An exocrine protein elicitor of attenuated gibellulopsis nigrescens and its application

Abstract

The invention relates to the field of plant protection and biological control, especially relates to an exocrine protein elicitor‘ of attenuated, Gibellulopsis nigrescens. The amino acid sequence is shown in SEQ ID NO: l, and the nucleotide sequence is shown in SEQ ID NO: 2. The invention discovers the application of a new type of protein elicitor PeGDl with unknown function protein, the protein elicitor PeGDl is a new type of protein elicitor, which can be used, to improve potato resistance and induce potato defense response by activating ROS burst, th accumulation, CAT, SOD and other related defense enzyme activities in potato. At the same time, it mainly activates the signaling pathway of salicylic acid in potato to resist potato Verticillium wilt, so as to realize the prevention and control of potato verticilliunl wilt, and, provide new materials for improving the disease resistance of potato and the biological control of potato Verticillium wilt. (+ Fig. 5)

Description

P206l / NL AN EXOCRINE PROTEIN ELICITOR OF ATTENUATED GIBELLULOPSIS NIGRESCENS AND ITS APPLICATION TECHNICAL FIELD The invention relates to the field of plant protection and biological control, in particular to an exocrine protein elicitor of attenuated Gibellulopsis nigrescens and its application. BACKGROUND ART Attenuated strain can inhibit the harm of pathogen to host plant, so as to achieve the purpose of green and environmentally friendly biological control, it is one of the most potential meth ods to prevent and control plant diseases. Many studies at home and abroad have shown that there may be two mechanisms for the crossprotection of attenuated strain. One is that the attenuated strain may occupy the spatial site first during the pre inoculation process and form a spatial competition with the viru lent strain, which results in a decrease in morbidity. The other is the posttranscriptional gene silencing (PTGS) of the host plant, that is, the gene of the host plant in response to the at tenuated strain is silenced after transcription, so that an invad ing pathogenic strain becomes the target of the mechanism, and the pathogenic RNA is degraded. However, selecting the suitable atten uated isolating strain from fungal community as potential antago nist usually requires a long period of identification and screen ing. In addition, using genetic engineering technique to convert the pathogenic bacteria into new nontoxic strain can easily and quickly obtain the biocontrol agents, but the edited organism, even if they do not contain foreign DNA, its safety and whether it conforms to the rules for the establishment of transgenic organism remain to be studied. The elicitor is able to induce plant disease resistance by mobilizing the disease resistance potential of the plant itself rather than directly inhibiting the pathogen like fungicides and antibiotics, so as to resist the infection of the pathogen, it is a new plant protective agent (plant vaccine) development research. In recent years, the isolation and identification of elicitor with new functions from pathogenic bacteria has become the focus of scientists at home and abroad. However, there are few reports on the isolation of protein elicitor from attenuated pathogenic bac teria. Therefore, in view of the problems of increasingly serious occurrence of land infectious diseasespotato Verticillium wilt in potato producing area, the environmental pollution and food safety problems caused by excessive use of pesticides, it is urgent to explore and screen a green, environmentally friendly new material to reduce the use of fungicides and achieve the purpose of green and environmentally friendly biological control. SUMMARY In order to solve the problems existing in the existing tech nology, the invention provides an exocrine protein elicitor of at tenuated Gibellulopsis nigrescens and its application. In order to achieve the above invention purpose, the inven tion provides the following technical scheme: The invention provides an exocrine protein elicitor PeGDl of attenuated Gibellulopsis nigrescens, and its amino acid sequence is shown in SEQ ID NO: 1. The invention also provides a coding gene of the exocrine protein elicitor PeGDl of the attenuated Gibellulopsis nigrescens. The invention also provides an exocrine protein elicitor PeGDl of attenuated Gibellulopsis nigrescens and an application of the coding gene of exocrine protein elicitor PeGDl of the attenu ated Gibellulopsis nigrescens according to claim 2 in improving plant disease resistance. Preferably, the effective concentration of PeGDl is 0.125~l.000mg / mL. Preferably, the enhancement of plant disease resistance com prises enhancement of plant resistance and / or induction of plant defense response. Preferably, the improvement of plant resistance is to enhance the resistance of plant to Verticillium dahliae pathogen, and the induction of plant defense response is to induce the reduction of plant diseases caused by verticillium dahliae pathogen. Preferably, the improvement of plant resistance is achieved by activating ROS burst, th accumulation and increasing the ac tivity of defense enzyme, comprising one or more of CAT, SOD, POD and PAL enzymes. Preferably, the improvement of plant resistance also compris ing increasing the content of SA, JA / ET and the relative expres sion quantity of its related signaling pathway genes. Compared with the existing technology, the invention has the following technical effects: The application of an exocrine protein elicitor PeGDl with unknown function is discovered in the invention, comprising the activation of ROS burst, th accumulation and the increasing of CAT, SOD, POD, PAL related defense enzyme activity in potato, the activation of salicylic acid signaling pathway in potato and the activation of jasmonic acid / ethylene signaling pathway in the po tato, the activation of jasmonic acid / ethylene signaling pathway and relative expression quality of related synthetic genes of sig nal pathway, thereby improving the resistance of potato to Verti cillium wilt, realizing the prevention and control of potato to Verticillium wilt, and accumulating new materials for the biologi cal control of potato Verticillium wilt. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly explain the technical scheme in the embodiment or existing technology of the invention, the following will briefly introduce the drawings needed to be used in the em bodiments or existing technology description. Obviously, the draw ings in the following description are only the embodiments of the invention. For ordinary technicians in this field, other drawings can be obtained according to the provided drawings without paying creative labor. EIG.l shows that the crude protein induces HR reaction of po tato leaves and Tris (20mM) is used as a control in Embodiment 1; EIG.2 is the fractionation diagram of active protein chroma tography column in Embodiment 1; FIG.3 is the SDSPAGE test diagram in Embodiment 1; wherein marker: 250kD; EIG.4 is the analysis diagram of phylogenetic tree in Embodi ment 1; FIG.5 is the PCR amplification map of the target gene of exo crine protein elicitor PeGDl in Embodiment 2; wherein M is Marker: 5000bp, 1 is PeGDl; FIG.6 is the PCR diagram of bacterial solution in Embodiment 2; wherein M is Marker: DL2000bp; number 17 is the DH5d strain number; FIG.7 is the result diagram of double enzyme digestion of pRESTCHPeGDl recombinant plasmid in Embodiment 2; among them, 1 is Marker, 2 is pRESTCHPeGDl; EIG.8 is the expression identification diagram of exocrine protein elicitor PeGDl in Embodiment 2; wherein the control 1 and 2 are the total protein of the bacteria before IPTG induction, and the strains 16 are the total protein of the bacteria after IPTG induced protein expression. FIG.9 is the purification and SDSPAGE electrophoresis dia gram in Embodiment 2; wherein A is the purification result of the recombinant protein of exocrine protein elicitor PeGDl, and B is the SDSPAGE electrophoresis map; FIG.10 is the allergic response map of potato leaves induced by exocrine protein elicitor PeGDl with different concentrations in Embodiment 3, in which BAKl is a positive control, PBS is a negative control, 1 represents that the protein concentration is l25ug / mL, 2 represents 250ug / mL, 3 represents 500ug / mL, 4 repre sents lOOOug / mL; FIG.11 is the map of potato resistance to Verticillium wilt induced by exocrine protein elicitor PeGDl in Embodiment 3. FIG.l2 is the result diagram of potato reactive oxygen spe cies burst and programmed cell death induced by exocrine protein elicitor PeGDl in Embodiment 3, wherein A is reactive oxygen spe cies burst and B is programmed cell death; FIG.l3 shows the changes of potato th and related defense enzyme activity induced by exocrine protein elicitor PeGDl in Em bodiment 3; wherein A is the content of Hxh at different time points, BE is the change of CAT, SOD, POD and PAL enzyme activity at different time points; FIG.l4 shows the changes of SA content and relative expres sion quality of related synthetic genes in potato induced by exo crine protein elicitor PeGDl in Embodiment 3, wherein A is the SA content at different time points, and B is the relative expression quality of SA related synthetic genes at different time points. FIG.15 shows the changes of JA / ET content and relative ex pression quality of related synthetic genes in potato induced by exocrine protein elicitor PeGDl in Embodiment 3, wherein A is the JA content at different time points, B is the relative expression quality of JArelated synthetic genes at different time points, C is the ET content at different time points, and D is the relative expression quality of ETrelated synthetic genes at different time points. DETAILED DESCRIPTION OF THE EMBODIMENTS The following will further explain the invention in combina tion with the drawings and embodiments, the embodiments of the in vention are only used to describe the technical scheme of the in vention, and are not limited to the invention. The test materials used in the following embodiments, without special instructions, are purchased from the conventional biochemical reagent store. The quantitative tests in the following embodiments are set up three repeated tests, and the result takes the average value. The exocrine protein elicitor PeGDl (hereinafter referred to as 'protein elicitor PeGDl') of the attenuated Gibellulopsis ni grescens is disclosed in the embodiments of the invention, its nu cleotide sequence is shown in SEQ ID NO: 2 in the sequence table, and its amino acid sequence is shown in SEQ ID NO: 1. Embodiment 1 Isolation, purification and sequence analysis of protein elicitor PeGDl. 1. Test materials. The tested strain is G.nigrescens Vnl, which is derived from the strain preservation room of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University. The tested plant is the plantlet of potato Eeiwuruita group, and it is purchased from Saifeng Potato Seed Industry Co., Ltd.in Wuchuan County, Hohhot City. 2. Isolation and identification of protein elicitor of G.nigrescens Vn1. 2.1 Preparation of fermentation broth of G.nigrescens Vn1. The marginal hyphae of G.nigrescens Vn1 cultured for 10 days are taken by using the sterilized yellow tip, and the fungus cake is inoculated in an optimized Czapek's liquid medium containing lOOOmL of sterilization, six fungus cakes are placed in each 50mL medium, and then shaken and cultured at 25°C and 180r / min for 8days. After they are centrifuged at l2,000r / min and 4°C for 15 min, the supernatant is taken for later use. 2.2 Extraction of crude protein. The fermentation broth of G.nigrescens Vn1 is filtered through a 0.22um filter and then precipitates with 80% ammonium sulfate at 4°C overnight, subsequently it is centrifuged at l2,000r / min and 4°C for 15min, and the precipitate is suspended in 20mM TrisHCl buffer (pH 7.4) and desalted at 4°C for 24h. The crude protein is obtained by the centrifugation at l3,000r / min and 4°C for 20min with a lOkD ultrafiltration centrifuge tube and then the crude protein is stored at 80°C for later use. 2.3 Detection of biological activity The potato with 7 leaves is used as the material, 50uL(lumol / L) crude protein solution is injected into the leaves from the back of the leaves, and the protein buffer is used as the control, each treatment is repeated three times. After 24hours of treatment, the formation of necrotic spots of allergic reaction is observed to determine whether the protein elicitor is active. The result is shown in FIG.1, after 24 hours, a typical HR allergic reaction is observed on the leaves, which indicates that the crude protein contains the protein type that can cause potato allergic necrosis. 3. Isolation and purification of protein elicitor of G.nigrescens Vn1. The crude protein obtained in the above step 2.2 is separated and purified by AKTApure protein purification instrument, anion chromatography column HP Q Hi TrapTM (5mL) and Superdex 200 in crease 10 / 300GL molecular sieve according to the instructions of the instruments, the specific operation is as follows: (1) After the crude protein stored at 80°C is dissolved on ice, the AKTA pure protein purification instrument is started and connected, first, the crude protein is fractionated with an anion chromatography column HP Q Hi TrapTM (5mL) and the protein under the A280 absorption peak is collected. When the flow rate of the instrument is lmL / min, the protein sampling volume is set to the volume at the peak, and the sampling volume is 100uL; (2) The chromatography column is washed with 5 times the vol ume of 20% alcohol, after reaching the equilibrium, the chromatog raphy column is balanced with 5 times the volume of TrisHCl load ing buffer with a pH of 7.4 and a concentration of 20mM, and the baseline is washed to parallel. (3) 500uL of the prepared crude protein solution is loaded through the loading well, and the target protein is eluted linear ly with the 20mM protein loading buffer TrisHCl, and the elution peaks of each protein are collected according to the display of AKTA pure. (4) After the target protein is collected, the chromatography column is recleaned with 20mM TrisHCI buffer and 20% ethanol, and the chromatography column is stored at 4°C. (5) 50uL of the collected protein is injected into the back of the potato leaves, after 24 hours, the HR reaction of the leaves is observed, and the protein that can produces HR reaction is selected for refractionation by using molecular sieve. (6) The fractionated protein that can produce HR reaction in the above step (5) is taken, and the molecular sieve Superdex 200increase 10 / 300GL is used to perform protein fractionation ac cording to the above (l)~(5) operation steps, but the flow rate is set to 0.5mL / min, protein sampling is determined according to the peak time, and the sampling volume is 100uL. (7) 50uL of protein collected in the step (6) is injected in to the back of the potato leaves again, after 24 hours, the HR re action of the leaves is observed, and the purity and size of the remaining protein are determined by SDSPAGE, after the electro phoresis, the purity of the protein is tested and the molecular weight is measured according to the Coomassie brilliant blue staining method. The result shows that there is an obvious peak in the 2030mL elution volume, and the A 280mAU value reaches 46 (see FIG.2), af ter the protein of this peak is collected, it is concentrated again with a concentrated tube and verified by SDSPAGE. According to the R250 Coomassie brilliant blue staining method, there is a band of about l7kD (see FIG.3), and the band is cut and sent to Seth Gene Technology (Qingdao) Co., Ltd.for mass spectrometry identification. 4. Mass spectrometry identification and sequence analysis of protein elicitor The amino acid sequence of the protein is obtained according to the de novo sequencing of mass spectrometry, the protein has a total of 153 amino acids. The sequence is compared with NCBI's Pblast, and the result shows that the protein elicitor has not been named and has no functional report. Compared with the align ment sequence, the sequence consistency rate is less than 60%. In this study, 33 proteins from different species are selected from the nonredundant (NR) database of NCBI Protein blast for phyloge netic tree analysis. It was found that PeG Dl had high homology with unnamed proteins from marine fungi ( Emericellopsis atlanti ). But it is only 58.39 %. It has a homologous sequence with Ver ticillium longisporum and Verticillium dahliae VDG2, respectively, but the sequence identity rate is only 53.85% (see EIG.4). There fore, the protein is identified as a new protein elicitor that can induce HR reaction of potato leaves, and it is named PeGDl (A pro tein elicitor from Gibellulopsis nigrescens), and its amino acid sequence was shown as SEQ ID NO.1. Embodiment 2 Cloning and sequencing of protein elicitor PeGDl gene. 1. Test materials. Gibellulopsis nigrescens Vn1: it is derived from the strain preservation room of the College of Horticulture and Plant Protec tion of Inner Mongolia Agricultural University. 2. Cloning and sequencing of protein elicitor PeGDl. The Gibellulopsis nigrescens Vn1 strain is grown on PDA me dium for 15 days, the hyphae is collected and the total RNA is ex tracted by Tiangen TRNzol Universal total RNA extraction kit, then, the specific primers GDlE and GDlR are designed according to the nucleotide sequence of the protein elicitor PeGD1 without signal peptide and stop codon, finally, the fulllength cDNA frag ment of the protein elicitor PeGD1 gene without signal peptide and stop codon is amplified by RTPCR. The nucleotide sequence of the protein elicitor PeGD1 is shown in SEQ ID NO. 2, the lst to 54th nucleotides at the 5' end of the sequence are signal peptide sequences, and the 460th to 462th nucleotides are stop codons, the signal peptides and stop codons need to be removed during protein expression. Primer 6.0 is used to design specific primers according to the gene sequence of protein elicitor PeGD1 without signal pep tide, the primers are as follows: GDlE (its nucleotide sequence is shown in SEQ ID NO.3): f5 ' -GCCCCCTCCCCTGCCGCCGAGGTCAA-3 ' ; GD1R (its nucleotide sequence is shown in SEQ ID NO.4): 5 ' -AGGGAGGGCCTGGACGGGGTAGC-3 ' ; The cDNA is used as the template (target sequence), and the primer pair composed of specific primers GDlF and GDlR is used for PCR amplification (target sequence is 405bp). The PCR amplification system is shown in Table 1: Name Content DNA <Hm0ng Forward primer GDI-F 1.251,11. 5 ><Q5 Reaction Bucr SpL lûlandNTPs 05pL QS HolSum High-FidelityDNA Polymerase 025% 5 ><05 High (;c Enhancer spL Supplementing Nuclcasc-Frec H_-O to 25|1L The PCR reaction procedure is as follows: predenaturation at 98°C for 30s, denaturation at 98°C for 5s, annealing at 72°C for 30s, extension at 72°C for 30s, 35 cycles, and final extension at 72°C for 2min. After the PCR reaction, the product is detected by 1% agarose hydrogel electrophoresis (see FIG.5), and the size of molecular weight is consistent with the size of target gene, which is 405bp. The PCR amplification product is recovered and connected to pMD19T according to the instruction of T cloning vector pMD19T, so that the PeGD1pMD19T recombinant plasmid is formed. Then the recombinant plasmid is transformed into E.coli DH5d strain, spread on the LB solid medium containing Amp and identified by colony PCR (see FIG.6), The positive clone strain 3 is selected and sent to Beijing Qingke Biotechnology Co., Ltd.to verify the positive clone by sequencing. Using the BLAST function of NCBI website, the de termined sequence is compared and analyzed, and the sequencing re sult is completely matched with the target sequence. Embodiment 3 Expression and purification of protein elicitor PeGDl. 1. Construction of gene engineering expression strain of pro tein elicitor PeGD1. (1) Extraction of recombinant plasmid PeGD1pMD19T. The recombinant plasmid PeGD1pMD19T is extracted according to the instruction by using the plasmid extraction kit of Generay company. (2) Primer design and target fragment amplification with dou ble enzyme digestion site: GD1BamH1F (its nucleotide sequence is shown in SEQ ID NO.5): ATATAGGAT CCGCCCCCTCCCCTGCCGCCGAG; GDlEcoRlR (its nucleotide sequence is shown in SEQ ID NO.6): GCGCA GAA TTCAGGGAGGGCCTGGAOGGGGTA; The PCR reaction system is as follows: Table 2: Name Content lpL GDI-BamHl-F 0.75pL GDI-EcoRl-R 0.75pL 2xTransTaqR HiFi PCR SuperMix ll 12.5pL AddingdngO to ZSpL The amplification conditions are as follows: predenaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing at 68°C for 30s, extension at 72°C for lmin, 40 cycles, final extension at 72°C for 10min, and preservation at 4°C. The amplified product is detected by 1% agarose hydrogel electrophoresis, and the target gene fragment with enzyme digestion site is recovered. (3) The pRESTCH vector is digested with restriction enzymes BamH I and EcoR I, and the linearized linear plasmid with enzyme digestion site is recovered by gel. (4) The purified target gene fragment with enzyme digestion site is ligated to the doubledigested vector pRSETCH by using T4 ligase. (5) 6uL ligation product is transformed into XLlBlue compe tent cell by heat shock at 42°C, and then coated on a solid LBAmp screening plate containing 60ug / mL Amp, and cultured at 37°C over night. (6) Monoclonal colony on solid LBAmp screening plate is se lected and added to 2mL LBAmp liquid medium containing lOOpg / mL Amp. After overnight culture at 37°C and 240r / min, plasmid is ex tracted by using a plasmid extraction kit. (7) Double enzyme digestion verification and seguencing. The extracted recombinant plasmid is verified by BamH1 and EcoR1 double digestion, and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The result shows that the pRSETCHPeGDl is conducted double enzyme digestion by using BamH I and EcoR I, so as to obtain a 3.lkb pRSETCH vector fragment and a 405bp tar get gene fragment (see FIG.7), the sequence is compared and ana lyzed by using the BLAST function of the NCBI website. And the se guencing result is completely matched with the target sequence af ter comparison. 2. Expression of protein elicitor PeGD1 recombinant protein. (1) The recombinant plasmid is transformed into BL21 compe tent cell. The recombinant expression plasmid PeGD1pRSETCHs trans formed into E.coli BL21 by heat shock at 42°C. (2) Expression of IPTG induced recombinant protein. 5~7 positive clones colonies are picked out and some of them are inoculated into 2mL LB medium containing lOOpg / mL ampicillin. After overnight culture at 7°C and 220rpm, lmL overnight culture is inoculated into a 50mL centrifuge tube containing lOmL LB medi um that is preheated at 37°C (containing 100ug / mL ampicillin), the culture is shaken at 37°C for 100min until the ODmo is 0.5~0.7, and lmL of the bacterial solution is left to the new EP tube and placed on ice as a preinduction control. IPTG is added (The con centration of storage solution is 0.5mol / L, and 20uL is added into 10 mL bacterial solution) until the final concentration is 1mmol / L, and then the culture is cultured at 30°C for 4h to induce protein expression. (3) SDSPAGE identification. lmL of the induced bacterial solution is taken in a new EP tube and centrifuged at l2,000rpm for 2min with the bacterial so lution before induction, the culture medium is discarded, and the bacteria is collected and lysed together. The precipitate is re suspended in 50uLPBS, fully vortexed until completely dispersed, and 5uL 10Xloading buffer (10% Bmercaptoethanol is added before use) is added, then it is washed in boiling water for 8min, and it is cooled on the ice for 5min. SDSPAGE electrophoresis: the PAGE gel with a separation gel concentration of 12% is used for gel running identification, and 100mA constant current electrophoresis is performed for 80min, the PAGE gel is stained in Coomassie brilliant blue staining solution for 2h, and decolorized in room temperature decolorization solu tion for 6h, the result is recorded on a gel imager, and the re sult shows that compared with the control group, the expression of strain 6 in the IPTG induction group is the most obvious, so the strain 6 is selected for largescale expression (see EIG.8). 3. Purification of PeGD1 recombinant protein The positive clone colony with high protein expression is se lected and a large amount of induced protein expressions are con ducted according to the above step 2 (2). The bacteria solution cultured by shaking is centrifuged at 12,000rpm and 4°C for 30min, and the precipitate is washed with PBS buffer (pH 7.4) for 3 times and then centrifuged again. The bacteria is resuspended with 0.lM PBS buffer, and the ice bath is broken for 40min (200W, broken for 4s, interval 3s) by ultrasonic cell crushing instrument, and then the bacteria is centrifuged at l2,000rpm for 20min, the superna tant is collected as the crude extract of the recombinant protein. The crude extract of the recombinant protein is purified by the method of separation and purification of the protein elicitor described in step 3 of Embodiment 1, and whether it is the target protein is identified by SDSPAGE, the result is shown in FIG.9. When the elution volume of the recombinant protein is 2030mL, there is an obvious absorption peak at A280. After recovery, it is verified by SDSPAGE to be l4kD, and the concentration of the pro tein elicitor PeGD1 recombinant protein is determined to be 1mg / mL by Brandford's method. In summary, the protein elicitor PeGD1 is conducted prokary otic expression under the induction of IPTG, the coding sequence of the protein elicitor PeGD1 obtained by using specific primer to conduct PCR amplification, which has no signal peptide and termi nator, is 405bp. The coding sequence is cloned and ligated into the T cloning vector pMDl9T. After colony PCR and sequencing ver ification, the gene is constructed into the expression vector pRSETCH to form a new vector pRSETCHPeGDl containing the pro tein elicitor PeGD1. After plasmid PCR, enzyme digestion and se guencing verification, it is transferred into E.coli BL21 for ex pression. After IPTG induction, the protein elicitor PeGD1 could be expressed at a concentration of 1mg / mL. In this embodiment, the specific enzyme digestion site is selected during the construction of the expression vector, this site is not included in the coding gene, at the same time, according to the characteristics of the gene, a protective base is added before the enzyme digestion site, which can make the gene form a correct reading frame and enzyme digestion easier. Study on the resistance of potato Verticillium wilt induced by protein elicitor PeGD1. 1. Test materials. The tested strain is V.dahliae Vd36 with the strongest tox icity. The tested plant is the plantlet of potato Eeiwuruita group, which is purchased from Saifeng Potato Seed Industry Co., Ltd.in Wuchuan County, Hohhot City. 2. Potato planting. Potato planting: a set of clean and sterile hydroponic pipe line planting system is prepared, and the pipeline is scrubbed with 75% alcohol. After the clean water is added to the nutrient solution pool, the Oktay's bactericide is also added, and the fi nal concentration is adjusted to 30ppm. The pipeline is rinsed five times, and after the water is poured out, the nutrient solu tion pool is refilled. Then the soilless culture nutrient solu tion is prepared and added to the 20L nutrient solution pool. The pH value of the nutrient solution is 6.8 and the EC value is 1.9. The formula of the nutrient solution for soilless culture is as follows: calcium nitrate 1360mg / L, potassium nitrate 1100mg / L, magnesium sulfate 500mg / L, ammonium dihydrogen phosphate 270mg / L, boric acid 3.0mg / L, manganese sulfate 1.6mg / L, zinc sulfate 0.28mg / L, copper sulfate 0.12mg / L, sodium molybdate 0.10mg / L, EDTA sodium ferric 20mg / L. The plantlets of potato group with highly consistent growth are selected and the bottle cap is opened in a sterile environment for 3 days, and then the plantlets are transplanted into a pre prepared hydroponic pipeline system (day / night temperature 26°C / 20°C, full light 14h, full dark 10h, humidity 65%). After 7 days of growth, the plantlets are transplanted into a small pot with ceramsite when the leaves of the plantlets grow to more than 10 leaves, wherein the ceramsite is sterilized in a autoclave at 121°C for 30min, and the small pot is soaked in 5% potassium per manganate for 10min. The planting process is carried out in a sterile environment. The HRinduced reaction of PeGD1 with different concentra tions on plant. The protein (concentration of 1mg / mL) obtained by prokaryotic expression in Embodiment 3 is diluted with 0.1M PBS buffer, and diluted 1 times, 2 times, 4 times and 6 times respectively, that is, the final concentration of protein solution is 1mg / mL, 0.5mg / mL, 0.25mg / mL and 0.125mg / mL respectively. Potato plantlets with 5~7 true leaves are selected, and the protein solutions dif ferent concentrations are injected into the leaves from the back of the leaves with 1mL syringe without needle. BAK1 is used as a positive control and PBS is used as a normal control. After 24 hours of injection, the HR reaction of the leaves is observed, the biological activity of the protein elicitor is determined and pho tographed under ultraviolet light. Each treatment is repeated 3 times, and each repetition is 3 plant leaves. The result shows that 50uL of purified protein elicitor PeGD1 with different concentrations (125pg / mL, 250pg / mL, 500pg / mL, 1000pg / mL) is injected into potato leaves, BAKl is positive con trol, and PBS is negative control. After 24 hours of injection, on potato leaves, with the increase of elicitor concentration, the HR reaction gradually increases, it is the weakest at the concentra tion of 125ug / mL, and it is the strongest at the concentration of 1000ug / mL (see FIG.10). 4.Determination of potato resistance to Verticillium wilt in duced by PeGD1 injection in stem. 50uL of 0.125mg / mL PeGD1 protein solution is injected into the stems of potato plantlets with 57 true leaves by 1mL disposa ble syringe with a needle. A total of 1 control group and 3 treat ment groups are set up in the experiment. The control group is in jected with 0.1M PBS buffer; treatment group 1 is injected with PeGD1 protein solution alone, treatment group 2 is inoculated with virulent strain Vd36 after 24h of injection of PeGD1 protein so lution, and treatment group 3 is inoculated with virulent strain Vd36 alone. The occurrence of potato verticillium wilt is ob served after 14 days. In vitro inoculation: the leaves of the above four treatment groups are collected and placed on a petri dish containing filter paper. The sterilized blue tip is used to beat the fungus cake from the cultured Vd36 PDA medium, and a fungus cake is placed on the back of each leaf. The inoculated petri dish is placed in the incubator, and the water is sprayed regularly to ensure the petri dish moist, and the incidence of potato leaves is observed after 7 days. According to the experimental result of step 3, 50uL of re combinant protein elicitor with a concentration of 125ug / mL is in jected into the potato from the potato stem. After 24 hours, the most virulent strain Vd36 is inoculated, and the incidence of po tato verticillium wilt is observed after 14 days. Pot experiment shows that the plants do not develop disease after injection of PeGD1 and PBS buffer alone. After 24 hours of injection of PeGD1, the plants are inoculated with the virulent strain Vd36, after 14 days, the plants almost do not develop disease, while Vd36 alone shows obvious symptoms of Verticillium wilt. In addition, the re sult of in vitro inoculation experiment is consistent with that of pot experiment (see FIG.11). 5. DAB and trypan blue staining DAB staining: DAB is dissolved in a solution containing 10mM NaQHPO4 and 0.05% Tween 20, and the final concentration of DAB is adjusted to 1mg / mL for later use. The potato leaves are immersed in DAB dye solution at 0,12,24,48h after treatment, after vacuum filtration for 20~30 min, the potato leaves are placed in 95% ethanol solution for 20min under dark condition and room temperature overnight. Then the decolorized potato leaves are scanned by scanner. Trypan blue staining: 2.5mg trypan blue is dissolved in lmL of lactophenol solution (lactic acid, glycerol, phenol, H2O mixed at a volume ratio of 1: 1: 1: 1), and a total of 100mL is prepared for later use. The potato leaves are immersed in trypan blue dye solution at 0,12,24,48h after treatment. After vacuum filtration for 25min, the potato leaves are placed in 95% ethanol solution for boiling water bath of 20min at dark condition and room temperature over night, and then the decolorized potato leaves are scanned by a scanner. The result shows that when the potato is first injected with PeGD1 in the stem for 24h, and then inoculated with Vd36 at 12h, the reactive oxygen species erupts and the programmed cell death is more serious. This indicates that PeGD1 induces the early de fense response of plantpathogen interaction (FIG.12). 6. Hydrogen peroxide accumulation and enzyme activity deter mination. The result of FIG.13 shows that the activities of HAM, CAT, SOD, POD and PAL are significantly higher than those at other time points at 12hpi after PeGD1 is injected first and then inoculated with virulent strain Vd36. After 24hpi, the activities of them begin to decrease and gradually stabilize. The control does not change, the changes of th accumulation and defense enzyme activi ty in this treatment group are consistent with the results of DAB staining and trypan blue staining in step 5. This indicates that the exocrine protein of attenuated G.nigrescens Vn1 can induce the resistance of potato to the virulent strain Vd36, and it ac tivates a series of physiological and biochemical reactions in po tato at 12hpi, such as the outbreak of reactive oxygen species and the enhancement of related defense enzyme activity. The content of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) in signaling pathway and the relative expression of related synthetic genes are determined. The SA content of potato leaves and the relative expression quality of related signaling pathway genes at different time points of PeGD1 + Vd36 in the treatment group are measured at 0,12,24 and 48hpi. As shown in FIG.14, the SA content in potato leaves of the treatment group (PeGD1 + Vd36) is higher than that of the control group at 12 and 24hpi. It reaches the highest at 12hpi and decreases gradually after 24hpi, which is consistent with the changes of ROS, be and related defense enzyme activi ties. The relative expression of PR resistance genes of SA signal ing pathway downstream is significantly upregulated at 12 and 24hpi, especially the relative expression quality of StPR1 b gene is significantly higher than that of other genes at 12,24 and 48hpi. In addition, the content of JA / ET and the relative expression quality of related signaling pathway genes in potato leaves are measured at different time points of PeGD1 + Vd36: 0,12,24 and 48hpi (see FIG.15). There is no significant difference in the con tent of JA between the treatment group PeGD1 + Vd36 and the con trol group, while the content of ET is significantly higher than that of the control at 24hpi. The relative expression quality of JA synthesis related gene PDFl.2 has a significant jump at 12hpi, while the relative expression quality of ET synthesis related genes ACS1, ACS3, ACS7, ACS9 and ER has a large jump at 24hpi. However, at 12hpi and other critical inoculation time points, the relative expression quality of JA / ETrelated genes is significant ly lower than that of SA. This indicates that the exocrine protein elicitor PeGD1 of G.nigrescens Vn1 may mainly activate the SA signaling pathway, and also weakly activate the JA / ET signaling pathway at 24hpi, thereby inducing the potato to resist the infec tion of the virulent strain Vd36. In summary, the protein elicitor PeGD1 described in this in vention has host selectivity. After 24 hours of injection of pro tein elicitor PeGD1 with different concentrations into potato leaves, only a concentration of 1000ug / mL can produce HR reaction. In potato leaves, with the increase of elicitor concentration, HR reaction also gradually increases. By measuring the content of SA, JA / ET and the relative expression quality of related synthetic genes in potato leaves inoculated with virulent strain Vd36 24 hours after injection of PeGD1 in potato stems at different time points, the result showed that PeGD1 induces systemic acquired re sistance in potato, and activates the SA signaling pathway in po tato at 12hpi, and weakly activates the JA / ET signaling pathway in potato at 24hpi, thus realizing the resistance of potato to viru lent strain Vd36. At the same time, the relative expression qual ity of the plant PR resistance gene StPR1b is significantly higher than that of the control at other time points except 0hpi. There fore, we speculates that this gene may play a major role in the process of potato resistance to pathogen. According to the above experimental results, it can be seen that when the plant is pre inoculated with the attenuated strain, the attenuated strain will secrete an exocrine protein elicitor. The protein elicitor recog nizes and interacts with the host plant, and then activates the plant's own defense response by stimulating a series of physiolog ical and biochemical reactions in the plant, when the virulent strain invades the plant, the plant can resist the invasion of the virulent strain. The above are only the preferred implementation methods of the invention. It should be pointed out that for ordinary techni cians in the technical field, some improvements and modifications can be made without breaking away from the principle of the inven tion. These improvements and modifications should also be regarded as the protection scope of the invention. 202310963314 .0 2023-08-01 Inner Mongolia Agricultural University Zhou Hongyou A weakly virulent exoprotein elicitor of Verticillium nigra and its application 6 153 AA PAT source 1..153 mol_type protein organism synthetic construct MQFSTAFLTALLSATALAAPSPAAEVKSMAASGTWTIESMKRTCDKADTPATEVRQHDSRQP HAPQTGVKCGAYTVKSPQHPASHAPQTGVKCGAYTVTSGWSGQFGAGNGFTTLSVADFAKK RIAWPAYTDKQLASGKVVTPNQSYPVQALP 462 DNA PAT source 1..462 mol_type other DNA organism synthetic construct atgcagttctccaccgccttcctcaccgccctcctctccgccaccgccctcgcggccccctcccctgccgccgaggtcaa gtccatggccgcctccggcacctggaccatcgagagcatgaagcgcacctgcgacaaggccgacacgcctgcaact gaagtccggcagcacgattcccgccagccacacgcgccccagaccggcgtcaagtgcggcgcctacaccgtcaaaa gtccgcagcaccccgccagccacgccccagacggcgtc ggccagttcggcgccggcaacggcttcacgaccctcagcgtcgcgactttgccaagaagcgcattgcctggccgcc tacaccgacaagcagctcgccagcggaaaggtcgttacgcccaaccagagccccgtccaggcctcccttaa 26 DNA PAT source 1..26 mol_type other DNA organism synthetic construct gccccctcccctgccgccgaggtcaa 23 DNA PAT source 1..23 mol_type other DNA organism synthetic construct agggagggcctggacggggtagc 32 DNA PAT source 1..32 mol_type other DNA organism synthetic construct atatagatccgccccctcccctgccgccgag 32 DNA PAT source 1..32 mol_type other DNA organism synthetic construct gcgcagaattcagggagggcctggacggggta CONCLUSIONS l. Vnl exocrine protein elicitor PeGDl from a weakly virulent black the roundworm fungus (Gibellulopsis nigrescens), with the characteristic whose amino acid sequence is shown as SEQ ID NO:l. 2. Coding gene of exocrine protein elicitor PeGDl of the weak poisonous black wheelbarrow fungus according to claim 1. 3. Application in improving disease resistance of potatoes of exocrine protein elicitor PeGDl of a weak viru spring black roundworm fungus according to claim 1 or coding gene of exocrine protein elicitor PeGDl of the weakly toxic black wheel branch fungus according to claim 2; the improvement of potato disease resistance refers to the increasing potato resistance and / or inducing potato fur defense reactions; the improvement of potato resistance is to improve the resistance of potatoes against the pathogen Verticillium dahliae improve, and the induction of potato defense response is to a reduction in the disease caused by the pathogen Inducing Verticillium dahliae on potatoes. 4. Use according to claim 3, characterized in that the effect tive concentration of exocrine protein elicitor PeGDl of a weak virulent black roundworm fungus is 0.1251,000 mg / ml. 5. Use according to claim 3, characterized in that the improved ring of potato resistance is achieved by ROS burst, th ac to activate accumulation and the activity of defense enzymes improve, including one or more CAT, SOD, POD and PAL enzymes. 6. Use according to claim 3, characterised in that the improved ring of the potato resistance further an increase in the ge stop at SA, JA / ET and the relative expression levels of their want signaling pathogens includes .