CN103923194B - The pathogen of Botrytis cinerea secreted protein exciton BcGS1 and application thereof - Google Patents

The pathogen of Botrytis cinerea secreted protein exciton BcGS1 and application thereof Download PDF

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CN103923194B
CN103923194B CN201310016741.4A CN201310016741A CN103923194B CN 103923194 B CN103923194 B CN 103923194B CN 201310016741 A CN201310016741 A CN 201310016741A CN 103923194 B CN103923194 B CN 103923194B
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bcgs1
pathogen
plant
botrytis cinerea
exciton
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CN103923194A (en
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邱德文
杨秀芬
张云华
曾洪梅
郭立华
袁京京
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Beijing Green Agrosino Corp
Institute of Plant Protection of Chinese Academy of Agricultural Sciences
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Institute of Plant Protection of Chinese Academy of Agricultural Sciences
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    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance

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Abstract

The present invention relates to a kind of purposes that can improve plant resistance to environment stress and defensive the pathogen of Botrytis cinerea secretory protein exciton and gene order and this albumen and gene thereof.This albumen exciton BcGS1, is its aminoacid sequence as SEQ? ID? shown in NO:2.May be used for improving plant resistance to environment stress and inducing plant defensive raction.Described plant optimization is tobacco or tomato.This albumen exciton significantly can improve the resistance of plant, and concentration is low, rapid-action, long action time.BcGS1 is that the disease resistance improving plant provides new approach, thus has broad application prospects in agriculture production.

Description

The pathogen of Botrytis cinerea secreted protein exciton BcGS1 and application thereof
Technical field
The present invention relates to biological technical field, particularly a kind of nucleotide sequence that can improve the aminoacid sequence and this protein of encoding that come from the pathogen of Botrytis cinerea (Botrytiscinerea) secreted protein exciton of plant resistance to environment stress and inducing plant defensive raction is extremely applied.
Background technology
Gray mold is a kind of fungal disease caused by Botrytis cinerea, can endanger 200 various plants such as fruit tree, vegetables, flowers, on the vegetables of greenhouse gardening, especially cause fruit rot, lose more serious.The pathogen of Botrytis cinerea is subordinate to Deuteromycotina, hyphomycetes, hyphomycetales, Staphlosporonites fungi on classification position.At present chemical prevention and breeding resistant variety are mainly taked to the control of Plant diseases.But they often also exist the drawback that cannot forgo.Last century, Plant-induced resistance received publicity along with the development of biotechnology, and exciton, as the Main Factors of inducing plant resistance, more receives publicity.Therefore find out from this pathogenic bacteria and have new activity, the active substance of New function has become the focus of domestic and international scientist concern.
The exciton that the pathogen of Botrytis cinerea produces is a kind of important effector molecule.It is by identify and the acceptor molecule acting on surface of Plant callus cell or subcellular components transmits the exciton signal of pathogenic bacteria, open vegeto-animal defence system, the local organization of inducing plant produces hypersensitive cell necrosis, and makes plant finally produce systemic acquired resistance by the systems communicate of local cells semiochemicals.
From the pathogen of Botrytis cinerea, many activeconstituentss that can cause Plant defense responses are isolated both at home and abroad at present.As calendar year 2001 V.Repka, Deng being separated a kind of exciton from the pathogen of Botrytis cinerea, this exciton is a kind of rough cell wall preparation, main component is protein and sugar, it can cause the oxidative burst of plant and the accumulation of pathogenesis-related proteins, improves activity (V.Repka. etc., the Vitis of phenylpropyl alcohol alkane approach relevant enzyme, 2001,40(4): 205-212); MohamedElOirdi in 2011 etc. have been separated a kind of exocellular polysaccharide from the pathogen of Botrytis cinerea, can effectively induce tomato to the resistance (MohamedElOirdi. etc. of gray mold by SA signal transduction pathway, ThePlantCell, 2011,23:2405 – 2421).
Applicant has also successively been separated two kinds of albumen exciton PebC1 and PebC2 from the pathogen of Botrytis cinerea, and their molecular weight is respectively 36kD and 14kD.Wherein PebC1 significantly can promote wheat seedling growth, improves drought resistance of wheat, induce tomato to the system resistant of gray mold.PebC1 full length gene 639bp, to encode 212 amino acid, in tomato body after PebC1 process, phenylalanine ammonia lyase (PAL), peroxidase (POD) and polyphenoloxidase (PPO) are active significantly strengthens (ZhangYunhua etc., MicrobiologicalResearch, 2010, (165), 142-151).
All documents published show, the pathogen of Botrytis cinerea can produce the defensive raction causing plant really, improve the exciton of plant resistance to environment stress, its character and kind different, but protein sequence and gene order are reported less, so find new albumen exciton, making mechanism and exciton mutually to announcement the pathogen of Botrytis cinerea and plant, to improve plant immunization resistance be very important.
Summary of the invention
Technical problem solved by the invention is to provide a kind of purposes that can improve plant resistance to environment stress and defensive the pathogen of Botrytis cinerea secretory protein exciton and gene order and this albumen and gene thereof.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
A kind of the pathogen of Botrytis cinerea (Botrytiscinerea) secreted protein exciton BcGS1, its aminoacid sequence is as shown in SEQIDNO:2.
Albumen exciton BcGS1 of the present invention may be used for improving plant resistance to environment stress and inducing plant defensive raction.Described plant is dicotyledons, is preferably tobacco, tomato, cucumber.When for induce reduce the harm of tobacco mosaic virus (TMV) to tobacco time, the induced concentration of described BcGS1 is 20 μ g/mL; When for inducing tomato or cucumber to improve the resistance to the pathogen of Botrytis cinerea, the induced concentration of described BcGS1 is 10 μ g/mL.
A kind of BcGS1 gene, nucleotides sequence is classified as one of what follows:
(1) polynucleotide sequence of coding protein as shown in SEQIDNO:2;
(2) with above-mentioned polynucleotide sequence according to the polynucleotide sequence of base pair complementarity principle complementation.
Described BcGS1 gene, its polynucleotide sequence is preferably as shown in SEQIDNO:1.Also can be other array configurations of coding amino acid whose codon of protein as shown in SEQIDNO:2.Described BcGS1 gene can be used for transforming expresses, the preferred pMD18-Tsimple of expression vector, and recombinant vectors can use intestinal bacteria Rossetta(DE3) express.Obtain the amalgamation and expression albumen (His-BcGS1) that molecular weight is about 83KD, Resistant reaction can be produced by the similar plant such as evoking tobacco, tomato, improve resistance capacity.Reduce tobacco mosaic virus (TMV) to the harm of tobacco, alleviate the harm of the pathogen of Botrytis cinerea to tomato.
Albumen exciton of the present invention can be the protein from the nutrient solution of the pathogen of Botrytis cinerea (Botrytiscinerea), the supernatant liquor of nutrient solution or thalline.Also can be express by genetic engineering means the recombinant protein transformed.There is identical effect.
Advantage of the present invention is, this albumen exciton significantly can improve the resistance of plant, and concentration is low, rapid-action, long action time.BcGS1 is that the disease resistance improving plant provides new approach, thus has broad application prospects in agriculture production.
Microorganism Deposit Information
Numbering CGMCCNo.7057, name Botrytiscinerea, on January 9th, 2013 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, phone 010-64807355.
Accompanying drawing explanation
Fig. 1 is the aminoacid sequence of albumen exciton BcGS1 peptide section.
Embodiment
For further illustrating the present invention, illustrate with the following Examples:
The cultivation of embodiment 1 the pathogen of Botrytis cinerea and fermented liquid preparation
By the pathogen of Botrytis cinerea (numbering CGMCCNo.7057, name Botrytiscinerea, on January 9th, 2013 is preserved in China Committee for Culture Collection of Microorganisms), be inoculated in PDA[and get peeled potatoes 200 grams, be cut into block, add water 1000 milliliters and boil 30 minutes, elimination potato ball, filtrate is complemented to 1000 milliliters, add glucose 20 grams, 15 grams, agar, dissolve rear packing, 121 DEG C of sterilizings 30 minutes] dull and stereotyped, cultivate 1 week for 25 DEG C, picking colony edge is inoculated in 200mL and fills the formula of examining [potassium primary phosphate 0.6g, saltpetre 0.7g, magnesium sulfate heptahydrate 0.25g, three water dipotassium hydrogen phosphate 0.125g, nitrocalcite 0.3g, boric acid 1mg, manganese sulfate monohydrate 1.5mg, Zinc Sulphate Heptahydrate 4mg, Sodium Molybdate Dihydrate 0.1mg, potassiumiodide 20 μ g, cupric sulfate pentahydrate 20 μ g, CoCL2 6H2O 20 μ g, FeNa 2eDTA8mg, nicotinic acid 1mg, pyridoxol 1mg, calcium pantothenate (calciumpanthotenate) 1mg, vitamin 1mg, l-asparagine 1g, glucose 20g, add deionized water to 1L] in the 500mL triangular flask of liquid nutrient medium, 25 DEG C, 180r/min cultivates 6 days, obtains the fermented liquid of the pathogen of Botrytis cinerea.
The preparation and determination methods of embodiment 2 crude protein exciton
The fermented liquid of gained in Example 1, at 4 DEG C, centrifugal 1h(or 13000rpm, 30min under 5000rpm condition), collect supernatant liquor, with filter membrane (Whatman) suction filtration twice of 0.45mm, until there is no thalline.Add ammonium sulfate powder, make ammonium sulfate final concentration in described supernatant liquor be 80% to precipitate target protein, 4 DEG C of dialysis 48h, be finally dissolved in ultrapure water by outer for the born of the same parents of acquisition gross protein, final protein concentration is 100 μ g/mL.Protein content BCA tMproteinassaykit(ThermoScientific) measure at wavelength 590nm place through GF-M2000 type microplate reader (Shandong Gaomi Caihong Analytical Instrument Co., Ltd.).
Bioactive monitoring: to grow 2 months, the tobacco with 5-7 sheet true leaf is material, take clear water as contrast.Crude protein strength of solution is diluted to 50 μ g/mL, utilizes the syringe of 1mL not with syringe needle, is injected into blade respectively by 50uL solution goes from the leaf back side.Whether observe through 24h has necrotic plaque to be formed.
Result: the transparent shape of injection place blade after 6h, appears water stain spot after injection site 4h, in the blade of injection crude protein exciton, after 12h, visible typical anaphylaxis, has necrotic plaque to be formed.Contrast there is no any reaction, the same with normal blade.
The separation and purification of embodiment 3 albumen exciton and biological activity determination
Using AKTAexplore10(GEHealthcare) gross protein of protein purification instrument to gained be further purified.Described in embodiment 2, crude protein is after lyophilize, is dissolved in 20mMHEPES(PH7.0) damping fluid.Sample is by HiTrapTMDEAEFF anion-exchange column (GEHealthcare), gradient elution (5% is carried out with NaCl, 20%, 30%, 50%, 100), the protein concentration of application is 50 μ g/mL, and result shows, the anaphylaxis causing tobacco that the protein penetrated in peak can be strong, be detected as single band through 12% denaturing polyacrylamide gel electrophoresis, illustrate that lipidated protein is high.This protein apparent molecular weight is 80kD(pI=4.72), by this albumen exciton called after BcGS1(ProteinElicitorofBotrytiscinerea).With the test of example 2 same method to different concns BcGS1(1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL) detect, 20 μ g/mL can cause typical allergic to react, and the protein concentration that most low energy causes allergic reaction is 10 μ g/mL.
Result: the BcGS1 of separation and purification can make tobacco form anaphylactoid necrotic plaque.
H 2o 2deposition
Blade treatment process such as example 2, BcGS1 concentration is 20 μ g/mL, and the time is 24h.Get process blade distilled water clean after be placed in 2mL pipe, get appropriate DAB(3,3 '-Diaminbenzidine) (Sigma) dye liquor (1mg.mL-1, pH3.8), with NaOH adjust pH to 5.8, add pretreated plant tissue respectively, room temperature keeps in Dark Place 8 hours.Remove dye liquor in each pipe subsequently, add 95% ethanol, boiling water bath number minute, remove each pipe liquid, then add dehydrated alcohol and boiling water bath till leaf green is sloughed completely, each pipe liquid of sucking-off again, to go to be immersed in 70% glycerine, drive the microscopic examination of iuntercellular bubble out of.
Result: have obvious brown precipitate matter to occur at the blade position of BcGS1 process, and the guard cell on the pore for the treatment of sites also occurs reddish-brown precipitation.
The acquisition of embodiment 4 albumen exciton BcGS1 peptide section sequence
The BcGS1 protein of purifying detects through two-dimensional electrophoresis, cut protein single-point and modify Trypsin matter enzyme (SequencingGradeModifiedTrypsin through 10ng/ μ l order-checking level respectively, Promega) 30 DEG C, pH7.5 enzymolysis 30min ~ 1h, ESI-MS/MS be the Q-TOF2 orthogonal acceleration esi-msn instrument of Micromass company of Britain.Outfit is received and is risen spraying source.All mensuration is all carried out under positive ion mode.The tandem mass spectrum fragment of mass spectrograph Glu-fib corrects, and mass accuracy is less than 0.1Da.Atomization gas is nitrogen, and collision gas is argon gas.Source temperature 80 DEG C, taper hole voltage 50V.TOF acceleration voltage 9.1kV.MCP detector voltage is 2150V.Capillary voltage 800V.Obtain multiple peptide section sequences of this protein, the aminoacid sequence in these sequences and NCBI coded by gi154316440 matches (http://www.ncbi.nlm.nih.gov/protein/gi%7C154316440).As shown in Figure 1, what represent with underscore in Fig. 1 is consistent peptide section sequence.
The clone of embodiment 5 protein elicitor BcGS1 encoding gene
Degeneracy according to mass spectrum sequencing result and codon designs 4 pairs of primers, through screening verification finally selected primer:
BcGS1-forward 5 '-ATGGCTTCTTCTCTGCTTTCCTC-3(is as shown in SEQIDNO:3) and the reverse 5 '-TTACCAGCTACCACTAACAGTAGCAGT-3(of BcGS1-as shown in SEQIDNO:4), and with the mRNA of Botrytis cinerea bacterial strain extracting for template, select HiFi reverse transcription polysaccharase to carry out PT-PCR(94 ° of C3min; 94 ° of C30s, 55 ° of C30s, 72 ° of C2min, 35cycles; 72 ° of C10min; 4 ° of C ∝), amplify the DNA fragmentation of 2019bp, comprising the entire open reading frame of 2019bp, by its called after BcGS1 gene, the sequence of this gene is as shown in SEQIDNO:1.
The prokaryotic expression of embodiment 6BcGS1 gene and purifying
(1) structure of expression vector
The Auele Specific Primer of design albumen exciton gene BcGS1, introduce the restriction enzyme site cutting protection base with enzyme, forward primer: 5 '- gAATTCaTGGCTTCTTCTCTGCTTTCCTC-3 ', (underscore is the cleavage sequence of EcoRI), reverse primer: 5 '- gCGGCCGCtTACCAGCTACCACTAACAGTAGCAGT-3 ', (underscore is the cleavage sequence of NotI), amplification total length BcGS1 gene (94 ° of C3min; 94 ° of C30s, 55 ° of C30s, 72 ° of C2min, 35cycles; 72 ° of C10min; 4 ° of C ∝).First BcGS1 gene object fragment is cloned into pMD18-Tsimple carrier, after EcoRI and NotI enzyme is cut, BcGS1 fragment is cloned into the EcoRI/NotI site of pET-28a (+) carrier (Novagen), heat-shock transformed to intestinal bacteria Trans10 (Transgene), picking positive colony, shake bacterium and extract plasmid, enzyme is cut and sequence verification.
(2) abduction delivering
By in step (1) obtain expression vector thermal excitation and be transformed in intestinal bacteria Rossetta (DE3).Plasmid extraction and conversion express the method for host strain with (1), after checking is correct, express.By recombinant strains activated overnight correct for checking, get the bacterial strain of pET28a empty plasmid in contrast simultaneously.Get respectively in the 100mLLB liquid nutrient medium that 1mL incubated overnight liquid joins containing 100 μ g/mLKan (1% inoculum size), 37 DEG C, 200r/min shaking culture 2 ~ 3h is cultured to OD 600be 0.6 ~ 0.8.Adding inductor IPTG(final concentration is 0.1mmol/L), 22.5 DEG C, 220r/min continues shaking culture 24h, abduction delivering target protein.High speed centrifugation, collects thalline and adds damping fluid.After ultrasonication thalline, 4 DEG C of high speed centrifugations, collect supernatant.Get 20 μ L supernatant liquors, add 5 μ L5 × SDS sample-loading buffer (sex change) resuspended thalline, heat 10min in boiling water bath, the centrifugal 10min of 13000r/min, get supernatant and carry out SDS-PAGE detection, Coomassie brilliant blue R250 dyes, and observes expression.
Result: detect through SDS-PAGE, obtain the amalgamation and expression albumen (His-BcGS1) that a molecular weight is about 83KD, in the same size with predicted molecular weight.
(3) purifying of recombinant protein
Utilize explorer10 protein purification instrument carries out the purifying of recombinant protein.Select HitrapchelatingColumn post to carry out affinity chromatography, first balance affinity column with cleaning buffer solution; Get step (2) centrifugal after recombinant protein liquid 5mL sample introduction, flow velocity is 1mL/min, and after drip washing to baseline, elution buffer carries out wash-out; The each elution peak collected, 4 DEG C of dialysed overnight in the Tris-HCL damping fluid (PH7.4) of a large amount of volume, carry out the purity of SDS-PAGE electrophoresis and Coomassie brilliant blue staining examine albumen subsequently.
Biological activity determination: adopt method in embodiment 2 to measure recombinant protein exciton to the activity of tobacco.
Result: the amalgamation and expression albumen (His-BcGS1 recombinant protein) obtaining the about 83KD of purifying
The defensive raction that embodiment 7 recombinant protein (His-BcGS1) causes on tobacco
(1) HR that recombinant protein (His-BcGS1) causes on tobacco reacts
With the syringe of 1mL not with syringe needle, by about 50uL, 50 μ g/mL amalgamation and expressions albumen (His-BcGS1), inject different tobacco leafs from the leaf back side.With pET-28a (+) the empty plasmid expressing protein (damping fluid is 20MmTris-HCL, pH7.4) of same concentrations in contrast, the reaction on blade is observed after 24h.
As the method in example 3, observe recombinant protein induction H 2o 2accumulation on process blade.
Result: recombinant protein (His-BcGS1) can cause tobacco leaf to produce HR reaction; His-BcGS1 makes process blade produce H 2o 2accumulation, form reddish-brown precipitation, the symptom caused with native purified BcGS1 is basically identical.
(2) recombinant protein (His-BcGS1) improves resistance related gene transcriptional level
Adopt semiquantitive RT-PCR, tobacco resistance related gene transcriptional level is analyzed.Get required sample liquid nitrogen to pulverize, add Trizol by 50-100mg/mL, room temperature 5min leaves standstill, and the centrifugal 5min of 12000rpm, abandons precipitation; By 200uL chloroform/mLTrizol, concussion mixing 15min places, 4 DEG C, and the centrifugal 15min of 12000rpm, draws upper strata, add 0.5mL Virahol/mLTrizol, mixing ,-20 DEG C of standing 20min; Centrifugal, 75% ethanol rinse, centrifugal segregation ethanol, 5-10min dries.Add DEPC process water, survey RNA concentration, furnishing same concentrations.
The synthesis of the first chain cDNA, adopts the TransScriptTwo-StepRT-PCRKit of TransGen company.Get 500ng total serum IgE, according to test kit specification sheets, with TransScriptRT/RIEnzymeMix, with AnchoredOligo(dT) 18 be primer, hatch 30min for 42 DEG C, 85 DEG C of heating 5min make enzyme deactivation.Get 1uL reverse transcription product and be PCR, β-actin is internal standard gene, is PCR (PR1-a gene F:5 '-TGGATGCCCATAACACAGC-3 ' R:5 '-AATCGCCACTTCCCTCAG-3 ' with resistance related gene special primer; PR1-b gene F:5 '-GATGTGGGTTGATGAGAAGC-3 ' R:5 '-CTCCAATTACCAGGTGGATC-3 '; NPR1 gene F:5 '-ACATCAGCGGAAGCAGTAG-3 ' R:5 '-GTCGGCGAAGTAGTCAAAC-3 ', respectively as shown in SEQIDNO:5-10) observe the expression of resistance related gene.
Result: through the visible His-BcGS1 of Semiquatitative RT-PCR assay result after process tobacco leaf 24 hours, can the transcriptional expression of induction of resistance genes involved.
Example 8 recombinant protein (His-BcGS1) strengthens dicotyledons disease resistance
(1) induction reduces TMV to the harm of tobacco
Better effect is had to Resistance In Tobacco TMV by the known 20 μ g/mL amalgamation and expression albumen of preliminary experiment.By the syringe of His-BcGS1 (the 20 μ g/mL) solution after 50 μ L, purifying by needle-less, inject tobacco leaf from the back side.Compare with Tris-HCL damping fluid, often process 10 strains, repeat 3 times.The inoculation method of TMV is: get the sick leaf of fresh TMV and add 0.01mol/L phosphoric acid buffer (pH=7.4) and grind to form homogenate, sick juice gravity is that every gram of sick leaf adds 15mL phosphoric acid buffer.Determine best inoculation time.
Respectively at after recombinant protein process 1,3,5 and 7d, at a upper leaf frictional inoculation TMV of process leaf.In latter 3 days of inoculation, investigation withered spot number amount, calculated withered spot inhibiting rate.
Inhibiting rate (%)=[(necrotic plaque number/diameter-treatment group necrotic plaque number/diameter)/necrotic plaque number/diameter] × 100
Result is as following table.
The significance of difference of different letter representation when ρ < 0.01 level in table.
The above results shows, recombinant protein exciton His-BcGS1 can induce and reduce TMV to the harm of tobacco, and after protein induced 3d, its inducing effect is better, can reach 58.55% to the inhibiting rate of TMV.
(2) induce Arabidopis thaliana to the resistance of the pathogen of Botrytis cinerea
With the Arabidopsis thaliana Seedlings of His-BcGS1 solution (concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL) the spraying process 6-8 leaf age after purifying, compare with Tris-HCL damping fluid, often process 10 strains, repeat 3 times.Spray inoculation the pathogen of Botrytis cinerea spore suspension (10 after induction 3d 5individual/mL), moisturizing 24h, 7d carry out the investigation of disease level, calculate Induced resistant effect.
Protein concentration (μ g/mL) Disease index (%) Inducing effect (%)
0 49.59±0.52A
1 32.36±0.85B 34.74
5 30.82±1.58B 37.85
10 23.08±0.96C 53.45
20 29.45±1.12B 40.61
The significance of difference of different letter representation when ρ < 0.01 level in table.
The above results shows, recombinant protein exciton His-BcGS1 can alleviate the harm of the pathogen of Botrytis cinerea to Arabidopis thaliana, and after protein induced 3d, the protein induced effect of 10 μ g/mL is best, and Induced resistant effect can reach 53.45%.Arabidopis thaliana (Arabidopsisthaliana), has another name called mouse ear mustard, Arabic mustard, Arabian cron.This slight flowering plant is at plant science, comprises one of model animals in genetics and development of plants research.This description of test recombinant protein exciton His-BcGS1 can alleviate the harm of the pathogen of Botrytis cinerea to Arabidopis thaliana, then substantially can illustrate that recombinant protein exciton His-BcGS1 improves the resistance of most plants and all have certain effect.Continue experiment as follows.
(3) resistance of tomato raising to the pathogen of Botrytis cinerea is induced
By the tomato seedling of His-BcGS1 solution (concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL) the spraying process 6-8 leaf age after purifying, compare with Tris-HCL damping fluid, often process 10 strains, repeat 3 times.Spray inoculation the pathogen of Botrytis cinerea spore suspension (10 after induction 3d 5individual/mL), moisturizing 48h, 14d carry out the investigation of disease level, calculate Induced resistant effect.
Protein concentration (μ g/mL) Disease index (%) Inducing effect (%)
0 45.34±0.98A
1 36.85±0.79B 18.73
5 27.25±1.84C 39.90
10 16.65±1.37D 63.28
20 19.18±1.24D 57.70
The significance of difference of different letter representation when ρ < 0.01 level in table.
The above results shows, recombinant protein exciton His-BcGS1 can alleviate the harm of the pathogen of Botrytis cinerea to tomato, and after protein induced 3d, the protein induced effect of 10 μ g/mL is best, and Induced resistant effect can reach 63.28%.
(4) inducing cucumber is to the resistance of the pathogen of Botrytis cinerea
With the cucumber seedling of His-BcGS1 solution (concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL) the spraying process 6-8 leaf age after purifying, compare with Tris-HCL damping fluid, often process 10 strains, repeat 3 times.Spray inoculation the pathogen of Botrytis cinerea spore suspension (10 after induction 3d 5individual/mL), moisturizing 48h, 14d carry out the investigation of disease level, calculate Induced resistant effect.
Protein concentration (μ g/mL) Disease index (%) Inducing effect (%)
0 54.45±1.32A
1 45.64±0.97B 16.17
5 35.74±1.64C 34.36
10 22.53±1.38D 58.62
20 25.13±1.29D 53.84
The significance of difference of different letter representation when ρ < 0.01 level in table.
The above results shows, recombinant protein exciton His-BcGS1 can alleviate the harm of the pathogen of Botrytis cinerea to cucumber, and after protein induced 3d, the protein induced effect of 10 μ g/mL is best, and Induced resistant effect can reach 58.62%.
As can be seen from above-mentioned experiment, recombinant protein exciton His-BcGS1 improves the resistance of tobacco, cucumber, tomato and Arabidopis thaliana and induced defense responses all has certain effect, proves that albumen exciton BcGS1 is very effective to dicotyledons.
Above-described example is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that the common engineering technical personnel in this area make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.

Claims (4)

1. the pathogen of Botrytis cinerea secreted protein exciton BcGS1 is improving plant to the application in the resistance of tobacco TMV and the pathogen of Botrytis cinerea, and wherein, the aminoacid sequence of described the pathogen of Botrytis cinerea secreted protein exciton BcGS1 is as shown in SEQIDNO:2.
2. application according to claim 1, is characterized in that: described plant is dicotyledons.
3. application according to claim 2, is characterized in that: described dicotyledons is tobacco, tomato or cucumber.
4. application according to claim 3, is characterized in that: when for induce reduce the harm of tobacco mosaic virus (TMV) to tobacco time, the induced concentration of described BcGS1 is 20 μ g/mL; When for improving resistance to the pathogen of Botrytis cinerea of tomato, cucumber, the induced concentration of described BcGS1 is 10 μ g/mL.
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