CN113105532A

CN113105532A - Aspergillus oryzae elicitor protein SGP1, short peptide and application thereof

Info

Publication number: CN113105532A
Application number: CN202110361820.3A
Authority: CN
Inventors: 宋天巧; 刘永锋
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-07-13
Anticipated expiration: 2041-04-02
Also published as: CN113105532B

Abstract

The invention discloses a ustilaginoidea virens elicitor protein SGP1, a short peptide and application thereof, wherein the elicitor protein and the elicitor short peptide can obviously activate plant defense reaction, improve the disease resistance of plants, enhance the resistance of the plants to pathogenic bacteria infection and effectively prevent or reduce the occurrence of diseases. The elicitor protein and the elicitor short peptide provide a new way for improving the disease resistance of plants, provide resources for development and application of biological pesticides in future, can serve as a novel microbial protein pesticide to prevent damage of plant diseases and insect pests, provide a basis for transgenic plants of the gene in future, and have wide application prospects in agricultural production.

Description

Aspergillus oryzae elicitor protein SGP1, short peptide and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to an ustilaginoidea virens exciton SGP1 and application thereof.

Background

At present, the main measures for preventing and controlling plant diseases are chemical prevention and breeding of disease-resistant varieties. However, the use of chemical agents often raises environmental pollution and food safety issues; the pathogenic bacteria have small species variation to bring drug resistance and overcome the problems of disease-resistant genes and the like, thus reducing the control effect; the effects of the two disease prevention and control strategies are seriously affected, and the development of environment-friendly, broad-spectrum, durable and efficient disease prevention and control strategies is needed. With the understanding of the interaction between plants and pathogens and the development of biotechnology, the plant immunity and resistance induced by pathogenic bacteria provide a basis for the development of novel disease-resistant strategies. Plant immunity includes pathogen-associated molecular patterns (PAMPs) induced immunity (PTI) secreted by pathogenic bacteria and effector-induced immunity (ETI) secreted by pathogenic bacteria. Compared with ETI, PAMPs induced immunity (PTI) is a kind of broad-spectrum immunity and is not easy to overcome by pathogenic bacteria, so that PAMPs as important factors for inducing plant resistance become an important development direction for researching and developing biological pesticides. Novel PAMPs with broad-spectrum immune function are separated and identified from pathogenic bacteria, and are expected to be developed into a novel disease-resistant strategy.

Pathogen-associated molecular patterns (PAMPs) refer to conserved molecular structures secreted by pathogenic bacteria, which are recognized by host plants to induce plant immunity. PAMPs are usually specific for pathogenic bacteria, are not present in the host cell, and are important for the adaptive survival of pathogenic bacteria. Plants are able to recognize PAMPs via Pattern Recognition Receptors (PRRs) on the membrane, activating PAMPs-induced immunity. The defense responses induced by PAMPs recognized by the plant pattern recognition receptor comprise MAPKs kinase signal pathway activation, active oxygen burst, disease course related gene expression, cell ion leakage, cell death and the like. The PAMPs-induced immunity ultimately enables the plants to acquire resistance.

However, very few PAMP molecules have been identified from fungi that are conserved among different fungi and that are short peptide stretches of conserved amino acids that induce a series of immune responses and mediate resistance. Therefore, the conservative PAMP molecules and the short amino acid peptide segments are identified in the fungi, so that a theoretical basis is provided for disclosing the interaction mechanism of plants and pathogens, and an effective protein source is provided for developing broad-spectrum, efficient and environment-friendly immune protein biological pesticides.

Disclosure of Invention

According to the principle that the protein secreted by pathogenic bacteria can be identified by plants to induce the immune response of the plants, the laboratory collects the liquid culture solution of the aspergillus oryzae, extracts the protein, sends the extracted protein to a company for mass spectrometry, and then determines the secretory component of the aspergillus oryzae through bioinformatics analysis. Proteins containing a signal peptide, conserved domains and present in pathogenic bacteria but absent from plants were identified as candidate proteins. The coding sequences of 79 candidate proteins are respectively constructed on a plant transient expression vector PVX, agrobacterium is transformed and tobacco is injected, finally, an exciton protein SGP1 capable of inducing plant defense reaction and improving plant disease resistance is identified, and the exciton protein SGP1 is found to have a conserved structural domain.

Based on the above, the first object of the present invention is to provide an aspergillus oryzae elicitor protein SGP1 and its amino acid sequence, an SGP1 gene encoding the protein and its nucleotide sequence, a vector containing an SGP1 gene and a host cell containing the vector, which can induce plant defense responses and improve plant disease resistance, and applications of the elicitor protein SGP 1.

The second purpose of the invention is to provide an Aspergillus oryzae elicitor short peptide SNP22 capable of inducing plant defense response and improving plant disease resistance and an amino acid sequence thereof, an SNP22 gene encoding the short peptide and a nucleotide sequence thereof, a vector containing an SNP22 gene, a host cell containing the vector, and application of the elicitor short peptide.

The third purpose of the invention is to provide a kind of elicitor short peptide which is homologous with the ustilaginoidea virens elicitor short peptide SNP22 and can also induce plant defense reaction and improve plant disease resistance, an amino acid sequence thereof, a gene for coding the short peptide and a nucleotide sequence thereof, a vector containing the gene, a host cell containing the vector and application of the elicitor short peptide.

The Aspergillus oryzae elicitor protein SGP1 is the protein of the following 1) or 2):

1) a protein consisting of an amino acid sequence of SEQ ID NO. 2 in the sequence list;

2) the protein which is derived from the SEQ ID NO. 2 and has the function of the elicitor protein, wherein the protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the SEQ ID NO. 2 amino acid sequence in the sequence table.

The Aspergillus oryzae elicitor protein coding gene SGP1 is a DNA molecule of the following 1) or 2) or 3) or 4):

1) a DNA molecule shown as SEQ ID NO. 1 in the sequence list;

2) a nucleotide sequence encoding the protein of claim 1;

3) a DNA molecule which hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and codes for the Aspergillus oryzae elicitor protein SGP 1;

4) the nucleotide sequence has more than 90 percent of homology with the nucleotide sequence of 1) or 2) and the coded protein has the nucleotide sequence for inducing plant defense response and improving plant disease resistance.

The exciton short peptide is the protein of the following 1) or 2):

1) a protein consisting of an amino acid sequence of SEQ ID NO. 4 in the sequence list;

2) the protein which is derived from the SEQ ID NO. 4 and has the function of the elicitor protein, wherein the protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the SEQ ID NO. 4 amino acid sequence in the sequence table.

The promoter short peptide coding gene is a DNA molecule of 1) or 2) or 3) or 4) as follows:

1) a DNA molecule shown as SEQ ID NO. 3 in the sequence list;

2) a nucleotide sequence encoding the peptide fragment of claim 3;

3) a DNA molecule which is hybridized with the DNA sequence defined in 1) or 2) under strict conditions and codes the short exciton peptide;

The present invention also includes a vector containing any of the above genes.

The vector of the present invention makes the target gene available for transformation and expression, and the expression vector is preferably pHMTc, that is, the vector is recombinant vector with target gene inserted into pHMTc.

The genetically engineered host cell of the invention comprises any of the vectors described above.

The genetically engineered host cell of the invention, wherein the recombinant vector can be expressed by escherichia coli Rosetta.

The application of the exciton protein or exciton short peptide and the coding gene thereof as the exciton protein to stimulate the plant defense reaction and anaphylactic reaction and/or stimulate the expression of plant disease resistance genes and/or as the protein pesticide to defend the invasion of plant diseases and insect pests belongs to the protection scope of the invention.

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):

2) a protein consisting of an amino acid sequence of SEQ ID NO. 4 in the sequence list;

3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of 1) or 2) and has the function of a elicitor protein and is derived from SEQ ID NO. 2 or SEQ ID NO. 4.

The elicitor proteins protect against plant disease by spraying or injecting the proteins onto plant tissues.

The invention has the advantages and beneficial effects that:

(1) the elicitor protein and the elicitor short peptide can obviously activate the immune system of plants, improve the disease resistance of the plants, strengthen the resistance of the plants to infection of pathogenic bacteria and effectively prevent or reduce the occurrence of diseases.

(2) The elicitor protein and the elicitor short peptide provide a new way for improving the disease resistance of plants, provide resources for development and application of biological pesticides in future, can serve as a novel microbial protein pesticide to prevent damage of plant diseases and insect pests, provide a basis for transgenic plants of the gene in future, and have wide application prospects in agricultural production.

Drawings

FIG. 1 is a gel electrophoresis diagram of crude extract protein of SGP1 prokaryotic expression.

FIG. 2 is a diagram of protein gel electrophoresis after SGP1 purification.

FIG. 3 is a graph showing the results of SGP 1-induced cell death and placental blue staining.

FIG. 4 is a graph showing the results of SGP1 inducing the production of Nicotiana benthamiana reactive oxygen species.

FIG. 5 is a graph showing the results of SGP1 inducing the expression of the Ben's tobacco defense gene.

FIG. 6 is a graph showing the results of SGP1 inducing resistance of B.benthamiana to P.nicotianae.

FIG. 7 is a graph showing the results of SGP1 inducing rice blast resistance.

FIG. 8 is a graph showing the result of SNP22 in inducing reactive oxygen species in Nicotiana benthamiana leaves.

FIG. 9 is a diagram showing the result of SNP22 inducing expression of genes involved in the defense of Bunsen tobacco.

FIG. 10 shows that SNP22 induces the expression of rice leaf defense genes.

FIG. 11 is a graph showing the result of SNP22 inducing resistance of B.benthamiana to P.nicotianae.

FIG. 12 is a graph showing the results of SNP22 in inducing rice resistance to Pyricularia oryzae.

FIG. 13 is a graph showing the results of SNP22 in inducing resistance of rice to P.albugineus.

FIG. 14 is a graph showing the results of the SNP22 mutant and the homologous short peptide inducing the production of active oxygen in plants.

For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.

Detailed Description

In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.

Example 1 prokaryotic expression and purification of SGP1 protein

The present example discloses a related protein elicitor SGP1, the nucleotide sequence of which is shown as SEQ ID NO. 1 in the sequence table, and the amino acid sequence of which is shown as SEQ ID NO. 2.

(1) Identification of SGP1 protein elicitor

According to the principle that the protein secreted by pathogenic bacteria can be identified by plants to induce the immune response of the plants, the laboratory collects the liquid culture solution of the aspergillus oryzae, extracts the protein, sends the extracted protein to a company for mass spectrometry, and then determines the secretory component of the aspergillus oryzae through bioinformatics analysis. Proteins containing a signal peptide, conserved domains and present in pathogenic bacteria but absent from plants were identified as candidate proteins. The coding sequences of 79 candidate proteins are respectively constructed on plant transient expression vectors PVX, agrobacterium is transformed and tobacco is injected, and finally one SGP1 capable of inducing allergic necrosis of the nicotiana benthamiana cells is used as a potential protein exciton.

(2) Prokaryotic expression vector construction

Designing a specific primer for encoding the gene of the SGP1 exciton of the aspergillus oryzae, wherein the specific primer comprises the following components in percentage by weight: 5'-ctgtattttcagggcgaattcACCGCCGACTTCGACCCC-3', as shown in SEQ ID NO:5, reverse primer: 5'-caggtcgactctagaggatccTTACAGAGCAAGAACGGCGG-3', as shown in SEQ ID NO:6, using the cDNA of Aspergillus oryzae as template to amplify the full-length SGP1 gene (94 ℃ for 5 min; 94 ℃ for 30s,58 ℃ for 30s,72 ℃ for 30s,34 cycles; 72 ℃ for 5 min). After the pHMTc vector was digested with EcoR I and BamH I, the SGP1 fragment was cloned into the pHMTc vector by recombinant cloning. The recombinant vector was transformed into E.coli strain DH5 alpha, plates containing ampicillin were plated and positive clones were screened by colony PCR. Extracting plasmid from the screened positive clone shake bacteria to obtain the recombinant vector.

As a result: and (3) obtaining the prokaryotic expression vector of SGP1 through colony PCR verification and sequencing verification.

(3) Induction of protein expression

The recombinant vector is transformed into an escherichia coli Rosetta expression strain, a plate containing ampicillin and chloramphenicol is coated, and positive clones are screened by colony PCR for induced expression. Positive clones were inoculated into 4ml of LB medium containing ampicillin and chloramphenicol, shaken at 37 ℃ overnight and then transferred to 200ml of LB medium containing both of the above-mentioned resistances and cultured to OD values of 0.6 to 0.8. After addition of 200. mu.L of 0.1M IPTG, the bacteria were shaken overnight at 28 ℃. 10ml of the suspension was centrifuged, and the supernatant and the precipitate were collected, respectively, wherein the precipitate was suspended with 600. mu.L SDS (10%), boiled to clear at 95 ℃ (10min), placed on ice until turbidity became 12000rpm, and centrifuged for 15min, and the supernatant was taken as the precipitated protein after induction. Adding 6 SDS loading buffer solution into the supernatant and the precipitated protein respectively, heating for 10min in a boiling water bath, then carrying out SDS-PAGE electrophoresis and Coomassie brilliant blue R250 staining, and observing the protein expression condition.

As a result: the SGP1 fusion protein with a molecular weight of about 68kD was present in both supernatant and pellet as determined by SDS-PAGE, as shown in FIG. 1.

(4) Recombinant protein purification

The overnight induced mycelia were centrifuged in a 50ml centrifuge tube at 8000rpm for 10min at 4 ℃ to collect mycelia. 10-20ml of lysine buffer (NaH) was used for the culture₂PO₄50mM, NaCl 300mM, Imidazole 10mM, pH 8.0), 20. mu.L of lysozyme was added and the suspension was left on ice for 30 min. After ultrasonication for 1s, 3s and 30min, centrifuging at 4 deg.C and 10000rpm for 70min, and collecting supernatant. After washing a 400. mu.L nickel column with 10ml lysis buffer by centrifugation at 3100rpm for 5min at 4 ℃ for 2 times, the above supernatant was added and incubated in a shaker at 4 ℃ for 2 h. After incubation at 4 ℃ at 3210rpm, 5min the supernatant was removed and 10ml of 20mM wash buffer (NaH) was added₂PO₄50mM, NaCl 300mM, Imidazole 20mM, pH 8.0) and centrifuged 3 times at 3100rpm and 4 ℃. 10ml of 50mM wash buffer (NaH) was added to the centrifuge tube₂PO₄50mM NaCl 300mM, Imidazole 50mM, pH 8.0), 3 washes, and then 250. mu.L of precipitation buffer (NaH) was added₂PO₄50mM, NaCl 300mM, Imidazole 250mM, pH 8.0) was eluted 10 times. Adding 10 μ L of protein solutionThe protein was purified by loading the buffer 6 × SDS, heating in a boiling water bath for 10min, performing SDS-PAGE and staining with Coomassie Brilliant blue R250. The purified protein was replaced with Tris buffer without imidazole using Millipore Amicon Ultra-15c ultrafiltration tubes for subsequent assays.

As a result: obtaining the purified single SGP1 recombinant protein. The purified SGP1 fusion protein is consistent with the predicted molecular weight and has a single band as shown in FIG. 2 by SDS-PAGE detection.

Example 2 SGP1 Induction of plant defense responses

(1) Inducing tobacco allergic reaction

SGP1 protein concentrations were adjusted to 1.5nM, 7.5nM, 15nM, 30nM, 150nM, 300nM, 1.5. mu.M. About 4 weeks of Nicotiana benthamiana was taken, and the SGP1 recombinant protein was injected into Nicotiana benthamiana leaves from the back side thereof using a 1ml syringe without a needle, and simultaneously, 1.5. mu.M MBP was used as a control, and the allergic necrosis reaction was observed 48 hours after the injection. Meanwhile, after the leaves were photographed, placenta blue staining observation was performed.

As a result: the protein with the concentration of more than 30nM can induce the phenomenon of allergic cell death visible to the naked eye on the Nicotiana benthamiana; placental blue staining stained the hypersensitive response sites blue, as shown in figure 3.

(2) Induction of ROS production in tobacco leaves

1 mu M SGP1 protein was injected into Nicotiana benthamiana leaves, mock treatment was used as a control, the treated leaves were taken after 6h and 12h, respectively, and put into DAB staining solution (1mg/ml, pH 3.8), after treatment at room temperature in the dark for 8h, the staining solution was removed, absolute ethanol was added to decolorize the leaves, and after the green color of the leaves was completely removed, the leaves were taken out and photographed.

The tobacco leaves are beaten into a plurality of lobular discs by a puncher, then the lobular discs are put into a 96-well plate containing deionized water, after standing overnight, the deionized water is changed into reaction liquid (luminol 35.4 mu g/mL; peroxosidase 10 mu g/mL) containing 1 mu M SGP1 recombinant protein, the reaction liquid is quickly put into a GLOMAX96 micropore measuring plate, and the relative generation amount of ROS is measured.

As a result: after 12h of treatment of the leaves with the SGP1 recombinant protein, a clear brown deposit appeared at the injection site, indicating that SGP1 recombinant protein induces ROS production in burley tobacco leaves, as shown in fig. 4A. It was found by luminol assay that, starting from about 20 minutes of treatment with SGP1 recombinant protein, SGP1 recombinant protein induced ROS production and peaked at about 200 minutes and continued for about 10 hours, as shown in fig. 4B.

(3) Induction of resistance-related Gene expression

After 1 mu M SGP1 recombinant protein and mock were injected into Nicotiana benthamiana leaves, samples were taken at 1d and 2d, respectively, RNA was extracted by a plant RNA extraction kit, and genomic DNA was removed to obtain high-purity RNA. First strand cDNA was synthesized by reverse transcription kit. According to the quantitative kit specification, 2 mu L of reverse transcription product is taken as a template, and then the expression levels of the Nicotiana benthamiana resistance related gene such as Lipoygene (LOX) and salicylic acid signal related genes PR1B and PR2B are measured by a real-time fluorescent quantitative PCR method by using EF-1 alpha as an internal reference gene.

The primers used were as follows:

NbEF1 a-QF: 5'-AGAGGCCCTCAGACAAAC-3', as shown in SEQ ID NO: 7;

NbEF1 a-QR: 5'-TAGGTCCAAAGGTCACAA-3', as shown in SEQ ID NO: 8;

LOX-QF: 5'-AAAACCTATGCCTCAAGAAC-3', as shown in SEQ ID NO: 9;

LOX-QR: 5'-ACTGCTGCATAGGCTTTGG-3', as shown in SEQ ID NO: 10;

PR 1B-QF: 5'-GTGGACACTATACTCAGGTG-3', as shown in SEQ ID NO: 11;

PR 1B-QR: 5'-TCCAACTTGGAATCAAAGGG-3', as shown in SEQ ID NO: 12;

PR 2B-QF: 5'-AGGTGTTTGCTATGGAATGC-3', as shown in SEQ ID NO: 13;

PR2B-QR: 5'-TCTGTACCCACCATCTTGC-3' as shown in SEQ ID NO: 14;

as a result: fluorescent quantitative PCR results show that the SGP1 protein can significantly induce the expression of Lipoxygene (LOX) and the genes PR1B and PR2B related to the salicylic acid signal after the injection of

Nicotiana benthamiana

1d and 2d, as shown in FIG. 5.

Example 3 SGP1 Induction of disease resistance in tobacco and Rice

(1) Inducing resistance of tobacco to phytophthora nicotianae

And (3) spraying the SGP1 recombinant protein and mock onto the Benzenbachia tabacum leaves for 24 hours, inoculating 100 zoospores of phytophthora nicotianae, preserving moisture, culturing for 48 hours, and counting the diameter of lesion spots.

As a result: after the tobacco leaves are treated by the SGP1 recombinant protein, the diameter of a lesion spot can be reduced by about 0.9cm, which shows that the SGP1 recombinant protein can induce the resistance of the tobacco leaves to phytophthora nicotianae, as shown in FIG. 6.

(2) Inducing resistance of rice to Magnaporthe grisea

After SGP1 recombinant protein and mock were sprayed on rice leaves for 24 hours, spore liquid of Pyricularia oryzae (5X 10)⁴spores/mL), and after carrying out moisture-preserving culture at 28 ℃ for 7d, counting the number of the five types of lesion spots. Type 1, needle size brown spot; type 2, 1.5 mm brown spots; type 3, 2-3 mm gray spots with brown edges; type 4, many oval gray patches longer than 3 mm; type 5, infection with 50% or more leaf area of combined lesions.

As a result: after the rice leaves are treated by the SGP1 recombinant protein, the incidence degree of the Magnaporthe grisea can be obviously reduced, which shows that the SGP1 recombinant protein can induce the resistance of rice to the Magnaporthe grisea, and is shown in figure 7.

EXAMPLE 4 preparation of exciton oligopeptide SNP22

(1) Discovery of exciton short peptide SNP22

According to sequence comparison, the laboratory discovers that the SGP1 sequence of the protein exciton has a conserved structural domain, the laboratory truncates the protein to obtain a short peptide SNP22, the amino acid sequence of the short peptide SNP22 is shown as a sequence table SEQ ID NO. 4, and the nucleotide sequence for coding the short peptide SNP22 is shown as a sequence table SEQ ID NO. 3.

(2) Synthesis of exciton short peptide SNP22

The exciton short peptide SNP22 is directly synthesized by Kinseri company.

Example 5 elicitor short peptide SNP22 induces a plant defense response

(1) Inducing the production of Reactive Oxygen Species (ROS) of Benzilian tobacco

Injecting 1 mu M SNP22 short peptide into Nicotiana benthamiana leaves, performing mock treatment as a control, taking the treated leaves after 6h and 12h respectively, putting the treated leaves into DAB staining solution (1mg/ml, pH 3.8), performing light-shielding treatment at room temperature for 8h, removing the staining solution, adding absolute ethyl alcohol for decoloring, taking out the leaves after the green color of the leaves is completely removed, and taking a picture.

A puncher is used for beating the Nicotiana benthamiana leaves into a plurality of small leaf discs, then the small leaf discs are placed into a 96-well plate containing deionized water, after standing overnight, the deionized water is changed into reaction liquid (luminol 35.4 mu g/mL; peroxosidase 10 mu g/mL) containing 1 mu M SNP22 short peptide, the reaction liquid is rapidly placed into a GLOMAX96 micropore measuring plate, and the relative generation amount of ROS is measured.

As a result: after 12h of SNP22 short peptide treatment of the leaves, a distinct brown deposit appeared at the injection site, indicating that SNP22 short peptide induces the generation of reactive oxygen species ROS in Nicotiana benthamiana leaves, as shown in FIG. 8A. It was found by luminol assay that, starting at about 20 minutes after SNP22 short peptide treatment, SNP22 induced ROS production immediately, peaking at about 200 minutes, and continuing for about 10 hours, as shown in fig. 8B.

(2) Induction of Byssinia PR Gene expression

After 1 mu M SNP22 short peptide and Mock were injected into Nicotiana benthamiana leaves, samples were taken for 24h and 48h, respectively, RNA was extracted by a plant RNA extraction kit, and genomic DNA was removed to obtain high purity RNA. First strand cDNA was synthesized by reverse transcription kit. According to the quantitative kit specification, 2 mu L of reverse transcription product is taken as a template, and then the expression levels of the Nicotiana benthamiana resistance related gene such as Lipoygene (LOX) and salicylic acid signal related genes PR1B and PR2B are measured by a real-time fluorescent quantitative PCR method by using EF-1 alpha as an internal reference gene. The primers used were as follows:

NbEF1 a-QF: 5'-AGAGGCCCTCAGACAAAC-3', as shown in SEQ ID NO: 7;

NbEF1 a-QR: 5'-TAGGTCCAAAGGTCACAA-3', as shown in SEQ ID NO: 8;

LOX-QF: 5'-AAAACCTATGCCTCAAGAAC-3', as shown in SEQ ID NO: 9;

LOX-QR: 5'-ACTGCTGCATAGGCTTTGG-3', as shown in SEQ ID NO: 10;

PR 1B-QF: 5'-GTGGACACTATACTCAGGTG-3', as shown in SEQ ID NO: 11;

PR 1B-QR: 5'-TCCAACTTGGAATCAAAGGG-3', as shown in SEQ ID NO: 12;

PR 2B-QF: 5'-AGGTGTTTGCTATGGAATGC-3', as shown in SEQ ID NO: 13;

PR2B-QR: 5'-TCTGTACCCACCATCTTGC-3' as shown in SEQ ID NO: 14;

as a result: fluorescent quantitative PCR results show that the SNP22 short peptide can significantly induce the expression of Lipoxygene (LOX) and the genes PR1B and PR2B related to the salicylic acid signal after the injection of

Nicotiana benthamiana

1d and 2d, as shown in FIG. 9.

(3) Induction of rice leaf PR Gene expression

After 1 mu M SNP22 short peptide and mock treatment of rice leaves, sampling is carried out for 6h, 12h, 24h and 48h respectively, RNA is extracted by a plant RNA extraction kit, and genome DNA is removed to obtain high-purity RNA. First strand cDNA was synthesized by reverse transcription kit. According to the instruction of the quantitative kit, 2 mu L of reverse transcription product is taken as a template, then the expression quantity of the rice defense related genes Os04g10010 and PR10 is measured relative to the induction multiple after mock treatment by a real-time fluorescent quantitative PCR method and utilizing Actin as an internal reference gene after SNP22 treatment. The primers used were as follows:

rice _ actin-F: 5'-CTCTCCCCCATGCTATCCTTCG-3', as shown in SEQ ID NO: 15;

rice _ actin-R: 5'-AATGAGTAACCACGCTCCGTCA-3', shown as SEQ ID NO: 16;

04g10010_ F: 5'-AAATGATTTGGGACCAGTCG-3', as shown in SEQ ID NO: 17;

04g10010_ R: 5'-GATGGAATGTCCTCGCAAAC-3', as shown in SEQ ID NO: 18;

qOsPR10-F: 5'-CCTCAGCCATGCCATTCAG-3' as shown in SEQ ID NO: 19;

qOsPR10-R: 5'-CTTGTCCACGTCCAGGAACTC-3' as shown in SEQ ID NO: 20;

as a result: fluorescent quantitative PCR results show that the rice defense gene expression can be remarkably improved after the rice leaves are treated by the SNP22, as shown in figure 10.

Example 6 elicitor short peptide SNP22 induces disease resistance in tobacco and rice

(1) Inducing resistance of tobacco to phytophthora nicotianae

SNP22 and mock are injected to the Benzenbachia tabacum leaf from the back of the leaf for 24 hours, and then 100 zoospores of phytophthora nicotianae are inoculated for moisture preservation and culture for 48 hours, and then the diameter of the lesion spots is counted.

As a result: after SNP22 treatment of Nicotiana benthamiana leaves, the lesion diameter can be reduced remarkably, which shows that SNP22 can induce resistance of Nicotiana benthamiana to Phytophthora nicotianae, as shown in FIG. 11.

(2) Inducing resistance of rice to Magnaporthe grisea

SNP22 and mock were sprayed on rice leaves for 24 hours, and then spore liquid (5X 10) of Pyricularia oryzae was inoculated⁴spores/mL), and after carrying out moisture-preserving culture at 28 ℃ for 7d, counting the number of the five types of lesion spots. Type 1, needle size brown spot; type 2, 1.5 mm brown spots; type 3, 2-3 mm gray spots with brown edges; type 4, many oval gray patches longer than 3 mm; type 5, infection with 50% or more leaf area of combined lesions.

As a result: after the SNP22 is used for treating rice leaves, the morbidity degree of the rice blast fungi can be remarkably reduced, and the SNP22 is shown to induce the resistance of the rice to the rice blast fungi, and is shown in figure 12.

(3) Induction of resistance of rice to bacterial blight

And (3) spraying SNP22 and mock on rice leaves for 24 hours, inoculating rice bacterial blight, carrying out moisture-keeping culture at 28 ℃ for 7 days, and counting the diameter of diseased spots.

As a result: after the SNP22 is used for treating rice leaves, the morbidity degree of the bacterial blight of the rice can be obviously reduced, and the SNP22 can induce the resistance of the rice to the bacterial blight of the rice is shown in a figure 13.

Example 7 SNP22 mutant and homologous short peptide induce plant immunity

(1) Elicitor short peptide SNP22 mutant inducing plant active oxygen generation

The conserved amino acid site of SNP22 was mutated to form a mutant containing a single amino acid site mutation, and its ability to induce the production of reactive oxygen species in plants was determined.

Beating the Ben tobacco leaves into pieces by a puncherDrying the small leaf disk, putting the small leaf disk into a 96-well plate containing deionized water, standing overnight, changing the deionized water into reaction liquid (luminol 35.4 mu g/mL; peroxosidase 10 mu g/mL) containing SNP22 or mutant short peptide thereof with different concentrations, quickly putting the small leaf disk into a GLOMAX96 micropore determination plate, determining the relative generation amount of ROS, and calculating EC₅₀The value is obtained.

As a result: after mutations at D23 (amino acid 23 of SGP1 is D), Y26, P28, I34, G37, Y40, T41, and V42, SNP22 still has the ability to induce the production of active oxygen in plants, although it is somewhat reduced, as shown in fig. 14.

(2) Elicitor short peptide SNP22 homologous short peptides in other species induce plant active oxygen production

By matching 22 amino acid short peptides of SNP22 with SGP1 in other fungus species (sclerotinia sclerotiorum, ergot, root rot, verticillium dahliae and rice blast), corresponding 22 amino acid short peptides in other species are determined, and the ability of the short peptides to induce the generation of ROS is determined. The ID, species and sequence of the homologous short peptide from other species are shown in the table below.

As a result: homologous amino acid short peptides corresponding to SNP22 in Sclerotinia sclerotiorum, Clavicepia ergota, Rhizopus oryzae, Verticillium verticillium, Magnaporthe grisea also induced the production of ROS, although the induction capacity was different, as shown in FIG. 14.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

SEQUENCE LISTING

<110> agricultural science and academy of Jiangsu province

<120> ustilaginoidea virens elicitor protein SGP1, short peptide and application thereof

<130> 1

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 750

<212> DNA

<213> Ustilaginoides virens

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accgtcacct ggagttctcc cgccaagtac atggatggca ccgtctccat cgagctgatt 180

ggcggcaaaa cccaaaacac ccagcagcac attgccgaca ttgcttctgg catcaagaac 240

agcgccaaca agtacacttg gaacgtggat gcttcgcttg gagccgaggc tgtctacggc 300

ctcgtcttca agctcgagag caattcctcc atcttccagt actccaaccc cttccatatc 360

agggctgccg ccaagccggc ccccagcagc gccagtcaaa ctgtcaccgt gacccagccg 420

cccggcgtca agaccatcat cctctccggc acgattccca cctccacctc cagctctgcg 480

gctcccacca gcaccatctg cacctctagc acctccagca cctccagcac ctccatctac 540

aagactgtgc agtacaacgt caccgtttgc ccgaccactc tggtcccgag cgccagccag 600

tacgtccccg tcaatgccaa cagctccact ggcatcgttt cggtttcgtc accaactgct 660

cctgctcctg ttcccactgc cgccgctgct gctattcgca tcggctccct cggcatcctt 720

ggtgtcgtcg ccgccgttct tgctctgtaa 750

<210> 2

<211> 249

<212> PRT

<213> Ustilaginoides virens

<400> 2

Met Arg Phe Ser Val Ala Ala Val Leu Ala Phe Ala Ala Ser Ala Ile

1 5 10 15

Ala Gln Thr Ala Asp Phe Asp Pro Ile Tyr Thr Pro Asn Ser Gly Glu

20 25 30

Thr Ile Asp Ala Gly Ala Pro Tyr Thr Val Thr Trp Ser Ser Pro Ala

35 40 45

Lys Tyr Met Asp Gly Thr Val Ser Ile Glu Leu Ile Gly Gly Lys Thr

50 55 60

Gln Asn Thr Gln Gln His Ile Ala Asp Ile Ala Ser Gly Ile Lys Asn

65 70 75 80

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

85 90 95

Ala Val Tyr Gly Leu Val Phe Lys Leu Glu Ser Asn Ser Ser Ile Phe

100 105 110

Gln Tyr Ser Asn Pro Phe His Ile Arg Ala Ala Ala Lys Pro Ala Pro

115 120 125

Ser Ser Ala Ser Gln Thr Val Thr Val Thr Gln Pro Pro Gly Val Lys

130 135 140

Thr Ile Ile Leu Ser Gly Thr Ile Pro Thr Ser Thr Ser Ser Ser Ala

145 150 155 160

Ala Pro Thr Ser Thr Ile Cys Thr Ser Ser Thr Ser Ser Thr Ser Ser

165 170 175

Thr Ser Ile Tyr Lys Thr Val Gln Tyr Asn Val Thr Val Cys Pro Thr

180 185 190

Thr Leu Val Pro Ser Ala Ser Gln Tyr Val Pro Val Asn Ala Asn Ser

195 200 205

Ser Thr Gly Ile Val Ser Val Ser Ser Pro Thr Ala Pro Ala Pro Val

210 215 220

Pro Thr Ala Ala Ala Ala Ala Ile Arg Ile Gly Ser Leu Gly Ile Leu

225 230 235 240

Gly Val Val Ala Ala Val Leu Ala Leu

245

<210> 3

<211> 66

<212> DNA

<213> Ustilaginoides virens

<400> 3

gaccccatct acacccccaa cagtggcgag acgattgatg ctggcgcgcc ctacaccgtc 60

acctgg 66

<210> 4

<211> 22

<212> PRT

<213> Ustilaginoides virens

<400> 4

Asp Pro Ile Tyr Thr Pro Asn Ser Gly Glu Thr Ile Asp Ala Gly Ala

1 5 10 15

Pro Tyr Thr Val Thr Trp

20

<210> 5

<211> 39

<212> DNA

<213> Artificial Synthesis

<400> 5

ctgtattttc agggcgaatt caccgccgac ttcgacccc 39

<210> 6

<211> 41

<212> DNA

<213> Artificial Synthesis

<400> 6

caggtcgact ctagaggatc cttacagagc aagaacggcg g 41

<210> 7

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 7

agaggccctc agacaaac 18

<210> 8

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 8

taggtccaaa ggtcacaa 18

<210> 9

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 9

aaaacctatg cctcaagaac 20

<210> 10

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 10

actgctgcat aggctttgg 19

<210> 11

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 11

gtggacacta tactcaggtg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 12

tccaacttgg aatcaaaggg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 13

aggtgtttgc tatggaatgc 20

<210> 14

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 14

tctgtaccca ccatcttgc 19

<210> 15

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 15

ctctccccca tgctatcctt cg 22

<210> 16

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 16

aatgagtaac cacgctccgt ca 22

<210> 17

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 17

aaatgatttg ggaccagtcg 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 18

gatggaatgt cctcgcaaac 20

<210> 19

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 19

cctcagccat gccattcag 19

<210> 20

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 20

cttgtccacg tccaggaact c 21

<210> 21

<211> 22

<212> PRT

<213> Sclerotinia sclerotiorum

<400> 21

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

1 5 10 15

Val Leu Asp Ile Thr Trp

20

<210> 22

<211> 22

<212> PRT

<213> Magnaporthe oryzae

<400> 22

His Ala Leu Val Lys Pro Val Arg Asp Glu Val Ile Pro Ala Gly Lys

1 5 10 15

Pro Tyr Thr Leu Glu Trp

20

<210> 23

<211> 22

<212> PRT

<213> Verticillium dahliae

<400> 23

Asn Pro Val Asn Lys Pro Thr Pro Asn Glu Lys Ile Pro Ala Gly Ser

1 5 10 15

Thr Tyr Lys Ile Glu Trp

20

<210> 24

<211> 22

<212> PRT

<213> Fusarium solani

<400> 24

Asp Pro Ile Phe Lys Pro Glu Ala Trp Gln Ser Val Ala Ala Gly Gln

1 5 10 15

Ser Phe Gln Ile Thr Trp

20

<210> 25

<211> 22

<212> PRT

<213> Claviceps purpurea

<400> 25

Asp Pro Ile Tyr Thr Pro Lys Lys Asp Glu Val Val Ala Ala Gly Ser

1 5 10 15

Pro Leu Val Ile Thr Trp

20

Claims

1. An Aspergillus oryzae elicitor protein SGP1, characterized by being a protein of 1) or 2) as follows:

2. A Aspergillus oryzae elicitor protein coding gene SGP1, characterized in that it is a DNA molecule of 1) or 2) or 3) or 4) as follows:

1) a DNA molecule shown as SEQ ID NO. 1 in the sequence list;

2) a nucleotide sequence encoding the protein of claim 1;

3. An elicitor short peptide, which is a protein of the following 1) or 2):

4. An exciton short peptide coding gene is a DNA molecule of the following 1) or 2) or 3) or 4):

1) a DNA molecule shown as SEQ ID NO. 3 in the sequence list;

2) a nucleotide sequence encoding the peptide fragment of claim 3;

5. A vector containing the gene of claim 2 or 4.

6. A host cell comprising the vector of claim 5.

7. A method for improving disease resistance of plants, characterized in that the following elicitor proteins are used to prevent plant diseases: the exciton protein is a protein shown in the following 1) or 2) or 3):