic transformation, so as to improve crop field resistance.The present invention can be used in crop breeding disease resistance improvement aspect.

Description

It is a kind of improve disease resistance of plant gene GmAP1 and its application

Technical field

The invention belongs to molecular biology of plants and genetically engineered plant field, specifically, the present invention relates to one kind to mention The gene of high disease resistance of plant and its application.

Background technique

The crop epidemic disease as caused by phytophthora was once referred to as " plant pestilence ", and rapid onset, harm weight lack and effectively kill Microbial inoculum still seriously threatens the grain-production safety in the whole world at present.Wherein soybean phytophthora (Phytophthora sojae) causes Soybean phytophthora root rot be destructive disease on Soybean production, hundred million dollars of 10-20 of loss is caused in the whole world every year^[1].By The late blight of potato caused by phytophthora infestans (Phytophthora infestans) is the important disease in potato production, often Economic loss caused by year in worldwide is up to 3,000,000,000 dollars^[2].Soybean phytophthora root rot passes for the first time in the later period eighties Enter China, then quickly diffusion in most of china area, and becomes one of northeast and the Major Diseases of Fujian soybean main producing region. The germ is distributed in a province in Chinese Heilungkiang, Jiangsu, Henan, Anhui etc. more than 20 at present, to local Soybean production Cause different degrees of harm.Therefore, Crop Improvement disease resistance, to effective control crop epidemic disease to which Ensuring Food Safety is raw Production has great importance.

Currently, mainly taking the generation for carrying out prevention and control crop epidemic disease using the strategy of disease-resistant variety in agricultural production.Disease-resistant product Containing a kind of disease-resistant gene for being directed to specific phytophthora microspecies in kind, this genoid encodes one kind and contains nucleotide binding site (NBS) and the ill-resistant protein of full asphalt mixture (LRR)^[3].But this kind of ill-resistant protein has high specificity, can only identify The albumen for a kind of virulent gene coding that specific certain phytophthora microspecies contain^[4].And phytophthora passes through lose during evolution The strategies such as these virulent genes are lost or are mutated, the microspecies for many escape plant disease-resistant albumen identifications that make a variation out lead to disease-resistant gene Failure is to break out crop epidemic disease^[5].Therefore, the gene that there is broad spectrum durable disease resistance to phytophthora is researched and developed, is to improve to make The effective way of object disease resistance.

Plant is encountering infecting some disease-resistance substances of Shi Huixiang cell exocrine and resisting invading for pathogen for pathogen Dye^[6].These disease-resistance substances include proteolytic enzyme and some resistance small molecules etc..These extracellular proteases are in resistance mechanism In play an important role, can act on pathogen be secreted into extracellular some toxic proteins make its degradation lose toxicity^[7]。 The gene of several coding extracellular proteases, such as cysteine proteinase RCR3 and serine have been cloned into plant at present Protease P 69B^[8,9].Protease can act on different toxic proteins, and pathogen is secreted into these extracellular toxicity eggs It is white very conservative in different phytophthoras, therefore the disease resistance of plant that the extracellular protease of these plant secretions is mediated has Broad spectrum activity and persistence.Aspartic protease is a kind of very important proteolytic enzyme, it is widely present in various plants In, and take part in various biological process.But the resistant effect for resisting phytophthora in plant to it at present is still unclear.

Bibliography

[1]Tyler,B.M.,Phytophthora sojae:root rot pathogen of soybean and model oomycete.Mol Plant Pathol,2007.8(1):1-8.

[2]Fry,W.,Phytophthora infestans:the plant(and R gene)destroyer.Mol Plant Pathol,2008.9(3):385-402.

[3]Kamoun,S.,E.Huitema,and V.G.Vleeshouwers,Resistance to oomycetes:a General role for the hypersensitive response? Trends Plant Sci, 1999.4 (5): 196- 200.

[4]Raffaele,S.and S.Kamoun,Genome evolution in filamentous plant pathogens:why bigger can be better.Nat Rev Microbiol,2012.10(6):417-30.

[5]Cui,L.K.,et al.,Analysis of polymorphism and transcription of the effector gene Avr1b in Phytophthora sojae isolates from China virulent to Rps1b.Molecular Plant Pathology,2012.13(2):114-122.

[6]Doehlemann,G.and C.Hemetsberger,Apoplastic immunity and its suppression by filamentous plant pathogens.New Phytologist,2013.198(4):1001- 1016.

[7]van der Hoorn,R.A.L.,Plant proteases:From phenotypes to molecular mechanisms.Annual Review Of Plant Biology,2008.59:191-223.

[8]Misas-Villamil,J.C.,R.A.van der Hoorn,and G.Doehlemann,Papain-like cysteine proteases as hubs in plant immunity.New Phytol,2016.212(4):902-907.

[9]Tian,M.Y.,et al.,AKazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B.Journal Of Biological Chemistry,2004.279(25):26370-26377.

Summary of the invention

One of the objects of the present invention is to provide a kind of gene GmAP1.

The second purpose of the present invention is to provide a kind of recombinant expression carriers containing GmAP1 gene.

The three of the object of the invention are to provide the application of gene GmAP1.

Specifically details are as follows for the content of present invention:

The present invention provides a kind of gene GmAP1, the gene source in soybean, nucleotide sequence as shown in SEQ ID NO.1, Or there is 70% or more homology with sequence SEQ ID NO.1.Preferably, nucleotide sequence is as shown in SEQ ID NO.1, or with Sequence SEQ ID NO.1 has 80% or more homology, it is further preferred that nucleotide sequence is as shown in SEQID NO.1, or There is 85% or more homology with sequence SEQ ID NO.1, it is furthermore preferred that nucleotide sequence is as shown in SEQ ID NO.1, or with Sequence SEQ ID NO.1 has 90% or more homology, or further has 95% or more homology with sequence SEQID NO.1.

The present invention also provides the segment of cryptiogene GmAP1 a kind of, nucleotides sequence is classified as SEQ ID NO.2.

Have the present invention also provides a kind of protein of gene GmAP1 coding or compared with the protein of gene GmAP1 coding There is the protein that can be improved disease resistance of plant of the amino acid sequence of not small 70% similitude, preferably there is not small 80% phase Like the protein that can be improved disease resistance of plant of the amino acid sequence of property；It is further preferred with not small 85% similitude The protein that can be improved disease resistance of plant of amino acid sequence, or the energy of the amino acid sequence with not small 90% similitude The protein of disease resistance of plant is enough improved, or amino acid sequence with not small 95% similitude can be improved plant disease-resistant The protein of property.

In a kind of specific embodiment, the present invention provides the protein of coding of gene GmAP1 described in one kind, ammonia Base acid sequence is shown in SEQ ID NO.3 or the amino acid sequence of SEQ ID NO.3 is by one or several amino acid residues Replace and/or deletion and/or addition and can provide disease resistance of plant can be improved plant disease-resistant as derived from SEQ ID NO.3 The protein of property.

In a kind of specific embodiment, the present invention also provides a kind of to have compared with the protein of gene GmAP1 coding There are its amino acid sequence of the protein of amino acid sequence of not small 70% similitude, for example, SEQ ID NO.4, SEQ IDNO.5 Or SEQ ID NO.6.

The amino acid sequence that is encoded using gene of the invention can design and be conducive to artificial synthesized codon optimization The nucleic acid sequence expressed in plant.

The present invention also provides a kind of recombinant expression carriers including the gene GmAP1.

Expression vector of the present invention is preferably plant transformation plasmid, can be expression vector pBin::eGFP, pCambia Or pTF101.1 etc..

In a kind of specific embodiment, the recombinant expression carrier is that gene GmAP1 is inserted into containing the end C- Carrier pBin::GmAP1-eGFP obtained in the binary vector pBin::eGFP restriction enzyme site SmaI of eGFP.

It, can be plus any enhancing before its transcription initiation nucleotide when using the gene constructed recombinant expression carrier Type promoter or constitutive promoter；In addition, enhancing also can be used when using gene constructed recombinant expression carrier of the invention Son, including translational enhancer or transcriptional enhancer.

Transgenic cell line and recombinant bacterium containing gene GmAP1 described above.

The primer pair of the overall length or any segment that expand the gene GmAP1 also belongs to protection scope of the present invention.

The present invention also provides a kind of recombination silent carriers of segment including cryptiogene GmAP1.

The silent carrier is preferably plant transformation plasmid, can be silent carrier pFGC5941.

In a kind of specific embodiment, the recombination silent carrier is to be inserted into the specific fragment of GmAP1 Carrier pFGC::GmAP1 obtained in silent carrier pFGC restriction enzyme site AscI and BamHI.

A kind of transformant is imported obtained by host cell by the recombinant expression carrier, and the host cell is preferably big Coli cell or agrobatcerium cell.

For the ease of transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be carried out Processing can produce the enzyme of color change or the gene of luminophor, resistant as the coding that can express in plant is added Antibiotic marker or anti-chemical reagent label etc..

The present invention also provides the protein or recombinant expression carrier of the gene GmAP1 or its coding or transformant to mention Application in high plant immune resistance or disease resistance.

The present invention also provides the protein or recombinant expression carrier of the gene GmAP1 or its coding or transformant to plant Object improves pathogenic bacteria immune resistance or improves the application in disease caused by Genes For Plant Tolerance pathogenic bacteria.

Further, pathogenic bacteria of the present invention are the pathogenic bacteria that can infect main food and industrial crops, Ke Yiwei Oomycetes, fungi or bacterium, the plant disease as caused by phytophthora, sickle-like bacteria, rice blast fungus etc..

The present invention also provides the protein or recombinant expression carrier of the gene GmAP1 or its coding or transformant in plant Application in breeding.

The present invention also provides the protein or recombinant expression carrier of the gene GmAP1 or its coding or transformant to import The application in the kind of significant disease resistance/or increase yield, preferably importing soybean, tobacco, tomato or potato are obtained after plant Application in the middle kind for being enhanced disease resistance and/or increasing yield.

This research finds that aspartic protease GmAP1 participates in soybean and resists soybean epidemic disease to the analysis of bleeding outside soya cells Mould infection processs, and the gene pairs difference phytophthora all shows disease-resistant function.It is connect on the soybean for being overexpressed GmAP1 gene Kind of soybean phytophthora, caused by soybean phytophthora biomass substantially reduce.And soybean epidemic disease is inoculated on the soybean of silencing GmAP1 gene It is mould, caused by soybean phytophthora biomass dramatically increase.In addition, being inoculated with Phytophthora capsici on the tobacco for being overexpressed GmAP1 gene, make At scab be also substantially reduced.GmAP1 gene pairs soybean disease resistance plays very important effect, grinds to GmAP1 in soybean Study carefully the correlative study that can drive the extracellular ill-resistant protein of many other plants such as tomato and potato.These researchs will be explained preferably Bright plant can provide outstanding disease-resistant gene resource to the disease-resistant function of phytophthora for resistant gene engineering breeding.

Beneficial effects of the present invention

The protein of gene GmAP1 coding of the present invention is by being secreted into extracellular performance resistant effect, to enhance plant Object disease resistance.Being overexpressed in plant will not influence plant growth character, especially have broad spectrum activity to the disease-resistant of phytophthora, can be with Plant is significantly increased to the disease resistance of phytophthora, the present invention can be used in crop breeding disease resistance improvement aspect, be expected to improve Plant subtracts medicine to achieve the purpose that increase production to the disease resistance of epidemic disease.

Detailed description of the invention

The hair showed after the hairy piece-root grafting kind soyabean phytophthora of Fig. 1 pBin::GFP and pBin::GmAP1-GFP genetically engineered soybean Disease symptoms.

Fig. 2 real-time fluorescence quantitative PCR detects the hairy piece-root grafting kind of pBin::GFP and pBin::GmAP1-GFP genetically engineered soybean Soybean phytophthora biomass after soyabean phytophthora.

The hairy radixin detection of expression of Fig. 3 pBin::GFP and pBin::GmAP1-GFP genetically engineered soybean.Western The expression quantity of blot detection control GFP and GmAP1-GFP, detection antibody is anti-GFP.

The morbidity disease showed after the hairy piece-root grafting kind soyabean phytophthora of Fig. 4 pFGC::GFP and pFGC::GmAP1 processed soybeans Shape.

Silencing soybean hairy middle GmAP1 gene expression detection of Fig. 5 pFGC::GmAP1.Real-time fluorescence quantitative PCR detection is heavy The silent middle GmAP1 gene expression amount of hairy of soybean, wherein pFGC::GFP is check plant.

Fig. 6 real-time fluorescence quantitative PCR detects the hairy piece-root grafting kind soybean epidemic disease of pFGC::GFP and pFGC::GmAP1 processed soybeans Soybean phytophthora biomass after mould.

The morbidity disease showed after Fig. 7 pBin::GFP and pBin::GmAP1-GFP transgene tobacco inoculation phytophthora blight of pepper Shape.

Lesion diameter measures after Fig. 8 pBin::GFP and pBin::GmAP1-GFP transgene tobacco is inoculated with phytophthora blight of pepper.

The detection of Fig. 9 pBin::GFP and pBin::GmAP1-GFP transgene tobacco protein expression.Western blot detection The expression quantity of GFP and GmAP1-GFP is compareed, detection antibody is anti-GFP.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Primer is by Nanjing Genscript Biotechnology Co., Ltd. involved by the embodiment of the present invention Synthesis.

The clone of embodiment 1.GmAP1 gene and Sequence structure analysis

Soybean (Glycinemax) seed is directly inoculated in the flowerpot equipped with wet vermiculite, greenhouse (25 DEG C, 14h illumination/ 10h is dark) culture, extraction of the 1 week old plant for RNA.

Total RNAs extraction: using soybean hypocotyl as material, the extracting of total serum IgE is pressed using Omega company RNA extracts kit It operates as directed, detects its rna content and quality with spectrophotometer.

Reverse transcription generates the first chain: taking 0.7 μ g RNA to make template, according to Takara company PrimeScript reverse transcriptase Matched reagent operation instruction carries out cDNA synthesis, is settled to 20uL reaction.Take suitable reverse transcription product for subsequent gene Clone PCR.

Using the first chain of cDNA as RT-PCR template, conventional method is PCR, expands GmAP1 genetic fragment or full-length gene:

PCR primer extension increasing sequence:

Upstream primer: SEQ ID NO.7

(5’-CGATAGGGTACCCCCATGGGAAACATGCCAAAT-3’)

Downstream primer: SEQ ID NO.8

(5’-GGATCCGTCGACCCCTGCTGCCTCAGCAAA-3’),

50uL reaction system is, wherein 5 × buffer 10uL, 2.5mM dNTPs 4uL, Takara PrimerSTARTaq enzyme 0.5uL, template cDNA1uL add water to 50uL；PCR amplification program be 98 DEG C initial denaturation 5 minutes, 98 DEG C Denaturation 30 seconds, 58 DEG C are annealed 30 seconds, and 72 DEG C extend 2 minutes, are recycled 35 times, and last 72 DEG C extend 10 minutes；On Ago-Gel It is separated by electrophoresis, ethidium bromide (EB) dyeing photograph, record is as a result, simultaneously gel extraction GmAP1PCR product.Use Agarose Gel DNAPurification Kit (TaKaRa) recycles electrophoretic band.The PCR product root of the GmAP1 of gel extraction SmaI digestion is operationally connected to according to explanation according to CloneExpress II One Step Cloning Kit (Vazyme) PBin::GmAP1-eGFP plasmid is obtained on pBin::eGFP carrier, converts competent escherichia coli cell JM109, is applied LB and (is contained Card receives 50ug/mL) plate, 37 DEG C of bacterium colony PCR verifying three clones of picking after culture 16 hours grasp according to plasmid extraction kit Make (Takara) and extract plasmid, send Nanjing Jin Sirui company to be sequenced, sequence such as SEQ IDNO.1.Correct plasmid electric shock is sequenced to turn Change into Agrobacterium K599 or GV3101, applies LB and (receive mycin 50ug/mL containing card, streptomysin 50ug/mL or receive mycin containing card 50ug/mL, rifampin 50ug/mL) plate, 28 DEG C of bacterium colony PCR verifying pickings after culture 48 hours correctly clones the subsequent reality of progress It tests.

The building of 2. silent carrier pFGC::GmAP1 of embodiment:

Soybean total serum IgE is extracted, synthesizes cDNA using reverse transcription.According to the primers amplification portion of the cDNA of GmAP1 Fragment section is used for the building of GmAP1 gene silencing vector.

PFGC-AscI-GmAP1-1F upstream primer: SEQ ID NO.9

5’-ttacaattaccatggggcgcgccAATGAGCTTTGTGAAAAATT-3’

PFGC-AscI-GmAP1-2R downstream primer: SEQ ID NO.10

5’-ttaaatcatcgattgggcgcgccTGCTGCCTCAGCAAATCCGA-3’

PFGC-BamHI-GmAP1-3R upstream primer: SEQ ID NO.11

5’-aatttgcaggtatttggatccTGCTGCCTCAGCAAATCCGA-3’

PFGC-BamHI-GmAP1-4F downstream primer: SEQ ID NO.12

5’-ctctagactcacctaggatccAATGAGCTTTGTGAAAAATT-3’

The primer amplification of SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.12 are used respectively The segment of GmAP1.The partial gene sequence that PCR amplification length is 297bp is carried out, PCR amplification program is that 98 DEG C of initial denaturations 5 are divided Clock, 98 DEG C are denaturalized 30 seconds, and 58 DEG C are annealed 30 seconds, and 72 DEG C extend 0.5 minute, are recycled 35 times, and last 72 DEG C extend 10 minutes；PCR Product carries out electrophoretic separation ethidium bromide (EB) dyeing photograph on 1% Ago-Gel, and record is as a result, simultaneously gel extraction PCR Product.Electrophoretic band is recycled with Agarose Gel DNA Purification Kit (TaKaRa).Gel extraction PCR product according to ClonExpress II One StepCloning Kit (Vazyme) according to explanation be operationally connected to AscI and PFGC::GmAP1 plasmid is obtained on the pFGC carrier of BamHI double digestion, converts competent escherichia coli cell JM109, applies LB (receiving 50ug/mL containing card) plate, 37 DEG C of bacterium colony PCR after culture 16 hours verify picked clones, grasp according to plasmid extraction kit Make (Takara) and extracts pFGC::GmAP1 plasmid, sequence such as SEQID NO.2.It is electroporated to Agrobacterium that correct plasmid is sequenced In K599, apply LB (kanamycins 50ug/mL, streptomysin 50ug/mL) plate, 30 DEG C culture 48 hours after bacterium colony PCR verifying choose Correct clone is taken to carry out subsequent experimental.

It is overexpressed and silencing GmAP1 gene in 3. soybean of embodiment

The hairy method for transformation of soybean (Kereszt et al., 2007) established with reference to Kereszt et al..

Kereszt,A.,Li,D.,Indrasumunar,A.,Nguyen,C.D.,Nontachaiyapoom,S., Kinkema,M.,and Gresshoff,P.M.(2007).Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.Nature Protocols 2,948-952.

Specific steps are as follows:

1) soya seeds (closing the susceptible varieties such as rich 47) kind in wet vermiculite, is placed in greenhouse (25 DEG C, 16h light According to/8h dark) culture, after about 7 days, removing bean cotyledon can sterilize for testing.Cotyledon differentiation capability is strong at this time.

2) after planting beans 6 days, picking Agrobacterium K599 single bacterium is fallen on containing antibiotic (50ug/mL kanamycins and 50ug/mL Streptomysin) liquid LB in, cultivated for 24 hours under the conditions of 28 DEG C.

3) cotyledon sterilizes: cutting off the preferable soybean cotyledon of growing way with blade, by cotyledon with 75% ethanol postincubation 1min, connects With 10%NaClO handle 10min, later with it is sterile wash 3 times.

4) it prepares thallus suspension liquid: in super-clean bench, taking 2mL bacterium solution, 5000rpm is centrifuged 3min, with configured buffer (component: 10mM 2- [N-morpholino] ethanesulfonic acid, 10mM MgCl2,200 μM acetosyringone PH 5.6) washing thalline twice, finally by bacterium solution OD₆₀₀Value is adjusted to 0.6, spare.

5) bean cotyledon is handled: putting on sterile gloves, bean cotyledon is cut from cotyledon petioles, petiole is leaned among cotyledon lower epidermis A wound is cut with aseptic operation blade in end.

6) it connects bacterium: the bean cotyledon epicuticle handled well is placed on downward on MS culture medium, in wound drop bacterium solution (15- 20 μ L), appropriate bacterium solution forms the vesicle slightly swelled.

7) sealed membrane closed petridish is used, greenhouse (25 DEG C, 16h illumination/8h dark) culture, about 3 Zhou Hou presidents are placed in Hairy root out.Pollution condition, the ware for pollution of transferring at any time are paid attention to daily.

8) growth of hair root: in incubation, be inoculated with bacterium solution at wound browning color (2-3 days), then can centre or Entire wound grows callus, callus graininess or is linked to be sheet (starting to grow within 7 days or so), callus need through It crosses to send out roots in one to two week.

9) hairy Preliminary Identification can be identified by Stereo microscope (Leica MZ FLIII) Fluirescence observation.

Embodiment 4. is overexpressed on the Soybean Root Hairs with silencing GmAP1 gene and is inoculated with soyabean phytophthora

Soybean hairy of performance strong fluorescence is screened using body formula fluorescence microscope (Leica MZ FLIII).Picking table Up to having purpose gene and control growing way comparable hairy, on the filter paper after sterile water is wet and discharge together, by fresh cultured The soybean phytophthora bacterial strain mycelia block of label red fluorescence be placed on the tips of two kinds of Hairy roots, cultivate 36-48h under the conditions of 25 DEG C Afterwards, it takes pictures (Fig. 1, Fig. 4) under the microscope in fluorescence microscopy, sampling freezes, the biology after infecting for subsequent measurements soybean phytophthora Measure (Fig. 2, Fig. 6) and be overexpressed GmAP1 Soybean Root Hairs protein expression level detection (Fig. 3) or silencing GmAP1 gene it is big Beans root hair gene expression detects (Fig. 5).

Soybean phytophthora biomass detects after 5. soybean phytophthora of embodiment infects Soybean Root Hairs

Soybean phytophthora is collected to infect the Soybean Root Hairs of the overexpression after 36h or silencing GmAP1 gene to carry out soybean phytophthora raw Object amount detection.The extracting of genome, according to illustrating to operate, uses spectrophotometer using TIANGEN company genome extraction kit Detect its content and quality.

Real-time fluorescence quantitative PCR reaction:

Primer before quantitative: SEQ ID NO.13

5’-ACTGCACCTTCCAGACCATC-3’

Primer after quantitative: SEQ ID NO.14

5’-CCACCACCTTGATCTTCATG-3’

PCR reaction system includes gDNA 5uL, SYBR Premix Ex Taq II (Tli RNase H Plus) 10uL, Front and back primer sings 0.4uL, ROX Reference Dye II 0.4uL, water 13.8uL.Response procedures: I:95 degree 30 seconds, II:95 Degree 5 seconds, 60 degree 34 seconds, step II react 40 recycle.Solubility curve analysis program be: 95 degree 15 seconds, 60 degree 1 minute, 95 degree 15 seconds.Data analysis uses 2- Δ Δ CT method, and testing result is as shown in Fig. 2, Fig. 6.Bibliography: Livak, K.J., and Schmittgen,T.D.(2001).Analysis of relative gene expression data usingreal- time quantitative PCR and the 2-ΔΔCT method.Methods 25,402-408.

Embodiment 6. is overexpressed protein level detection on the Soybean Root Hairs of GmAP1 gene

Collect the inspection that soybean phytophthora infects the Soybean Root Hairs progress GmAP1 protein expression level of the overexpression GmAP1 after 36h It surveys.Protein extract (component are as follows: 150mM NaCl, 50mM Tris- is added in grinding after the Soybean Root Hairs liquid nitrogen flash freezer of collection 1.0% (v/v) protease inhibitor cocktail of HCl pH 7.5,1.0% (v/v) NP-40, and) mix as 30 minutes on ice.18000g is collected by centrifugation supernatant 80uL and 5 times of albumen sample-loading buffers of 20uL boiling water bath after mixing is added 5 minutes.10uL sample is taken to be separated by electrophoresis on PAGE gel, 120V is run glue 1.5 hours.After reaction by albumen Sample is gone on pvdf membrane, is incubated for sealer with 5%PBST milk.It is small that diluted GFP primary antibody (Abmart) incubation 2 of 1:5000 is added Shi Houyong PBST washes film 5 minutes three times, and the diluted mouse of 1:10000 is then added and resists (LI-COR, irdye 800,926-32210) Film is washed 5 minutes three times with PBST after being incubated for 30 minutes, is swept film and is taken pictures (Fig. 3).

Silencing level detects on the Soybean Root Hairs of 7. silencing GmAP1 gene of embodiment

The Soybean Root Hairs that collection soybean phytophthora infects the silencing GmAP1 gene after 36h carry out GmAP1 gene silencing level Detection.The extracting for mentioning total serum IgE uses Omega company RNA extracts kit according to illustrating to operate, and detects its RNA with spectrophotometer Content and quality.

Reverse transcription generates the first chain: taking 0.7ug RNA to make template, according to Takara company PrimeScript reverse transcriptase Matched reagent operation instruction carries out cDNA synthesis, is settled to 20uL reaction.Reverse transcription product carries out 10 times of dilutions for real with water When quantitative PCR reaction detection gene silencing efficiency.

Real-time fluorescence quantitative PCR reaction:

Primer before quantitative: SEQ ID NO.15

5’-GGAATTGCTCTTGGCTGA-3’

Primer after quantitative: SEQ ID NO.16

5’-GATTCTGCCTCAGCTGGTT-3’

PCR reaction system includes cDNA 5uL, SYBR Premix Ex Taq II (Tli RNase H Plus) 10uL, Front and back primer sings 0.4uL, ROX Reference Dye II 0.4uL, water 13.8uL.Response procedures: I:95 degree 30 seconds, II:95 Degree 5 seconds, 60 degree 34 seconds, step II react 40 recycle.Solubility curve analysis program be: 95 degree 15 seconds, 60 degree 1 minute, 95 degree 15 seconds.Data analysis uses 2- Δ Δ CT method, and testing result is as shown in Figure 5.Bibliography: Livak, K.J., and Schmittgen,T.D.(2001).Analysis of relative gene expression data using real- timequantitative PCR and the 2-ΔΔCT method.Methods 25,402-408.

Embodiment 8. is overexpressed on the tobacco of GmAP1 gene and is inoculated with phytophthora blight of pepper

1. Agrobacterium is cultivated

Transfection pBin::GmAP1-eGFP carrier (GmAP1 gene binary expression vector, partial order are picked from the plate respectively Agrobacterium GV3101 single bacterium of the column such as SEQ ID NO.17) and pBin::eGFP carrier (partial sequence such as SEQ ID NO.18) Fall and silencing suppressor P19, be inoculated into 2mL LB liquid medium respectively (Kan 50ug/mL, Rif 50ug/mL) in 28 DEG C on constant-temperature table, 200rpm overnight incubation to OD600 is 2.0.The GV3101 agrobacterium liquid 5000g being incubated overnight is centrifuged 3 minutes collection thallus.With buffer (component: 10mM 2- [N-morpholino] ethanesulfonic acid, 10mM MgCl2,200 μM of acetosyringone pH 5.6) thalline were collected by centrifugation after suspension bacteria liquid.After repetition washes 2 times, buffer is used Dilute bacterium solution.The Agrobacterium 1:1 of P19 and pBin::GmAP1-eGFP or pBin::eGFP is mixed, and ultimate density is respectively 0.3.

2. tobacco expressed GmAP1

The Agrobacterium prepared is injected into tobacco leaf with syringe, tobacco is in greenhouse (21-23 DEG C, 14h light after injection According to/10h dark) culture.

The detection of 3.GmAP1 albumen cumulative amount

Collect the detection that the tobacco leaf of injection two days later carries out albumen cumulative amount.By the tobacco leaf liquid nitrogen flash freezer of collection Afterwards grinding be added protein extract (component are as follows: 150mM NaCl, 50mM Tris-HCl pH 7.5,1.0% (v/v) NP-40, And 1.0% (v/v) protease inhibitor cocktail) it mixes as 30 minutes on ice.18000g is collected by centrifugation 5 times of albumen sample-loading buffers of 20uL boiling water bath 5 minutes after mixing are added in clear liquid 80uL.Take 10uL sample in SDS-PAGE It is separated by electrophoresis on gel, 120V is run glue 1.5 hours.Protein sample is gone on pvdf membrane after reaction, uses 5%PBST Milk is incubated for sealer.It is added after the diluted GFP primary antibody (Abmart) of 1:5000 is incubated for 2 hours and washes film 5 minutes three times with PBST, with Film 5 is washed with PBST after the diluted mouse of 1:10000 anti-(LI-COR, irdye 800,926-32210) being added afterwards incubation 30 minutes to divide Clock three times, sweeps film and takes pictures (Fig. 9).

4) it is overexpressed GmAP1 and significantly increases tobacco to phytophthora blight of pepper disease resistance.

Blade inoculation Phytophthora capsici two days later is injected, disease symptom observation is carried out after inoculation 2 days and is taken pictures (Fig. 7), is used Ruler measures and records lesion diameter (Fig. 8).Compared with negative control, the tobacco for being overexpressed GmAP1 is equal after being inoculated with phytophthora There is scab conspicuousness to reduce, these results confirm that being overexpressed GmAP1 conspicuousness improves tobacco to the disease-resistant of different phytophthoras Property.

Sequence table

<110>Agricultural University Of Nanjing

<120>a kind of gene GmAP1 for improving disease resistance of plant and its application

<160> 18

<170> SIPOSequenceListing 1.0

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<211> 1524

<212> DNA

<213>soybean (Glycine max)

<400> 1

atgggaaaca tgccaaatgt ggttgtgttg tgtttctgtc tctggaccct gttgtttcct 60

cttgtgttct gtgcacccaa cgatgggttg cgtaggattg gacttaaaaa ggtgaagttg 120

gacaccgatg acgttgtggg gtttaaggaa tttcggtctt caattagaaa gcatcacctt 180

cagaacatcc ttggaggcgc tgaggacacc gatgttgttg cgctgaagaa ttacttggat 240

gctcagtatt acggtgaaat agccattggt acccctcctc agaagttcac tgtgattttt 300

gacaccggta gctctaattt gtgggtgcca tcatccaaat gttacttctc ggttgcatgt 360

ttcatgcacg ctaggtacag gtctagccag tccagtacct acagggaaaa tggtacttct 420

gctgcaattc aatatggtac tggagcaatt tctggtttct ttagcaatga cgatgtcaaa 480

gttggtgaca tagttgtaaa ggaccaggaa tttattgaag caactagaga acctggagtt 540

acatttgtgg ctgctaagtt tgatggtata ctaggacttg gatttcaaga gatatcagtt 600

ggatatgctg ttccagtgtg gtacactatg gttgaacaag gtcttgtaaa ggatccagta 660

ttttcatttt ggctaaatcg gaaaccagaa gaagagaatg gtggggagct tgtttttggt 720

ggtgctgatc ctgctcacta caagggcaaa cacacttatg taccagtgac acgaaaagga 780

tattggcagt ttgacatggg agatgtgctt atttctggta aacccactgg atattgtact 840

aatgactgtt cagcaattgc agactctgga acttctttgt tagctggtcc aacgactgta 900

attaccatga taaatcaagc aatcggagca gctggagttg taagcaaaga atgcaggtcc 960

gttgtcaatc aatatggaca aacaatcttg gaattgctct tggctgaagc aaagccaaaa 1020

aagatctgct cacaaattgg attgtgtacc tttgatggga ctcatggtgt tagcatgggt 1080

atcgagagtg tggtggataa gaatgaaaaa aaatcatctg gtggcattcg ggatgctggt 1140

tgttctgcat gtgagatggc tgttatttgg atgcagaacc agctgaggca gaatcagaca 1200

gaagatagga taatagacta tgccaatgag ctttgtgaaa aattgcctaa cccaatggga 1260

ccatcatccg ttgactgtgg aaagctctct tcaatgccta ttgtttcctt tactattggt 1320

ggaaaagttt ttgacctttc ccctgaggag tatatactga aggtgggtga aggtcctgaa 1380

gcccaatgca ttagtggctt tactgctttg gatgttcctc ctcctcgtgg tcctctatgg 1440

atccttggag atgtcttcat ggggcgctat cacaccatct ttgattatgg taagttgaga 1500

gtcggatttg ctgaggcagc atag 1524

<210> 2

<211> 297

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

aatgagcttt gtgaaaaatt gcctaaccca atgggaccat catccgttga ctgtggaaag 60

ctctcttcaa tgcctattgt ttcctttact attggtggaa aagtttttga cctttcccct 120

gaggagtata tactgaaggt gggtgaaggt cctgaagccc aatgcattag tggctttact 180

gctttggatg ttcctcctcc tcgtggtcct ctatggatcc ttggagatgt cttcatgggg 240

cgctatcaca ccatctttga ttatggtaag ttgagagtcg gatttgctga ggcagca 297

<210> 3

<211> 507

<212> PRT

<213>soybean (Glycine max)

<400> 3

Met Gly Asn Met Pro Asn Val Val Val Leu Cys Phe Cys Leu Trp Thr

1 5 10 15

Leu Leu Phe Pro Leu Val Phe Cys Ala Pro Asn Asp Gly Leu Arg Arg

20 25 30

Ile Gly Leu Lys Lys Val Lys Leu Asp Thr Asp Asp Val Val Gly Phe

35 40 45

Lys Glu Phe Arg Ser Ser Ile Arg Lys His His Leu Gln Asn Ile Leu

50 55 60

Gly Gly Ala Glu Asp Thr Asp Val Val Ala Leu Lys Asn Tyr Leu Asp

65 70 75 80

Ala Gln Tyr Tyr Gly Glu Ile Ala Ile Gly Thr Pro Pro Gln Lys Phe

85 90 95

Thr Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ser

100 105 110

Lys Cys Tyr Phe Ser Val Ala Cys Phe Met His Ala Arg Tyr Arg Ser

115 120 125

Ser Gln Ser Ser Thr Tyr Arg Glu Asn Gly Thr Ser Ala Ala Ile Gln

130 135 140

Tyr Gly Thr Gly Ala Ile Ser Gly Phe Phe Ser Asn Asp Asp Val Lys

145 150 155 160

Val Gly Asp Ile Val Val Lys Asp Gln Glu Phe Ile Glu Ala Thr Arg

165 170 175

Glu Pro Gly Val Thr Phe Val Ala Ala Lys Phe Asp Gly Ile Leu Gly

180 185 190

Leu Gly Phe Gln Glu Ile Ser Val Gly Tyr Ala Val Pro Val Trp Tyr

195 200 205

Thr Met Val Glu Gln Gly Leu Val Lys Asp Pro Val Phe Ser Phe Trp

210 215 220

Leu Asn Arg Lys Pro Glu Glu Glu Asn Gly Gly Glu Leu Val Phe Gly

225 230 235 240

Gly Ala Asp Pro Ala His Tyr Lys Gly Lys His Thr Tyr Val Pro Val

245 250 255

Thr Arg Lys Gly Tyr Trp Gln Phe Asp Met Gly Asp Val Leu Ile Ser

260 265 270

Gly Lys Pro Thr Gly Tyr Cys Thr Asn Asp Cys Ser Ala Ile Ala Asp

275 280 285

Ser Gly Thr Ser Leu Leu Ala Gly Pro Thr Thr Val Ile Thr Met Ile

290 295 300

Asn Gln Ala Ile Gly Ala Ala Gly Val Val Ser Lys Glu Cys Arg Ser

305 310 315 320

Val Val Asn Gln Tyr Gly Gln Thr Ile Leu Glu Leu Leu Leu Ala Glu

325 330 335

Ala Lys Pro Lys Lys Ile Cys Ser Gln Ile Gly Leu Cys Thr Phe Asp

340 345 350

Gly Thr His Gly Val Ser Met Gly Ile Glu Ser Val Val Asp Lys Asn

355 360 365

Glu Lys Lys Ser Ser Gly Gly Ile Arg Asp Ala Gly Cys Ser Ala Cys

370 375 380

Glu Met Ala Val Ile Trp Met Gln Asn Gln Leu Arg Gln Asn Gln Thr

385 390 395 400

Glu Asp Arg Ile Ile Asp Tyr Ala Asn Glu Leu Cys Glu Lys Leu Pro

405 410 415

Asn Pro Met Gly Pro Ser Ser Val Asp Cys Gly Lys Leu Ser Ser Met

420 425 430

Pro Ile Val Ser Phe Thr Ile Gly Gly Lys Val Phe Asp Leu Ser Pro

435 440 445

Glu Glu Tyr Ile Leu Lys Val Gly Glu Gly Pro Glu Ala Gln Cys Ile

450 455 460

Ser Gly Phe Thr Ala Leu Asp Val Pro Pro Pro Arg Gly Pro Leu Trp

465 470 475 480

Ile Leu Gly Asp Val Phe Met Gly Arg Tyr His Thr Ile Phe Asp Tyr

485 490 495

Gly Lys Leu Arg Val Gly Phe Ala Glu Ala Ala

500 505

<210> 4

<211> 507

<212> PRT

<213>soybean (Glycine max)

<400> 4

Met Gly Asn Met Ser Asn Val Val Val Phe Cys Phe Cys Leu Trp Thr

1 5 10 15

Leu Leu Phe Ser Leu Val Phe Cys Ala Pro Asn Asp Gly Leu Gly Arg

20 25 30

Ile Gly Leu Lys Lys Val Lys Leu Asn Thr His Asp Val Glu Gly Leu

35 40 45

Lys Glu Phe Arg Ser Ser Ile Arg Lys His His Leu Gln Asn Ile Leu

50 55 60

Gly Gly Ala Glu Glu Thr Asp Val Val Ala Leu Lys Asn Tyr Leu Asp

65 70 75 80

Ala Gln Tyr Tyr Gly Glu Ile Ala Ile Gly Thr Pro Pro Gln Lys Phe

85 90 95

Thr Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ser

100 105 110

Lys Cys Tyr Phe Ser Ile Ala Cys Phe Met His Ala Arg Tyr Arg Ser

115 120 125

Ser Gln Ser Ser Thr Tyr Arg Glu Asn Gly Thr Ser Ala Ala Ile Gln

130 135 140

Tyr Gly Thr Gly Ala Ile Ser Gly Phe Phe Ser Asn Asp Asp Val Lys

145 150 155 160

Val Gly Asp Ile Val Val Lys Asp Gln Glu Phe Ile Glu Ala Thr Arg

165 170 175

Glu Pro Gly Val Thr Phe Val Ala Ala Lys Phe Asp Gly Ile Leu Gly

180 185 190

Leu Gly Phe Gln Asp Ile Ser Val Gly Tyr Ala Val Pro Val Trp Tyr

195 200 205

Ser Met Val Glu Gln Gly Leu Val Lys Asp Pro Val Phe Ser Phe Trp

210 215 220

Leu Asn Arg Lys Pro Glu Glu Glu Asn Gly Gly Glu Leu Val Phe Gly

225 230 235 240

Gly Ala Asp Pro Ala His Tyr Lys Gly Lys His Thr Tyr Val Pro Val

245 250 255

Thr Arg Lys Gly Tyr Trp Gln Phe Asp Met Gly Asp Val Leu Ile Ala

260 265 270

Gly Lys Pro Thr Gly Tyr Cys Ala Asp Asp Cys Ser Ala Ile Ala Asp

275 280 285

Ser Gly Thr Ser Leu Leu Ala Gly Pro Thr Thr Val Val Thr Met Ile

290 295 300

Asn Gln Ala Ile Gly Ala Ser Gly Val Val Ser Lys Glu Cys Arg Ser

305 310 315 320

Val Val Asn Gln Tyr Gly Gln Thr Ile Leu Glu Leu Leu Leu Ala Glu

325 330 335

Ala Lys Pro Lys Lys Ile Cys Ser Gln Ile Gly Leu Cys Thr Phe Asp

340 345 350

Gly Thr His Gly Val Ser Met Gly Ile Glu Ser Val Val Asp Lys Asn

355 360 365

Glu Arg Lys Ser Ser Gly Ser Ile Arg Asp Ala Gly Cys Ser Ala Cys

370 375 380

Glu Met Ala Val Ile Trp Met Gln Asn Gln Leu Arg Gln Asn Gln Thr

385 390 395 400

Glu Asp Arg Ile Ile Asp Tyr Ala Asn Glu Leu Cys Asp Lys Leu Pro

405 410 415

Asn Pro Met Gly Gln Ser Ser Val Asp Cys Glu Lys Leu Ser Ser Met

420 425 430

Pro Ile Val Ser Phe Thr Ile Gly Gly Lys Val Phe Asp Leu Ser Pro

435 440 445

Gln Glu Tyr Ile Leu Lys Val Gly Glu Gly Pro Glu Ala Gln Cys Ile

450 455 460

Ser Gly Phe Thr Ala Leu Asp Val Pro Pro Pro Arg Gly Pro Leu Trp

465 470 475 480

Ile Leu Gly Asp Val Phe Met Gly Arg Tyr His Thr Ile Phe Asp Tyr

485 490 495

Gly Lys Leu Arg Val Gly Phe Ala Glu Ala Ala

500 505

<210> 5

<211> 514

<212> PRT

<213>soybean (Glycine max)

<400> 5

Met Gly Asn Lys Met Asn Ala Ile Ala Leu Cys Leu Leu Val Ser Thr

1 5 10 15

Leu Leu Val Ser Ala Val Tyr Cys Ala Pro Asn Ala Gly Leu Arg Arg

20 25 30

Ile Gly Leu Lys Lys Ile Lys Leu Asp Pro Lys Asn Arg Leu Ala Ala

35 40 45

Arg Val Gly Ser Lys Asp Val Asp Ser Phe Arg Ala Ser Ile Arg Lys

50 55 60

Phe His Leu Gln Asn Asn Phe Gly Gly Thr Glu Glu Thr Asp Ile Val

65 70 75 80

Ala Leu Lys Asn Tyr Leu Asp Ala Gln Tyr Tyr Gly Glu Ile Ala Ile

85 90 95

Gly Thr Ser Pro Gln Lys Phe Ala Val Ile Phe Asp Thr Gly Ser Ser

100 105 110

Asn Leu Trp Val Pro Ser Ser Lys Cys Thr Phe Ser Val Ala Cys Tyr

115 120 125

Phe His Ala Lys Tyr Lys Ser Ser Lys Ser Ser Thr Phe Lys Lys Asn

130 135 140

Gly Thr Ala Ala Ala Ile Gln Tyr Gly Thr Gly Ala Ile Ser Gly Phe

145 150 155 160

Phe Ser Tyr Asp Ser Val Arg Val Gly Glu Ile Val Val Lys Asn Gln

165 170 175

Glu Phe Ile Glu Ala Thr Arg Glu Pro Gly Val Thr Phe Leu Ala Ala

180 185 190

Lys Phe Asp Gly Ile Leu Gly Leu Gly Phe Gln Glu Ile Ser Val Gly

195 200 205

Asn Ala Ala Pro Val Trp Tyr Asn Met Val Asp Gln Gly Leu Leu Lys

210 215 220

Glu Pro Val Phe Ser Phe Trp Phe Asn Arg Asn Pro Glu Glu Glu Glu

225 230 235 240

Gly Gly Glu Ile Val Phe Gly Gly Val Asp Pro Ala His Tyr Lys Gly

245 250 255

Lys His Thr Tyr Val Pro Val Thr Arg Lys Gly Tyr Trp Gln Phe Asp

260 265 270

Met Gly Asp Val Leu Ile Gly Gly Lys Pro Thr Gly Tyr Cys Ala Asn

275 280 285

Gly Cys Ser Ala Ile Ala Asp Ser Gly Thr Ser Leu Leu Ala Gly Pro

290 295 300

Thr Thr Val Ile Thr Met Ile Asn His Ala Ile Gly Ala Ser Gly Val

305 310 315 320

Met Ser Gln Glu Cys Lys Thr Ile Val Ala Glu Tyr Gly Gln Thr Ile

325 330 335

Leu Asp Leu Leu Leu Ala Glu Thr Gln Pro Lys Lys Ile Cys Ser Arg

340 345 350

Ile Gly Leu Cys Ala Phe Asp Gly Thr His Gly Val Asp Val Gly Ile

355 360 365

Lys Ser Val Val Asp Glu Asn Glu Arg Lys Ser Leu Gly Gly His His

370 375 380

Gly Ala Ala Cys Pro Ala Cys Glu Met Ala Val Val Trp Met Gln Asn

385 390 395 400

Gln Leu Ser Arg Asn Gln Thr Gln Asp Gln Ile Leu Ser Tyr Ile Asn

405 410 415

Gln Leu Cys Asp Lys Met Pro Ser Pro Met Gly Glu Ser Ala Val Asp

420 425 430

Cys Gly Asn Ile Ser Ser Leu Pro Val Val Ser Phe Thr Ile Gly Gly

435 440 445

Arg Thr Phe Asp Leu Ser Pro Glu Glu Tyr Val Leu Lys Val Gly Glu

450 455 460

Gly Pro Val Ala Gln Cys Ile Ser Gly Phe Thr Ala Ile Asp Ile Pro

465 470 475 480

Pro Pro Arg Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Met Gly Arg

485 490 495

Tyr His Thr Val Phe Asp Phe Gly Lys Leu Arg Val Gly Phe Ala Asp

500 505 510

Ala Ala

<210> 6

<211> 514

<212> PRT

<213>soybean (Glycine max)

<400> 6

Met Gly Asn Arg Met Asn Ala Ile Ala Leu Cys Leu Leu Val Ser Ser

1 5 10 15

Leu Leu Leu Ser Ser Val Tyr Cys Ala Pro Asn Asp Gly Leu Arg Arg

20 25 30

Ile Gly Leu Lys Lys Ile Lys Leu Asp Pro Lys Asn Arg Leu Ala Ala

35 40 45

Arg Ile Gly Ser Lys Asp Val Asp Ser Phe Arg Ala Ser Ile Arg Lys

50 55 60

Phe His Leu Gln Asn Asn Phe Gly Gly Ser Glu Glu Thr Asp Ile Val

65 70 75 80

Ala Leu Lys Asn Tyr Leu Asp Ala Gln Tyr Tyr Gly Glu Ile Ala Ile

85 90 95

Gly Thr Ser Pro Gln Lys Phe Thr Val Ile Phe Asp Thr Gly Ser Ser

100 105 110

Asn Leu Trp Val Pro Ser Ser Lys Cys Thr Phe Ser Val Ala Cys Tyr

115 120 125

Phe His Ala Lys Tyr Lys Ser Ser Lys Ser Ser Thr Tyr Lys Lys Asn

130 135 140

Gly Thr Ala Ala Ala Ile Gln Tyr Gly Thr Gly Ala Ile Ser Gly Phe

145 150 155 160

Phe Ser Tyr Asp Ser Val Arg Val Gly Asp Ile Phe Val Lys Asn Gln

165 170 175

Glu Phe Ile Glu Ala Thr Arg Glu Pro Gly Val Thr Phe Leu Ala Ala

180 185 190

Lys Phe Asp Gly Ile Leu Gly Leu Gly Phe Gln Glu Ile Ser Val Gly

195 200 205

Asn Ala Val Pro Val Trp Tyr Asn Met Val Asp Gln Gly Leu Ile Lys

210 215 220

Glu Pro Val Phe Ser Phe Trp Phe Asn Arg Lys Pro Glu Glu Glu Glu

225 230 235 240

Gly Gly Glu Ile Val Phe Gly Gly Val Asp Pro Ala His Tyr Lys Gly

245 250 255

Lys His Thr Tyr Val Pro Val Thr Arg Lys Gly Tyr Trp Gln Phe Asp

260 265 270

Met Gly Asp Val Leu Ile Gly Gly Lys Pro Thr Gly Tyr Cys Ala Asp

275 280 285

Gly Cys Ser Ala Ile Ala Asp Ser Gly Thr Ser Leu Leu Ala Gly Pro

290 295 300

Thr Thr Val Ile Thr Met Ile Asn His Ala Ile Gly Ala Ser Gly Val

305 310 315 320

Met Ser Gln Glu Cys Lys Thr Val Val Ala Glu Tyr Gly Gln Thr Ile

325 330 335

Leu Asp Leu Leu Leu Ser Glu Thr Gln Pro Lys Lys Ile Cys Ser Arg

340 345 350

Ile Gly Leu Cys Ala Phe Asp Gly Thr Arg Gly Val Asp Val Gly Ile

355 360 365

Lys Ser Val Val Asp Glu Asn Glu Arg Lys Ser Ser Gly Gly His His

370 375 380

Gly Ala Ala Cys Pro Ala Cys Glu Met Ala Val Val Trp Met Gln Asn

385 390 395 400

Gln Leu Ser Arg Asn Gln Thr Gln Asp Gln Ile Leu Ser Tyr Ile Asn

405 410 415

Gln Leu Cys Asp Lys Met Pro Ser Pro Met Gly Glu Ser Ala Val Asp

420 425 430

Cys Gly Asn Ile Ser Ser Leu Pro Val Val Ser Phe Thr Ile Gly Gly

435 440 445

Arg Thr Phe Glu Leu Ser Pro Glu Glu Tyr Ile Leu Lys Val Gly Glu

450 455 460

Gly Pro Val Ala Gln Cys Ile Ser Gly Phe Thr Ala Ile Asp Ile Pro

465 470 475 480

Pro Pro Arg Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Met Gly Arg

485 490 495

Tyr His Thr Val Phe Asp Phe Gly Lys Gln Arg Val Gly Phe Ala Asp

500 505 510

Ala Ala

<210> 7

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cgatagggta cccccatggg aaacatgcca aat 33

<210> 8

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ggatccgtcg acccctgctg cctcagcaaa 30

<210> 9

<211> 43

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ttacaattac catggggcgc gccaatgagc tttgtgaaaa att 43

<210> 10

<211> 43

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

ttaaatcatc gattgggcgc gcctgctgcc tcagcaaatc cga 43

<210> 11

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

aatttgcagg tatttggatc ctgctgcctc agcaaatccg a 41

<210> 12

<211> 41

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

ctctagactc acctaggatc caatgagctt tgtgaaaaat t 41

<210> 13

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

actgcacctt ccagaccatc 20

<210> 14

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ccaccacctt gatcttcatg 20

<210> 15

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

ggaattgctc ttggctga 18

<210> 16

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gattctgcct cagctggtt 19

<210> 17

<211> 8065

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

atgcttgaca ctttatcact gataaacata atatgtccac caacttatca gtgataaaga 60

atccgcgcgt tcaatcggac cagcggaggc tggtccggag gccagacgtg aaacccaaca 120

tacccctgat cgtaattctg agcactgtcg cgctcgacgc tgtcggcatc ggcctgatta 180

tgccggtgct gccgggcctc ctgcgcgatc tggttcactc gaacgacgtc accgcccact 240

atggcattct gctggcgctg tatgcgttgg tgcaatttgc ctgcgcacct gtgctgggcg 300

cgctgtcgga tcgtttcggg cggcggccaa tcttgctcgt ctcgctggcc ggcgccagat 360

ctggggaacc ctgtggttgg catgcacata caaatggacg aacggataaa ccttttcacg 420

cccttttaaa tatccgatta ttctaataaa cgctcttttc tcttaggttt acccgccaat 480

atatcctgtc aaacactgat agtttgtgaa ccatcaccca aatcaagttt tttggggtcg 540

aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 600

ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgccatt 660

caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 720

ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc 780

acgacgttgt aaaacgacgg ccagtgaatt gttaattaag aattcgagct ccttgcatgc 840

ctgcaggtca acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca 900

gtctcagaag accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc 960

ctcggattcc attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt 1020

ggctcctaca aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc 1080

gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 1140

ccaaccacgt cttcaaagca agtggattga tgtgataaca tggtggagca cgacacactt 1200

gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat tgagactttt 1260

caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat ctgtcacttt 1320

attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg cgataaagga 1380

aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg 1440

aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt 1500

gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc 1560

tctatataag gaagttcatt tcatttggag aggacctcga gaattctcaa cacaacatat 1620

acaaaacaaa cgaatctcaa gcaatcaagc attctacttc tattgcagca atttaaatca 1680

tttcttttaa agcaaaagca attttctgaa aattttcacc atttacgaac gatagccggt 1740

accatgggaa acatgccaaa tgtggttgtg ttgtgtttct gtctctggac cctgttgttt 1800

cctcttgtgt tctgtgcacc caacgatggg ttgcgtagga ttggacttaa aaaggtgaag 1860

ttggacaccg atgacgttgt ggggtttaag gaatttcggt cttcaattag aaagcatcac 1920

cttcagaaca tccttggagg cgctgaggac accgatgttg ttgcgctgaa gaattacttg 1980

gatgctcagt attacggtga aatagccatt ggtacccctc ctcagaagtt cactgtgatt 2040

tttgacaccg gtagctctaa tttgtgggtg ccatcatcca aatgttactt ctcggttgca 2100

tgtttcatgc acgctaggta caggtctagc cagtccagta cctacaggga aaatggtact 2160

tctgctgcaa ttcaatatgg tactggagca atttctggtt tctttagcaa tgacgatgtc 2220

aaagttggtg acatagttgt aaaggaccag gaatttattg aagcaactag agaacctgga 2280

gttacatttg tggctgctaa gtttgatggt atactaggac ttggatttca agagatatca 2340

gttggatatg ctgttccagt gtggtacact atggttgaac aaggtcttgt aaaggatcca 2400

gtattttcat tttggctaaa tcggaaacca gaagaagaga atggtgggga gcttgttttt 2460

ggtggtgctg atcctgctca ctacaagggc aaacacactt atgtaccagt gacacgaaaa 2520

ggatattggc agtttgacat gggagatgtg cttatttctg gtaaacccac tggatattgt 2580

actaatgact gttcagcaat tgcagactct ggaacttctt tgttagctgg tccaacgact 2640

gtaattacca tgataaatca agcaatcgga gcagctggag ttgtaagcaa agaatgcagg 2700

tccgttgtca atcaatatgg acaaacaatc ttggaattgc tcttggctga agcaaagcca 2760

aaaaagatct gctcacaaat tggattgtgt acctttgatg ggactcatgg tgttagcatg 2820

ggtatcgaga gtgtggtgga taagaatgaa aaaaaatcat ctggtggcat tcgggatgct 2880

ggttgttctg catgtgagat ggctgttatt tggatgcaga accagctgag gcagaatcag 2940

acagaagata ggataataga ctatgccaat gagctttgtg aaaaattgcc taacccaatg 3000

ggaccatcat ccgttgactg tggaaagctc tcttcaatgc ctattgtttc ctttactatt 3060

ggtggaaaag tttttgacct ttcccctgag gagtatatac tgaaggtggg tgaaggtcct 3120

gaagcccaat gcattagtgg ctttactgct ttggatgttc ctcctcctcg tggtcctcta 3180

tggatccttg gagatgtctt catggggcgc tatcacacca tctttgatta tggtaagttg 3240

agagtcggat ttgctgaggc agcaggtacc cccgggatgg tgagcaaggg cgaggagctg 3300

ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 3360

agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 3420

tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 3480

gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 3540

atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 3600

acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 3660

atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 3720

cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 3780

cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 3840

atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 3900

agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3960

gggatcactc tcggcatgga cgagctgtac aagtaaggat cctctagatg aactagagtc 4020

cgcaaaaatc accagtctct ctctacaaat ctatctctct ctatttttct ccagaataat 4080

gtgtgagtag ttcccagata agggaattag ggttcttata gggtttcgct catgtgttga 4140

gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc aataaaattt 4200

ctaattccta aaaccaaaat ccagtgacaa gcttggcgcg ccagcttggc gtaatcatgg 4260

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 4320

ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 4380

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 4440

ggccaacgcg cggggagagg cggtttgcgt attgggccaa agacaaaagg gcgacattca 4500

accgattgag ggagggaagg taaatattga cggaaattat tcattaaagg tgaattatca 4560

ccgtcaccga cttgagccat ttgggaatta gagccagcaa aatcaccagt agcaccatta 4620

ccattagcaa ggccggaaac gtcaccaatg aaaccatcga tagcagcacc gtaatcagta 4680

gcgacagaat caagtttgcc tttagcgtca gactgtagcg cgttttcatc ggcattttcg 4740

gtcatagccc ccttattagc gtttgccatc ttttcataat caaaatcacc ggaaccagag 4800

ccaccaccgg aaccgcctcc ctcagagccg ccaccctcag aaccgccacc ctcagagcca 4860

ccaccctcag agccgccacc agaaccacca ccagagccgc cgccagcatt gacaggaggc 4920

ccgatctagt aacatagatg acaccgcgcg cgataattta tcctagtttg cgcgctatat 4980

tttgttttct atcgcgtatt aaatgtataa ttgcgggact ctaatcataa aaacccatct 5040

cataaataac gtcatgcatt acatgttaat tattacatgc ttaacgtaat tcaacagaaa 5100

ttatatgata atcatcgcaa gaccggcaac aggattcaat cttaagaaac tttattgcca 5160

aatgtttgaa cgatcgggga tcatccgggt ctgtggcggg aactccacga aaatatccga 5220

acgcagcaag atatcgcggt gcatctcggt cttgcctggg cagtcgccgc cgacgccgtt 5280

gatgtggacg ccgggcccga tcatattgtc gctcaggatc gtggcgttgt gcttgtcggc 5340

cgttgctgtc gtaatgatat cggcaccttc gaccgcctgt tccgcagaga tcccgtgggc 5400

gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac 5460

gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag 5520

gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca 5580

agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg 5640

aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg 5700

tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca 5760

ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcatcgccg 5820

tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct 5880

tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 5940

cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 6000

attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 6060

tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 6120

acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc 6180

agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 6240

gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 6300

aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 6360

cgaaacgatc cagatccggt gcagattatt tggattgaga gtgaatatga gactctaatt 6420

ggataccgag gggaatttat ggaacgtcag tggagcattt ttgacaagaa atatttgcta 6480

gctgatagtg accttaggcg acttttgaac gcgcaataat ggtttctgac gtatgtgctt 6540

agctcattaa actccagaaa cccgcggctg agtggctcct tcaacgttgc ggttctgtca 6600

gttccaaacg taaaacggct tgtcccgcgt catcggcggg ggtcataacg tgactccctt 6660

aattctccgc tcatgatcag attgtcgttt cccgccttca gtttgtgggc catcgccctg 6720

atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 6780

ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt 6840

gccgatttcg gaaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 6900

cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca 6960

ctggtgaaaa gaaaaaccac cccagtacat taaaaacgtc cgcaatgtgt tattaagttg 7020

tctaagcgtc aatttgttta caccacaata tatcctgcca ccagccagcc aacagctccc 7080

cgaccggcag ctcggcacaa aatcaccact cgatacaggc agcccatcag tccgggacgg 7140

cgtcagcggg agagccgttg taaggcggca gactttgctc atgttaccga tgctattcgg 7200

aagaacggca actaagctgc cgggtttgaa acacggatga tctcgcggag ggtagcatgt 7260

tgattgtaac gatgacagag cgttgctgcc tgtgatcaaa tatcatctcc ctcgcagaga 7320

tccgaattat cagccttctt attcatttct cgcttaaccg tgacaggctg tcgatcttga 7380

gaactatgcc gacataatag gaaatcgctg gataaagccg ctgaggaagc tgagtggcgc 7440

tatttcttta gaagtgaacg ttgacgatat caactcccct atccattgct caccgaatgg 7500

tacaggtcgg ggacccgaag ttccgactgt cggcctgatg catccccggc tgatcgaccc 7560

cagatctggg gctgagaaag cccagtaagg aaacaactgt aggttcgagt cgcgagatcc 7620

cccggaacca aaggaagtag gttaaacccg ctccgatcag gccgagccac gccaggccga 7680

gaacattggt tcctgtaggc atcgggattg gcggatcaaa cactaaagct actggaacga 7740

gcagaagtcc tccggccgcc agttgccagg cggtaaaggt gagcagaggc acgggaggtt 7800

gccacttgcg ggtcagcacg gttccgaacg ccatggaaac cgcccccgcc aggcccgctg 7860

cgacgccgac aggatctagc gctgcgtttg gtgtcaacac caacagcgcc acgcccgcag 7920

ttccgcaaat agcccccagg accgccatca atcgtatcgg gctacctagc agagcggcag 7980

agatgaacac gaccatcagc ggctgcacag cgcctaccgt cgccgcgacc ccgcccggca 8040

ggcggtagac cgaaataaac aacaa 8065

<210> 18

<211> 6397

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

atgcttgaca ctttatcact gataaacata atatgtccac caacttatca gtgataaaga 60

atccgcgcgt tcaatcggac cagcggaggc tggtccggag gccagacgtg aaacccaaca 120

tacccctgat cgtaattctg agcactgtcg cgctcgacgc tgtcggcatc ggcctgatta 180

tgccggtgct gccgggcctc ctgcgcgatc tggttcactc gaacgacgtc accgcccact 240

atggcattct gctggcgctg tatgcgttgg tgcaatttgc ctgcgcacct gtgctgggcg 300

cgctgtcgga tcgtttcggg cggcggccaa tcttgctcgt ctcgctggcc ggcgccagat 360

ctggggaacc ctgtggttgg catgcacata caaatggacg aacggataaa ccttttcacg 420

cccttttaaa tatccgatta ttctaataaa cgctcttttc tcttaggttt acccgccaat 480

atatcctgtc aaacactgat agtttgtgaa ccatcaccca aatcaagttt tttggggtcg 540

aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 600

ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgccatt 660

caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 720

ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc 780

acgacgttgt aaaacgacgg ccagtgaatt gttaattaag aattcgagct ccttgcatgc 840

ctgcaggtca acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca 900

gtctcagaag accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc 960

ctcggattcc attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt 1020

ggctcctaca aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc 1080

gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 1140

ccaaccacgt cttcaaagca agtggattga tgtgataaca tggtggagca cgacacactt 1200

gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat tgagactttt 1260

caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat ctgtcacttt 1320

attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg cgataaagga 1380

aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg 1440

aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt 1500

gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc 1560

tctatataag gaagttcatt tcatttggag aggacctcga gaattctcaa cacaacatat 1620

acaaaacaaa cgaatctcaa gcaatcaagc attctacttc tattgcagca atttaaatca 1680

tttcttttaa agcaaaagca attttctgaa aattttcacc atttacgaac gatagccggt 1740

acccccggga tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1800

gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1860

gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1920

tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1980

cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 2040

accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 2100

gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 2160

ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 2220

cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 2280

cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 2340

gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 2400

cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 2460

tacaagtaag gatcctctag atgaactaga gtccgcaaaa atcaccagtc tctctctaca 2520

aatctatctc tctctatttt tctccagaat aatgtgtgag tagttcccag ataagggaat 2580

tagggttctt atagggtttc gctcatgtgt tgagcatata agaaaccctt agtatgtatt 2640

tgtatttgta aaatacttct atcaataaaa tttctaattc ctaaaaccaa aatccagtga 2700

caagcttggc gcgccagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 2760

tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 2820

gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 2880

ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 2940

cgtattgggc caaagacaaa agggcgacat tcaaccgatt gagggaggga aggtaaatat 3000

tgacggaaat tattcattaa aggtgaatta tcaccgtcac cgacttgagc catttgggaa 3060

ttagagccag caaaatcacc agtagcacca ttaccattag caaggccgga aacgtcacca 3120

atgaaaccat cgatagcagc accgtaatca gtagcgacag aatcaagttt gcctttagcg 3180

tcagactgta gcgcgttttc atcggcattt tcggtcatag cccccttatt agcgtttgcc 3240

atcttttcat aatcaaaatc accggaacca gagccaccac cggaaccgcc tccctcagag 3300

ccgccaccct cagaaccgcc accctcagag ccaccaccct cagagccgcc accagaacca 3360

ccaccagagc cgccgccagc attgacagga ggcccgatct agtaacatag atgacaccgc 3420

gcgcgataat ttatcctagt ttgcgcgcta tattttgttt tctatcgcgt attaaatgta 3480

taattgcggg actctaatca taaaaaccca tctcataaat aacgtcatgc attacatgtt 3540

aattattaca tgcttaacgt aattcaacag aaattatatg ataatcatcg caagaccggc 3600

aacaggattc aatcttaaga aactttattg ccaaatgttt gaacgatcgg ggatcatccg 3660

ggtctgtggc gggaactcca cgaaaatatc cgaacgcagc aagatatcgc ggtgcatctc 3720

ggtcttgcct gggcagtcgc cgccgacgcc gttgatgtgg acgccgggcc cgatcatatt 3780

gtcgctcagg atcgtggcgt tgtgcttgtc ggccgttgct gtcgtaatga tatcggcacc 3840

ttcgaccgcc tgttccgcag agatcccgtg ggcgaagaac tccagcatga gatccccgcg 3900

ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa 3960

ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 4020

gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 4080

gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc 4140

tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc 4200

cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag 4260

gcatcgccat gggtcacgac gagatcatcg ccgtcgggca tgcgcgcctt gagcctggcg 4320

aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 4380

ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 4440

caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc 4500

tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc 4560

cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 4620

gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg 4680

gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 4740

cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 4800

gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atccagatcc ggtgcagatt 4860

atttggattg agagtgaata tgagactcta attggatacc gaggggaatt tatggaacgt 4920

cagtggagca tttttgacaa gaaatatttg ctagctgata gtgaccttag gcgacttttg 4980

aacgcgcaat aatggtttct gacgtatgtg cttagctcat taaactccag aaacccgcgg 5040

ctgagtggct ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg 5100

cgtcatcggc gggggtcata acgtgactcc cttaattctc cgctcatgat cagattgtcg 5160

tttcccgcct tcagtttgtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 5220

ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 5280

ctcgggctat tcttttgatt tataagggat tttgccgatt tcggaaccac catcaaacag 5340

gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag 5400

gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccccagta 5460

cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt ttacaccaca 5520

atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca caaaatcacc 5580

actcgataca ggcagcccat cagtccggga cggcgtcagc gggagagccg ttgtaaggcg 5640

gcagactttg ctcatgttac cgatgctatt cggaagaacg gcaactaagc tgccgggttt 5700

gaaacacgga tgatctcgcg gagggtagca tgttgattgt aacgatgaca gagcgttgct 5760

gcctgtgatc aaatatcatc tccctcgcag agatccgaat tatcagcctt cttattcatt 5820

tctcgcttaa ccgtgacagg ctgtcgatct tgagaactat gccgacataa taggaaatcg 5880

ctggataaag ccgctgagga agctgagtgg cgctatttct ttagaagtga acgttgacga 5940

tatcaactcc cctatccatt gctcaccgaa tggtacaggt cggggacccg aagttccgac 6000

tgtcggcctg atgcatcccc ggctgatcga ccccagatct ggggctgaga aagcccagta 6060

aggaaacaac tgtaggttcg agtcgcgaga tcccccggaa ccaaaggaag taggttaaac 6120

ccgctccgat caggccgagc cacgccaggc cgagaacatt ggttcctgta ggcatcggga 6180

ttggcggatc aaacactaaa gctactggaa cgagcagaag tcctccggcc gccagttgcc 6240

aggcggtaaa ggtgagcaga ggcacgggag gttgccactt gcgggtcagc acggttccga 6300

acgccatgga aaccgccccc gccaggcccg ctgcgacgcc gacaggatct agcgctgcgt 6360

ttggtgtcaa caccaacagc gccacgcccg cagttcc 6397

Claims (10)

1. it is a kind of improve disease resistance of plant gene GmAP1, nucleotide sequence as shown in SEQ ID NO.1 or with sequence SEQ ID NO.1 has 70% or more homology；Preferably, nucleotide sequence as shown in SEQ ID NO.1 or with sequence SEQ ID NO.1 has 80% or more homology；It is further preferred that nucleotide sequence as shown in SEQ ID NO.1 or with sequence SEQ ID NO.1 has 85% or more homology；It is furthermore preferred that nucleotide sequence is as shown in SEQ ID NO.1, or with sequence SEQ ID NO.1 has 90% or more homology.

2. the protein of the coding of gene GmAP1 described in claim 1 or the albumen with the coding of gene GmAP1 described in claim 1 Matter compares the protein of the amino acid sequence that can be improved disease resistance of plant with not small 70% similitude, preferred to have not The protein that can be improved disease resistance of plant of the amino acid sequence of small 80% similitude.

3. protein according to claim 2, it is characterised in that amino acid sequence is SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 or these amino acid sequences pass through the substitution of one or several amino acid residues And/or it is deleted and/or added and the protein as derived from them of disease resistance of plant can be provided.

4. a kind of recombinant expression carrier comprising gene GmAP1 described in claim 1.

5. recombinant expression carrier described in claim 4, it is characterised in that be that gene GmAP1 is inserted into pair containing the end C- eGFP Carrier pBin::GmAP1-eGFP obtained in first carrier pBin-GFP restriction enzyme site SmaI.

6. a kind of transformant, it is characterised in that it is imported obtained by host cell by recombinant expression carrier as claimed in claim 4, it is described The preferred Bacillus coli cells of host cell or agrobatcerium cell.

7. gene GmAP1, protein described in claim 2 or 3 described in claim 1, claim 4 or 5 the recombination table The application in plant immune resistance or disease resistance is being improved up to carrier or claim 6 transformant.

8. gene GmAP1, protein described in claim 2 or 3 described in claim 1, claim 4 or 5 the recombination table Pathogenic bacteria immune resistance is improved in plant up to carrier or claim 6 transformant or is improved in disease caused by Genes For Plant Tolerance pathogenic bacteria Application.

9. gene GmAP1, protein described in claim 2 or 3 described in claim 1, claim 4 or 5 the recombination table Up to the application of carrier or claim 6 transformant in plant breeding.

10. gene GmAP1, protein described in claim 2 or 3 described in claim 1, claim 4 or 5 the recombination table The application in the kind of significant disease resistance/or increase yield is obtained after importing plant up to carrier or claim 6 transformant, it is excellent Application in the kind for yield that choosing imports soybean, tobacco, tomato or potato are enhanced disease resistance and/or increased.