CN110452290B - Elicitor protein from Scopulariopsis fungus and application of coding gene thereof in biocontrol of vegetables - Google Patents

Abstract

The invention discloses an application of elicitor protein from a mycosphaerella fungus in vegetable biocontrol. The application is the application in inducing plants to generate disease resistance or improving the disease resistance of the plants. The elicitor protein is a protein having one of the following amino acid residue sequences: 1) SEQ ID of the sequence Listing

The amino acid residue sequence of (a); 2) the SEQ ID in the sequence table

The amino acid residue sequence is substituted and/or deleted and/or added by one or more amino acid residues and has the function of elicitor protein

Description

Elicitor protein from Scopulariopsis fungus and application of coding gene thereof in biocontrol of vegetables

Technical Field

The invention belongs to the field of molecular biology, and relates to an elicitor protein from a cladosporium fungus and application of a coding gene thereof in vegetable biocontrol, in particular to an effector protein from a cladosporium endophytic fungus, a coding gene thereof and application thereof.

Background

The protein elicitors are some proteins secreted by bacteria and fungi, and mainly comprise allergic protein (Harpin), Cryptogein (Cryptogein), Activator (Activator) and the like. Can stimulate plants to obtain resistance by inducing the expression of plant defense related genes and activating defense signal pathways in plants such as salicylic acid and the like, and enhance the autoimmune capacity of the plants to resist the infection of pathogenic microorganisms. Until now, many new elicitors of protein types have been discovered, but there is no report on elicitor proteins and their coding genes that can induce plants to generate disease resistance in M.virescens.

Disclosure of Invention

The laboratory separates the high-efficiency biocontrol strain from brachiaria blades, and the endophytic fungus Sarocladium brachiariae HND5 belongs to the genus Scopulariopsis. When the HND5 strain was subjected to whole genome sequencing and analyzed by bioinformatics, a gene encoding an exo-elicitor was found and identified from its genome, and the elicitor protein encoded by the gene was named SbES.

Based on the above, the invention aims to provide an elicitor protein from a fungus belonging to the genus Scopulariopsis and a coding gene thereof, and an application of the elicitor protein and the coding gene thereof in vegetable biocontrol, in particular an application of the elicitor protein and the coding gene thereof in inducing plants to generate disease resistance or improving the disease resistance of the plants, or an application in inducing the plants to generate defense response to diseases and insect pests. The elicitor protein is derived from a protein of a Sarocladium brachiariae (endophytic fungi) strain HND5, named SbES, and is 1) or 2) or 3) below:

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

3) the SEQ ID No. 1 amino acid residue sequence in the sequence table is substituted and/or lost and/or added by one or more amino acid residues, and the amino acid residue sequence has the function of stimulating the protoprotein and is composed of the amino acid sequence shown in SEQ ID No. 1: 1, or a derivative thereof.

SEQ ID No. 1 in the sequence table is composed of 387 amino acid residues.

In order to facilitate the purification of SbES encoded by SEQ ID No. 1, a tag as shown in Table 1 can be attached to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence Listing.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The SbES can be synthesized artificially, or can be obtained by synthesizing a coding gene and then performing biological expression. The gene encoding SbES can be obtained by deleting one or more amino acid residues from the DNA sequence represented by the 1 st to 1164 th nucleotides from the 5 ' end of sequence 2 in the sequence listing, and/or by performing missense mutation of one or more base pairs, and/or by connecting the coding sequence of the tag shown in table 1 above to the 5 ' end and/or 3 ' end thereof.

The sequence of the coding gene of the promoter protein (SbES) of the M.virens endophytic fungus Sarocladium brachiariae HND5 is as follows, and the cDNA of the promoter protein of the M.virens endophytic fungus Sarocladium brachiariae HND5 is the DNA molecule of 1) or 2) or 3) as follows:

1) DNA molecule shown in sequence 2 in the sequence table;

2) a DNA molecule which is hybridized with the DNA sequence defined in 1) under strict conditions and codes the elicitor protein of the endophytic fungus Sarocladium brachiariae HND5 of the Scopulariopsis;

3) the nucleotide sequence has more than 90 percent of homology with the nucleotide sequence of the sequence 2 in the sequence table, and the coded protein has the functions of improving the plant resistance and inducing the plant defense reaction activity.

The total length of the sequence 2 in the sequence table is 1164 nucleotides, which codes a protein with the length of 387 amino acids (the sequence 1 in the sequence table) and the molecular weight of about 39kD, namely, the protein is the promoter protein (SbES) of the endophytic fungus Sarocladium brachiariae HND5, 1-45 bases are SbES protein signal peptide coding sequences, 46-315 bases are propeptide coding sequences, and 316-1164 bases are secreted mature polypeptide coding sequences.

The nucleotide sequence of the genome gene of the promoter protein (SbES) of the endophytic fungus Sarocladium brachiariae HND5 of the Scopulariopsis is shown as 1), 2) or 3) as follows:

1) SEQ ID No: 3;

2) a nucleotide sequence that can hybridize with the DNA sequence of 1) under strict conditions;

3) and SEQ ID No: 3 has more than 90 percent of homology and the coded protein has the functions of improving the plant resistance and inducing the plant defense reaction activity.

The full length of the sequence 3 in the sequence table is 1375 nucleotides, the nucleotides 1-276 from the 5' end of the sequence 3 are the first exon, the nucleotide 277-341 is the first intron, the nucleotide 342-527 is the second exon, the nucleotide 528-595 is the second intron, the nucleotide 596-1102 is the third exon, the nucleotide 1103-1179 is the third intron, and the nucleotide 1180-1375 is the fourth exon, encoding the protein shown in the sequence 1 in the sequence table.

The stringent conditions can be hybridization and membrane washing at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS.

The coding gene can also be codon optimized nucleotide, and if the coding gene can be a sequence 4 in a sequence table, the sequence can express the exciton protein in pichia pastoris, and the expression quantity is increased and the expression activity is high through codon optimization.

In the application, the induced defense reaction is micro-HR reaction, the accumulation of hydrogen peroxide in leaves, the accumulation of callose in leaves and/or the expression of plant disease resistance gene.

The application of the protein or the coding gene thereof in preparing the medicament for preventing plant diseases and insect pests also belongs to the protection scope of the invention.

In the application, the plant diseases and insect pests are diseases, and the diseases and insect pests are preferably diseases caused by Corynespora cassiicola (Corynespora cassiicola).

The use of the elicitor protein and the coding gene thereof as an elicitor protein for stimulating plant defense reactions, allergic reactions and/or plant disease resistance gene expression and/or for protecting plants against the attack of pests and diseases as a protein pesticide belongs to the protection scope of the present invention, and the plants are vegetable plants, such as vegetable plants which can be infected by Corynespora spinosa (Corynespora cassicola), such as cucurbitaceae, solanaceae, cruciferae, leguminous vegetables, and the like, specifically such as cucumbers, eggplants, peppers, tomatoes, and the like, and preferably peppers.

The invention also provides a method for improving the disease resistance of plants, which uses the following elicitor proteins to control plant diseases:

the exciton protein is a protein shown in the following 1) or 2) or 3):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

3) the amino acid residue sequence described in 1) or 2) is substituted and/or deleted and/or added by one or more amino acid residues, and has the function of elicitor protein, and the protein has the sequence shown in SEQ ID No: 1-derived protein;

the plant disease is preferably a pepper disease caused by Corynespora polymorpha (Corynespora cassiicola);

the elicitor proteins are used to increase disease resistance in plants by spraying the protein onto the plant or by injection onto plant tissues.

The exciton protein can be obtained by in vitro synthesis, and the specific method is that a recombinant expression vector capable of expressing the exciton protein is transferred into yeast to obtain a transgenic recombinant bacterium for expressing the exciton protein; the yeast is preferably Pichia pastoris X-33 strain.

The recombinant expression vector for expressing the exciton protein is obtained by inserting the nucleotide fragment shown in the sequence 4 in the sequence table into a starting vector, and the starting vector is preferably pRICZA.

The elicitor protein has the activity of inducing pepper to generate resistance to corynespora leaf spot, can cause the micro-allergic necrosis reaction of pepper leaves, stimulates the active oxygen outbreak and callose accumulation of the pepper leaves, and can stimulate the function of disease resistance gene overexpression in the pepper, thereby being the elicitor protein with the activities of improving plant resistance and inducing plant defense reaction. It can induce plant defense reaction, and can activate cascade signal system in plant body by combining with receptor on plant cell surface or subcellular surface and induce defense gene expression to play a role in disease-resistant signal path.

Drawings

FIG. 1 is an SDS-PAGE protein gel image of His-SbES fusion proteins in example 2.

FIG. 2 is a graph showing the results of the activity of His-SbES fusion protein in inducing Corydaria capsicum to produce anti-Corydaria leaf spot in example 3.

FIG. 3 is the micro-HR reaction of His-SbES fusion protein on pepper leaves in example 4.

FIG. 4 is a graph showing that His-SbES fusion protein in example 4 induces reactive oxygen species accumulation on pepper leaves.

FIG. 5 is a graph showing that His-SbES fusion proteins induced callose accumulation on pepper leaves in example 4.

FIG. 6 shows the relative expression levels of the resistance-associated genes after spraying of His-SbES fusion proteins to leaves of Capsicum annuum in example 5.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 cloning of the Gene encoding the elicitor protein SbES and its cDNA

A high-efficiency biocontrol strain separated from Brachiaria brachycarpa leaves in the laboratory is identified as the endophytic fungus Sarocladium brachiariae in the genus Scopulariopsis and named Sarocladium brachiariae HND5(Liu X, Guo Z, Huang G: Sarocladium brachiariae sp. nov., an endophytic fungus isolated from Brachyraria Brachiaria brachycantha Mycosphere 2017,8:827 834.). The inventor of the invention finds and identifies the obtained elicitor protein gene which can possibly improve the plant resistance and induce the plant defense reaction activity by comparing and analyzing the elicitor sequence reported by the relatives fungi on the basis of the whole genome sequencing data of the strain.

The method for obtaining the full-length coding DNA and the full-length cDNA fragment of the exciton protein gene is as follows:

taking total DNA extracted from Sarocladium brachiariae HND5 strain as a template, designing specific primers SbES-F and SbES-R, and amplifying by utilizing a PCR technology to obtain a full-length coding DNA fragment of an SbES gene; total RNA extracted from Sarocladium brachiariae HND5 strain is used as template, specific primers SbES-F and SbES-R are designed, and RT-PCR technology is used to amplify to obtain the full-length cDNA segment of SbES gene. The primer sequences are as follows:

SbES-F：5′-ATGCGTTTCTCACTCGTCCT-3′；

SbES-R：5′-TTAACCAGAGGGGTTGCCGT-3′。

the Sarocladium brachiariae HND5 strain was grown on PDA medium for 3 days, and the genomic DNA was extracted after collecting the mycelia. PCR amplification was performed using genomic DNA as a template and a primer set consisting of SbES-F and SbES-R (target sequence: about 1375 bp). The PCR amplification system is as follows: 2.5 μ L of 10 XPCR buffer (Mg)²⁺free)，1.8μLMgCl₂(25mM), 0.5. mu.L of dNTPs (10mM), 0.2. mu.L of LTaq enzyme (5U/. mu.L), 1.0. mu.L of template genomic DNA, forward primer and reverse primer (10 m)M) 0.5. mu.L each, ddH₂O make up to 25. mu.L. The reaction procedure was as follows: after pre-denaturation at 94 ℃ for 5min, 32 cycles of 94 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 50s, and finally extension at 72 ℃ for 10 min. And recovering the PCR amplification product, connecting with a pMD18-T vector (TaKaRa), converting escherichia coli DH5 alpha, carrying out enzyme digestion identification on the plasmid, correctly cloning to Beijing Hua Dageney technology Limited company for sequencing, wherein the sequencing result is shown as a sequence 3 in the sequence table, and transcribing to generate a cDNA amplification fragment shown as a sequence 2 in the sequence table (both the sequence 2 and the sequence 3 can be obtained through artificial synthesis).

A7-day Sarocladium brachiariae HND5 strain was grown on PDA medium, and total RNA was extracted after collection of mycelia and reverse transcribed to give cDNA. PCR amplification was performed using a primer pair consisting of SbES-F and SbES-R (target sequence: about 1164bp) using cDNA as a template. The PCR amplification system is as follows: 2.5 μ L of 10 XPCR buffer (Mg)²⁺free)，1.8μLMgCl₂(25mM), 0.5. mu.L of dNTPs (10mM), 0.2. mu.L of LTaq enzyme (5U/. mu.L), 1.0. mu.L of template cDNA, 0.5. mu.L each of forward and reverse primers (10mM), and ddH2O to make up 25. mu.L. The reaction procedure was as follows: after pre-denaturation at 94 ℃ for 5min, the mixture is subjected to 32 cycles of 94 ℃ for 30s, 57 ℃ for 30s and 72 ℃ for 30s, and finally extended at 72 ℃ for 10 min. And recovering the PCR amplification product, connecting with a pMD18-T vector (TaKaRa), converting escherichia coli DH5 alpha, carrying out enzyme digestion identification on the plasmid, correctly cloning to Beijing Hua Dageney technology Limited company for sequencing, wherein the sequencing result is shown as a sequence 2 in the sequence table, and the sequencing result is the protein shown as a sequence 1 in the coding sequence table. The recombinant plasmid sequenced to show that the foreign insert was SEQ ID No. 2 was designated pMD 18-T/SbES.

The protein shown in sequence 1 of the sequence listing was designated as SbES protein. The gene encoding the SbES protein was designated as SbES gene, and its open reading frame was shown in sequence 2 of the sequence listing.

The full length of the sequence 3 in the sequence table is 1375 nucleotides, the nucleotides 1-276 from the 5' end of the sequence 3 are the first exon, the nucleotide 277-341 is the first intron, the nucleotide 342-527 is the second exon, the nucleotide 528-595 is the second intron, the nucleotide 596-1102 is the third exon, the nucleotide 1103-1179 is the third intron, and the nucleotide 1180-1375 is the fourth exon, encoding the protein shown in the sequence 1 in the sequence table.

Example 2 eukaryotic expression and purification of SbES proteins

cDNA sequence optimization based on pichia pastoris codon preference

In order to use Pichia pastoris X-33 strain to carry out heterologous expression of SbES protein, according to the codon preference of Pichia pastoris, a signal peptide and a propeptide coding gene of the SbES protein are excluded, and the nucleotide at position 316-1164 of the sequence 2 is subjected to codon optimization to obtain a sequence 4 which is coded by the protein shown by amino acid 106-387 at the amino terminal of the sequence 1 in the sequence list. Sequence 4 was synthesized by Beijing Hua big Biotechnology, Inc.

Second, eukaryotic expression vector construction

The primers SbES-PF and SbES-PR are used for matching, the sequence 4 is used as a template, and the amplified fragment is connected to a T vector and is named as pMD 18-T/SbESp; pMD18-T/SbESp was digested simultaneously with EcoRI and KpnI, and the fragments recovered were inserted into the same sites of pRICZA to obtain a recombinant vector designated pRICZA/SbES. Colony PCR and enzyme cutting identification results show that the expression vector pRICZA/SbES is successfully constructed. The protein expressed by the expression vector pRICZA/SbES is a protein (which carries a His label and is convenient to purify) consisting of an amino acid sequence at the 106 th and 387 th positions of SEQ ID No. 1 in a sequence table. The primer sequences used were as follows:

SbES-PF：5′-TCATGGCTTACACTACTCAATCTGC-3′；

SbES-PR：5′-GTTGAAAGCCAATCTGTCAGCAGTA-3′。

third, construction of eukaryotic expression strain of SbES protein

Extraction of pRICZA/SbES plasmid Using SacI for a single cleavage, completely linearizing the recombinant expression vector, and recovering the linearized plasmid fragment. Adding 10 mu g of linearized pRICZA/SbES plasmid into 80 mu L of pichia pastoris X-33 shock competent cells, uniformly mixing, adding into a 0.2cm electric transformation cup with 0 ℃ (placed on ice in advance), incubating on ice for 5min, placing the electric transformation tank on a Bio-Rad Pulser electric transformation instrument for electric transformation, and shocking by adopting transformation parameters set by pichia pastoris stored in the electric transformation instrument. Immediately after the electric shock, 1mL of 1M sorbital at 0 ℃ was added to the electroinversion tank, the mixture in the electroinversion tank was transferred to a sterilized 1.5mL centrifuge tube after rapid mixing with a pipette, and was incubated at 30 ℃ for about 1.5 hours. And (3) coating 200 mu L of culture solution on a YPDS plate containing 100 mu g/mL Zeocin, and after the bacterial solution is completely dried, inversely culturing the plate at 28 ℃ for 4-6 days to show that bacterial colonies grow out. And (5) obtaining the Pichia pastoris expression strain of the SbES protein after verification.

Fourthly, eukaryotic expression and protein purification of SbES protein

The Pichia pastoris expression strain of SbES protein was inoculated into a conical flask containing 50mL of BMGY medium and shake-cultured at 28 ℃ and 230r/min for about 20h (OD600 ═ 3.5 or so); collecting the shake-cultured bacterial liquid by using a 50mL sterile centrifuge tube, centrifuging for 5min at 25 ℃ under 2000g, removing supernatant, transferring the thallus into a conical flask containing 100mL BMMY culture medium, sealing the conical flask by using four layers of sterile gauze, performing shake culture at 28 ℃ under 230r/min, and performing induced expression; adding 100% methanol into the culture every 24h to make the final concentration 1%, and inducing for 120 h; 1L of the fermentation broth was collected, and the cells were resuspended in 100mL of PBS Buffer (pH 6.5), and 10. mu.L of DNase was added; the thallus is crushed by a Xinzhi ultrasonic cell crusher on ice water mixture, and the procedure is as follows: 150W, crushing for 5sec, pausing for 5sec, taking 30min totally, evenly mixing again at the interval of 10min in the whole process, after the crushing is finished, 12000r/min, centrifuging at 4 ℃ for 30min, and obtaining supernatant which is the crude extract of the recombinant protein.

Purifying the recombinant protein by using a HisTrap affinity column of GE Healthcare; after 10mL of 20mM imidazole is used for balancing the affinity column, the crude recombinant protein extract is subjected to column passing according to the flow rate of 2 mL/min; the affinity column was eluted sequentially with 40mL PBS Buffer containing 20mM, 40mM, 60mM, 80mM imidazole; eluting the affinity column by using 10mL PBS Buffer with the concentration of 200mM imidazole, collecting eluent, and detecting the protein quality by using SDS-PAGE electrophoresis; the recombinant protein solution was concentrated and replaced with PBS buffer without imidazole using a 10kDa Millipore Amicon Ultra-15 ultrafiltration tube.

The results are shown in FIG. 1, and a His-SbES fusion expression protein with a molecular weight of about 32kD was obtained by SDS-PAGE, in accordance with the predicted size. Moreover, His-SbES fusion protein is enriched in the supernatant, and the purified protein band is single and has no miscellaneous band. In figure 1, note that: m is Marker, lane 1 is heteroprotein washed with 20mM imidazole; lane 2 is heteroprotein washed with 40mM imidazole; lane 3 is heteroprotein washed with 60mM imidazole; lane 4 is heteroprotein washed with 80mM imidazole; lane 5 is the protein of interest washed with 200mM imidazole.

Example 3 His-SbES fusion protein induces resistance of Capsicum annuum to Correa leaf spot

Adjusting the concentration of the recombinant protein His-SbES to 0.1mg/mL, spraying 8 pepper seedlings with 4-5 main leaf stages twice at an interval of 3d, and selecting PBS buffer solution as a control. After 3 days, the suspension of spores of Corynespora cassiicola (isolated by a conventional method and identified as being strongly pathogenic to Capsicum) was inoculated at a concentration of 1X 10⁵Each cell/mL was examined for the onset of disease after 10 days at 30 ℃ and 90% relative humidity, and the experiment was repeated 3 times.

The experimental results are shown in FIG. 2, and the results show that CK is treated only by PBS buffer solution, and the fallen leaves are obvious and the number of scabs on the leaves is large after the spores of the polyspora spinosa are inoculated; after treatment with 0.1mg/mL His-SbES recombinant protein, pepper seedlings were substantially free of disease and had few lesions. The result shows that the His-SbES recombinant protein can effectively induce the pepper to generate resistance to the corynespora leaf spot.

Example 4 defense response of His-SbES fusion proteins on Capsicum

micro-HR reaction caused by His-SbES fusion protein on pepper

The micro-HR means that a small amount of plant cells die caused by an inducer, and the plant can limit invading pathogenic bacteria in a single cell through the micro-HR, so that the effect of preventing diseases is achieved, and the micro-HR is an index of plant disease resistance reaction. Trypan blue can stain dead cells but cannot stain live cells, and micro-HR can be detected by this principle.

50 μ L of purified SbES protein at a concentration of 0.1mg/mL was injected into pepper leaves using a 1mL needle-free syringe, protein Buffer PBS Buffer as a control. Taking the pepper leaves processed for 24h, and cutting with scissors. The pepper leaves are placed in trypan blue solution, and the trypan blue solution contains 15mg of trypan blue, 10mL of lactic acid (85%), 10mL of glycerin (98%), 10mL of water-saturated phenol and 10mL of ddH2O 10. Vacuumizing by a freeze dryer for about 5-10 min, completely introducing the solution into leaf tissues and cell gaps, and heating in a boiling water bath for 5min, wherein the leaves do not float on the surface. Placing the dyed pepper leaves in absolute ethyl alcohol, decoloring in a boiling water bath until the pepper leaves are completely transparent, repeating the treatment for 3 times, and observing and photographing under a Nikon microscope.

As a result, as shown in FIG. 3, the His-SbES fusion protein was able to induce micro-HR reaction in the leaves of Capsicum annuum. In FIG. 3, A is 4 XObjective control treatment, B is 20 XObjective control treatment, C is 40 XObjective control treatment, D is 4 XObjective SbES protein treatment, E is 20 XObjective SbES protein treatment, and F is 40 XObjective SbES protein treatment.

Secondly, His-SbES fusion protein induces accumulation of hydrogen peroxide in pepper leaves

The accumulation of hydrogen peroxide can inhibit the growth of invading pathogenic bacteria and induce the transduction of disease-resistant related signal path. Injecting 50 mu L of purified SbES protein with the concentration of 0.1mg/mL into pepper leaves, treating the pepper leaves for 24 hours, cleaning the treated leaves with clear water, placing the cleaned leaves into a 10mL centrifuge tube, adding 6mL of DAB dye (1mg/mL, pH 5.8) into the centrifuge tube, dyeing overnight at room temperature in a dark condition, removing dye liquor in each tube, adding 100% ethanol to remove chlorophyll, carrying out boiling water bath for several minutes, sucking the liquid in each tube by a pipette gun, adding absolute ethanol, carrying out boiling water bath until the leaves are completely green, sucking the liquid in each tube again, soaking the leaves in 70% glycerol, carefully removing intercellular bubbles by using tweezers, and observing by using a microscope. Protein Buffer PBS Buffer was used as a control, and each treatment was repeated 3 times.

A is 4X underobjective control treatment, B is 10X underobjective control treatment, C is 20X underobjective control treatment, D is 40X underobjective control treatment, E, I is 4X underobjective SbES protein treatment, F, J are 10X underobjective SbES protein treatment, G, K are 20X underobjective SbES protein treatment, H, L are 40X underobjective SbES protein treatment.

Third, His-SbES fusion protein induces accumulation of callose of pepper leaves

The accumulation of callose can strengthen the structural strength of plant cells and prevent the invasion of pathogenic bacteria.

50 μ L of purified SbES protein at a concentration of 0.1mg/mL was injected into pepper leaves, and the pepper leaves treated for 24h were taken and cut with scissors. Placing the leaves in Carnot solution (ethanol: glacial acetic acid ═ 3:1), vacuumizing for 10min, fixing overnight, soaking in 50% ethanol for 2h before dyeing to soften the leaf tissue, and washing with clear water. The leaves were stained overnight in 0.1% aniline blue staining solution (0.1g aniline blue in 100mL 0.15mol/L K2HPO4 Buffer) at pH 9.5, washed with clear water, and examined under a Nikon fluorescence microscope for callose accumulation, using protein Buffer PBS Buffer as a control, and each treatment was repeated 3 times.

As a result, as shown in FIG. 5, blue-white fluorescent substance accumulation was observed in protein SbES-treated leaves, whereas no blue-white fluorescent substance accumulation was observed in control pepper leaves treated with PBS Buffer protein Buffer, indicating that His-SbES fusion protein was able to induce accumulation of callose in pepper leaves. IV, His-SbES fusion protein for improving transcription level of pepper resistance related gene

After the purified recombinant SbES induced resistance protein with the concentration of 0.1mg/mL is sprayed on the pepper leaves, the pepper leaves are respectively taken at 0h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h, and the pepper leaves are sprayed by PBS Buffer in a contrast way. And (5) quickly freezing by using liquid nitrogen. RNA extraction is carried out by using an RNAprep Pure polyphenol polysaccharide plant total RNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing), synthesis of a reverse transcription cDNA first chain is carried out by using a FastQuant cDNA kit (Tiangen Biochemical technology Co., Ltd., Beijing), and relative quantitative real-time fluorescence PCR detection of gene expression quantity is carried out by using a SuperReal PreMix Plus (SYBR Green) kit (Tiangen Biochemical technology Co., Ltd., Beijing).

CaEIN2-F/CaEIN2-R, CaMYB306-F/CaMYB306-R, CaNPR1-F/CaNPR1-R, CaPAL-F/CaPAL-R, CaRBOH-A-F/CaRBOH-A-R, CaRBOH-C-F/CaRBOH-C-R, CaSAMES-F/CaSAMES-R, CaPDF1.2-F/CaPDF1.2-R, CaChia5-F/CaChia5-R and CaPR1-F/CaPR1-R are used for detecting the pairing of CaEIN2, CaMYB306, CaNPR1, CaPAL, CaRBOH-A, CaRBOH-C, CaSAMES, CaPDF1.2, CaChia5 and CaPR1 genes in sequence, and the CaEF-1 alpha gene is used as an internal reference gene. The primer sequences are shown in Table 2.

TABLE 2 primers used in this study

As shown in FIG. 6, the expression of the relevant resistance-associated genes was activated by treating pepper leaves with His-SbES fusion protein.

Sequence listing

<110> institute for environmental and plant protection of tropical agricultural academy of sciences in China

<120> elicitor protein from Scopulariopsis fungus and application of coding gene thereof in biocontrol of vegetables

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atcgtgaagc tcgctgccta caccacccag tcggccgccc catggggcct tgcccgtatc 360

tctactcgtc agcgtggtcc tactggatat acgtacgaca ccagcgctgg tcaaggaaca 420

tgttcctaca tcctcgacac tggcattcag gccagccacc ccttcggtgg acgagccttc 480

cagctcatct cctaccaagg cggcaatgcc gatggtaacg gtcatggcac tcacgttgcc 540

ggtaccattg gctccagaac ctacggtgtt gccaaggcta ccaccctgct cggtgtcaag 600

gtcctgagtg actcgggctc tggctccatc tctggcatca tctccggcat gaactatgtt 660

gtcagcgact ctcgcacccg tagctgccct cgcggtgcat tcgccaacat gtctctcggt 720

ggaggctact ccgcctcgct caacagcgct gccaagtcca tggtagacaa tggcgtcttc 780

cttgctgttg ctgccggtaa cgagaaccag aatgctgcca acgtatctcc cgcgtctgag 840

cccagcgtct gcacggtcgg cgccaccact tccactgatg cccgtgcttc cttctccaac 900

tacggcgctc ttgtcgatat cttcgctcct ggccagggta ttctgtctac ctggcccggc 960

agcagcacca acaccatctc tggcacctcc atggcctccc ctcatattgc tggtcttgcc 1020

gcttacctgg ctggccttga gggtaaccct ggtgcctcgg ccatgtgcgg acgtatcatc 1080

cagcttgcca ccactggtgt catcactggc ctgcccagcg gtaccgccga ccgcctcgcc 1140

ttcaacggca acccctctgg ttaa 1164

<210> 3

<211> 1375

<212> DNA

<213> Gliocladium (Sarocladium brachiariae)

<400> 3

atgcgtctct ctctcgtcct cgcccttctc cctgccgccc tcggtgctcc cacgaggcgc 60

gatgagcccg ctccccttca tgttcctcgt ggcgtcgaca gcctgatcaa ggacacctac 120

atcgtcaagt acaaggacat tactgccatg tctgctgtcg atgaaggcct caaggttctt 180

cccggcaagc ccgagcgtgt cttcaaaggt gccttcaagg gctttgctgg caagattgat 240

gccaagactc tggagctcct ccgtgatgat cccagtgtaa gtggtctcgt cccgtggtca 300

aagtgtagat aggataatgt ccctgacacg ttccatcaca ggtcgacttc attgagcagg 360

atgctatcgt gaagctcgct gcctacacca cccagtcggc cgccccatgg ggccttgccc 420

gtatctctac tcgtcagcgt ggtcctactg gatatacgta cgacaccagc gctggtcaag 480

gaacatgttc ctacatcctc gacactggca ttcaggccag ccaccccgta agcgctgcac 540

attgctccct cgcccatttc cctcggccca tactgacctg accttcatca tatagttcgg 600

tggacgagcc ttccagctca tctcctacca aggcggcaat gccgatggta acggtcatgg 660

cactcacgtt gccggtacca ttggctccag aacctacggt gttgccaagg ctaccaccct 720

gctcggtgtc aaggtcctga gtgactcggg ctctggctcc atctctggca tcatctccgg 780

catgaactat gttgtcagcg actctcgcac ccgtagctgc cctcgcggtg cattcgccaa 840

catgtctctc ggtggaggct actccgcctc gctcaacagc gctgccaagt ccatggtaga 900

caatggcgtc ttccttgctg ttgctgccgg taacgagaac cagaatgctg ccaacgtatc 960

tcccgcgtct gagcccagcg tctgcacggt cggcgccacc acttccactg atgcccgtgc 1020

ttccttctcc aactacggcg ctcttgtcga tatcttcgct cctggccagg gtattctgtc 1080

tacctggccc ggcagcagca ccgtgagttc acctccgaaa tcactgcaga ctcttcagag 1140

tttgcctgta tgcttgcttg tatactgaca tctcccccag aacaccatct ctggcacctc 1200

catggcctcc cctcatattg ctggtcttgc cgcttacctg gctggccttg agggtaaccc 1260

tggtgcctcg gccatgtgcg gacgtatcat ccagcttgcc accactggtg tcatcactgg 1320

cctgcccagc ggtaccgcca accgcctcgc cttcaacggc aacccctctg gttaa 1375

<210> 4

<211> 861

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gaattcatgg cttacactac tcaatctgct gctccatggg gtcttgctag aatttccact 60

agacagagag gtccaaccgg ttacacttac gatacttccg ctggtcaagg tacttgctcc 120

tacattttgg acactggtat ccaggcttct cacccatttg gtggtagagc tttccagctg 180

atttcctacc aaggtggtaa cgctgatggt aacggtcatg gtactcatgt tgctggtact 240

atcggttcca gaacttacgg tgttgctaag gccactacct tgttgggtgt taaggttttg 300

tctgactctg gttccggttc catctccggt attatttccg gtatgaacta cgtcgtgtcc 360

gactccagaa ctagatcttg tccaagaggt gctttcgcca acatgtctct tggtggtggt 420

tactctgctt ccttgaactc tgctgctaag tccatggttg acaacggtgt tttcttggct 480

gttgctgccg gtaacgagaa tcaaaacgct gctaacgttt ctccagcttc tgaaccatcc 540

gtctgtactg ttggtgctac tacttctact gacgctagag cttccttctc caactacggt 600

gctttggttg acattttcgc tccaggtcag ggtattttgt ctacttggcc aggttcctcc 660

actaacacca tttctggtac ttctatggct tccccacaca ttgctggttt ggctgcttat 720

ttggctggat tggaaggtaa cccaggtgct tctgctatgt gcggtagaat tatccagttg 780

gctaccaccg gtgtcatcac tggtttgcca tctggtactg ctgacagatt ggctttcaac 840

ggtaacccat ctggtggtac c 861

Claims (6)

1. An application of elicitor protein from cladosporium endophyticus or a coding gene thereof in improving disease resistance of pepper, wherein the elicitor protein from the cladosporium endophyticus is the protein shown in the following 1) or 2):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table; the disease resistance of the pepper is improved by improving the pepper to corynespora spinosa (Corynespora cassiicola) And (4) causing diseases.

2. An application of elicitor protein from cladosporium endophytic or coding gene thereof in inducing the pepper to generate defense reaction to diseases; the protein is a protein shown in the following 1) or 2):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in sequence table 1

The induced defense reaction is micro-HR reaction, the accumulation of hydrogen peroxide in leaves, the accumulation of callose in leaves and/or the expression of plant disease resistance gene; the disease is the pathogen of corynespora polystachya (A)Corynespora cassiicola) And (4) causing diseases.

3. An application of elicitor protein from cladosporium endophytic or its coding gene in preparing medicine for preventing pepper diseases and insect pests;

the protein is a protein shown in the following 1) or 2):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table 1;

the plant diseases and insect pests are diseases, and the diseases and insect pests are diseases caused by corynespora polystachya (A)Corynespora cassiicola) And (4) causing diseases.

4. Use according to any one of claims 1 to 3, characterized in that: the cDNA nucleotide sequence of the coding gene is shown as the following 1), 2) or 3):

1) SEQ ID No: 2;

2) SEQ ID No: 2 at the 5' end of the sequence at positions 316-1164;

3) SEQ ID No: 4.

5. Use according to any one of claims 1 to 3, characterized in that: the nucleotide sequence of the genome gene of the coding gene is shown as SEQ ID No: 3 is shown in the nucleotide sequence.

6. A method for improving disease resistance of plants, which uses the following proteins to control plant diseases:

the protein is a protein shown in the following 1) or 2):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

the plant disease is a pepper disease caused by Corynespora polymorpha (Corynespora cassiicola);

the way of using the protein to control plant diseases is to spray the protein on plants or to inject the protein on plant tissues;

the plant is capsicum.

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