Application of short peptide MP41 and protein MLC1 of Laodelphax striatellus elicitor in improving insect resistance of plants and insect resistance methods

Abstract

The invention provides an application of a short peptide MP41 and a protein MLC1 of a gray leaf hopper elicitor in improving the insect resistance of a plant and an insect resistance method, and belongs to the field of bioengineering. The gray leaf hopper elicitor protein MLC1 and the short peptide MP41 can obviously activate the plant immune system, provide the insect resistance of the plant, and effectively prevent or reduce the occurrence of insect pests; they provide a new way to improve the insect resistance of the plant, and provide resources for the development and application of biological pesticides in the future. They can be used as a new type of protein pesticide to defend against the invasion of insect pests, and also provide a basis for the future transgenic plants of the gene, and have broad application prospects in agricultural production.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to an application of a small brown planthopper elicitor short peptide MP41 and a protein MLC1 in improving the insect resistance of plants and an insect resistance method. Background Art

[0002] At present, the main measures for plant pest control are chemical control and breeding of insect-resistant varieties. However, the use of chemical agents often brings environmental pollution and food safety problems; insect gene mutations bring about resistance and overcome insect-resistant genes and other problems reduce the control effect; the effects of the two pest control strategies are seriously affected, and environmentally friendly, broad-spectrum, durable and efficient pest control strategies need to be developed. With people's understanding of the interaction between plants and herbivorous insect effectors and the development of biotechnology, the use of plant immunity and resistance induced by herbivorous insect effectors provides a basis for the development of new insect resistance strategies. Plant immunity includes immunity induced by pathogen-associated molecular patterns (PAMPs) / HAMPS (herbivore-associated molecular patterns) secreted by pathogens / herbivorous insects (PAMP / HAMP triggered immunity, P / HTI) and immunity induced by effectors (effectors) secreted by pathogens (effector triggered immunity, ETI). Compared with ETI, P / HAMPs-induced immunity (P / HTI) is a broad-spectrum immunity that is not easily overcome by insects. Therefore, P / HAMPs, as an important factor in inducing plant resistance, has become an important development direction for the research and development of biopesticides. Isolating and identifying new P / HAMPs with broad-spectrum immune functions from insects is expected to develop into a new anti-insect strategy.

[0003] Herbivorous insect-associated molecular patterns (HAMPs) refer to a class of substances that are conserved molecular structures secreted by herbivorous insects and can be recognized by host plants to induce plant immunity. HAMPs are usually unique to herbivorous insects, not present in host cells, and are very important for the adaptive survival of insects. Plants can recognize HAMPs through pattern recognition receptors (PRRs) on the membrane and activate HAMPs-induced immunity. The defense responses induced by plant pattern recognition receptors recognizing HAMPs include activation of MAPKs kinase signaling pathways, reactive oxygen bursts, expression of insect resistance-related genes, cell ion leakage, and cell death. HAMPs-induced immunity can ultimately enable plants to acquire resistance.

[0004] However, the HAMP molecules identified from insects are conserved in different insects, and there are very few HAMP molecules that can induce a series of immune responses and mediate resistance with a short peptide segment of a few conserved amino acids. Therefore, identifying conserved HAMP molecules and short amino acid peptide segments in insects not only provides a theoretical basis for revealing the mechanism of interaction between plants and herbivorous insects, but also provides an effective protein source for the development of broad-spectrum, efficient and environmentally friendly immune protein biopesticides. Summary of the invention

[0005] In order to solve the above defects and shortcomings in the prior art, the present invention provides an application of Laodelphax striatellus salivary sheath protein MLC1 and short peptide MP41 in improving the insect resistance of plants and an insect resistance method.

[0006] To solve the above technical problems:

[0007] The first object of the present invention is to provide an application of a short peptide MP41 or a homologous sequence thereof having an amino acid sequence as shown in SEQ ID NO:3 in improving plant insect resistance, and a nucleotide sequence encoding the short peptide MP41 or a homologous sequence thereof as shown in SEQ ID NO:4.

[0008] The second purpose of the present invention is to provide an application of the salivary sheath protein MLC1 of the gray leafhopper or its homologous protein in improving the insect resistance of plants. The protein MLC1 or its homologous protein includes a short peptide MP41, the amino acid sequence of which is shown in SEQ ID NO:1, and the nucleotide sequence encoding the protein MLC1 or its homologous protein is shown in SEQ ID NO:2.

[0009] Based on the principle that proteins secreted by insects can be recognized by plants and induce plant immune responses, the applicant collected the salivary sheaths of gray leafhoppers and extracted proteins. After mass spectrometry sequencing of the extracted proteins, the secretome of the salivary sheaths of gray leafhoppers was determined by bioinformatics analysis. Proteins with more than two unique peptides, containing conserved domains, and present in insects but not in plants were identified as candidate proteins. The coding sequences of 19 candidate proteins were respectively constructed into the plant transient expression vector pBINPLUS, transformed into Agrobacterium and injected into tobacco, and finally an elicitor protein MLC1 that can induce plant defense responses and improve plant insect resistance was identified, and it was found that it has a conserved domain. By truncating the protein, this laboratory found that the short peptide MP41 composed of 41 amino acids after truncation also has the performance of inducing plant defense responses and improving plant insect resistance. The applicant further found that the gene encoding MLC1 exists in different arthropods, and experimentally verified that the short peptides corresponding to MP41 in some homologous proteins of MLC1 in other arthropods have the property of inducing plant defense responses.

[0010] The third object of the present invention is to provide a vector comprising the above-mentioned short peptide MP41 or its homologous sequence, or the above-mentioned protein MP41 or its homologous protein.

[0011] The fourth object of the present invention is to provide a host cell expressing the above-mentioned vector.

[0012] The fifth object of the present invention is to provide a method for improving insect resistance of plants, comprising spraying or injecting a short peptide MP41 or a homologous sequence thereof, or a protein MLC1 or a homologous protein thereof onto plant tissues;

[0013] The amino acid sequence of the short peptide MP41 or its homologous sequence is shown in SEQ ID NO:3, and the amino acid sequence of the protein MLC1 or its homologous protein is shown in SEQ ID NO:1.

[0014] The beneficial technical effects achieved by the present invention include: the application of the gray leafhopper salivary sheath protein MLC1 and the short peptide MP41 provided by the present invention in improving the insect resistance of plants and the insect resistance method, wherein the protein MLC1 and the short peptide MP41 can significantly activate the plant immune system, provide the plant with insect resistance, and effectively prevent or reduce the occurrence of insect pests; a new approach is provided to improve the insect resistance of plants, resources are provided for the future development and application of biological pesticides, and it can be used as a new type of protein pesticide to defend against insect pests, and a basis is provided for the future transgenic plants of the gene, and it has broad application prospects in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the protein gel electrophoresis diagram of the crude extract of MLC1 prokaryotic expression of the present invention;

[0016] Figure 2 This is the protein gel electrophoresis diagram after MLC1 purification of the present invention;

[0017] Figure 3 This is a diagram showing the results of MLC1 inducing Nicotiana benthamiana cell death and reactive oxygen production;

[0018] Figure 4 This is a diagram showing the results of MLC1 inducing rice defense factors in the present invention;

[0019] Figure 5 This is a graph showing the results of MLC1 inducing rice resistance to Laodelphax striatellus and Nilaparvata lugens;

[0020] Figure 6 This is a graph showing the results of MLC1 inducing rice resistance to Chilo suppressalis;

[0021] Figure 7 This is a diagram showing the results of the MP41-induced Nicotiana benthamiana cell death and reactive oxygen production;

[0022] Figure 8 This is a graph showing the results of MP41 inducing the expression of defense-related genes in Nicotiana benthamiana;

[0023] Fig. 9 This is a diagram showing the results of MP41 inducing rice defense factors in the present invention;

[0024] Fig.10 This is a graph showing the results of the MP41 of the present invention inducing resistance of Nicotiana benthamiana to Bemisia tabaci;

[0025] Fig.11 This is a graph showing the results of the MP41-induced rice resistance to the brown planthopper and the brown planthopper;

[0026] Fig.12 This is a graph showing the results of the MP41-induced rice resistance to Chilo suppressalis;

[0027] Fig.13 This is a diagram showing the results of MP41 inducing cell death in different plants;

[0028] Fig.14 This is a diagram showing the results of the MP41 homologous short peptide of the present invention inducing tobacco cell death and reactive oxygen production;

[0029] Fig.15 This is a graph showing the field resistance results of rice induced by MLC1 of the present invention to three types of planthoppers. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0031] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0032] Example 1 Prokaryotic expression and purification of protein MLC1

[0033] (1) Identification of MLC1 protein elicitor: Based on the principle that proteins secreted by insects can be recognized by plants and induce immune responses in plants, the applicant collected the salivary sheaths of gray leafhoppers and extracted proteins. After sending the extracted proteins to the company for mass spectrometry sequencing, the secretome of the salivary sheaths of gray leafhoppers was determined through bioinformatics analysis. Proteins with more than two unique peptides, containing conserved domains, and present in insects but not in plants were identified as candidate proteins. The coding sequences of the 19 candidate proteins were constructed into the plant transient expression vector pBINPLUS, transformed into Agrobacterium and injected into tobacco. Finally, one of the proteins, MLC1, which can induce allergic necrosis of Nicotiana benthamiana cells, was selected as a potential protein elicitor. Its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:2 in the sequence listing.

[0034] The amino acid sequence of protein MLC1 is shown in SEQ ID NO:1:

[0035] MADVSSDDVEKCTFAYNIYDTEGSGQLDCFFLGDLLRALGLNPTQGLIEKLGGTKKKGEKTISFEEFLPVFSQARKDKDCGVYEDFIECLKLYDKAENGLMVGAELSHILLSLGERLNEKEVDEVLKDCLDPEDDDGMIPYIPFLARICEKPVPDDIKPVL。

[0036] The nucleotide sequence of protein MLC1 is shown in SEQ ID NO:2 in the sequence listing:

[0037] ATGGCAGATGTCAGCAGCGATGACGTTGAAAAGTGCACCTTCGCCTACAACATTTATGACACAGAGGGTAGCGGCCAACTAGATTGCTTCTTCCTTGGCGATCTGCTCAGAGCTCTGGGACTGAACCCAACTCAGGGCCTCATTGAGAAACTCGGAGGCACCAAGAAGAAGGGTGAGAAGACAATCTCATTCGAAGAGTTCCTGCCAGTGTTCAGCCAGGCGAGGAAGGACAAGGACTGCGGTGTCTACGAGGACTTCATTGAGTGCTTGAAGCTCTACGACAAGGCTGAGAACGGACTCATGGTTGGCGCTGAACTCTCACACATTCTCTTGTCTCTTGGTGAAAGGTTAAATGAGAAGGAAGTCGATGAGGTCCTTAAGGACTGCCTGGACCCTGAGGATGATGATGGCATGATCCCCTACATCCCATTCCTGGCTCGTATTTGCGAAAAACCAGTACCAGATGACATCAAGCCAGTATTGTAA。

[0038] (2) Construction of prokaryotic expression vector: Design specific primers encoding the MLC1 elicitor gene of gray leafhopper, the forward primer is shown in SEQ ID NO:5 and the reverse primer is shown in SEQ ID NO:6. Use the cDNA of gray leafhopper as a template to amplify the full-length MLC1 gene (94 °C 5min; 94 °C 30s, 58 °C 30s, 72 °C 30s,34 cycles; 72 °C 5min). After the pHMTc vector was digested with EcoR I and BamH I, the MLC1 fragment was cloned into the pHMTc vector by recombinant cloning. Then, it was transformed into Escherichia coli DH5α strain, coated with a plate containing ampicillin antibiotics, and positive clones were screened by colony PCR. The screened positive clones were shaken to extract the plasmid to obtain the recombinant vector. Among them:

[0039] SEQ ID NO: 5: 5'-ctgtattttcagggcgaattcatggcagatgtcagcagcgatg-3';

[0040] SEQ ID NO: 6: 5'-caggtcgactctagaggatccttacaatactggcttgatgtc-3'.

[0041] Results: The prokaryotic expression vector of MLC1 was obtained through colony PCR verification and sequencing verification.

[0042] (3) Induce protein expression: The prokaryotic expression vector of MLC1 was transformed into the E. coli Rosetta expression strain, coated on a plate containing ampicillin and chloramphenicol, and induced expression by screening positive clones through colony PCR. Inoculate the positive clone into 4 ml of LB medium containing ampicillin and chloramphenicol, shake at 37 degrees overnight, and then transfer to 200 ml of LB medium containing the above two resistances to culture until the OD value is 0.6-0.8. After adding 200 μL of 0.1M IPTG, shake at 28 °C overnight. Take 10 ml and centrifuge, collect the supernatant and precipitate separately, among which, the precipitate is suspended with 600 μL of SDS (10%), boiled at 95 °C until transparent (10 min), placed on ice until turbid, and centrifuged at 12000 rpm for 15 min, and the supernatant is taken as the induced precipitated protein. The supernatant and precipitated proteins were added with 6*SDS loading buffer, heated in a boiling water bath for 10 min, and then subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue R250 staining to observe protein expression.

[0043] Results: After SDS-PAGE detection, the protein MLC1 with a molecular weight of about 18.8KD was present in both the supernatant and the precipitate. Figure 1 shown.

[0044] (4) Recombinant protein purification: After overnight induction, the cells were centrifuged in a 50ml centrifuge tube at 8000rpm for 10min at 4℃ to collect the cells. The cells were suspended in 10-20ml lysis buffer (NaH2PO4 50mM, NaCl 300mM, Imidazole 10mM, PH=8.0), 20 μL lysozyme was added, and the cells were placed on ice for 30 min. After ultrasonic disruption for 1s, 3s, and 30min, the cells were centrifuged at 4℃, 10000rpm for 70min to collect the supernatant. A 400 μL nickel column was washed twice with 10ml lysis buffer by centrifugation at 4℃, 3100rpm for 5min, and then the supernatant was added and the cells were placed in a shaker and incubated at 4℃ for 2h. After incubation at 4°C, 3210 rpm, 5 min, remove the supernatant, suspend with 10 ml 20mM wash buffer (NaH2PO4 50mM, NaCl 300mM, Imidazole 20mM, PH=8.0), centrifuge 3 times at 4°C, 3100 rpm, 5 min. Add 10 ml 50mM wash buffer (NaH2PO4 50mM, NaCl300mM, Imidazole 50mM, PH=8.0) to the centrifuge tube, wash 3 times, and then add 250 μL elution buffer (NaH2PO4 50mM, NaCl 300mM, Imidazole 250mM, PH=8.0) to elute 10 times. Take 10 μL of protein solution and add 6*SDS loading buffer, heat in a boiling water bath for 10 min, then perform SDS-PAGE electrophoresis and Coomassie Brilliant Blue R250 staining to observe the protein purification. The purified protein was replaced with Tris buffer without imidazole using a Millipore Amicon Ultra-15c ultrafiltration tube for subsequent determination.

[0045] Results: The purified single protein MLC1 was obtained. After SDS-PAGE detection, the purified protein MLC1 was consistent with the predicted molecular weight and had a single band, such as Figure 2 shown.

[0046] Example 2 Protein MLC1 induces plant defense responses

[0047] (1) Inducing tobacco allergic reactions: The concentration of protein MLC1 was adjusted to 1 nM, 20 nM, 50 nM, 100 nM, 500nm, and 1 μM. Select Nicotiana benthamiana leaves of about 4 weeks old, and inject protein MLC1 into Nicotiana benthamiana leaves from the back of the leaves using a 1 ml syringe without a needle. At the same time, 1 μM EV was used as a control. After 48 hours of injection, the allergic necrosis reaction was observed.

[0048] The results are as follows Figure 3 As shown in the first row: Protein concentrations above 50 nM can induce visible allergic cell death in Nicotiana benthamiana.

[0049] (2) Induce ROS production in tobacco leaves: The concentration of protein MLC1 was adjusted to 1 nM, 20 nM, 50 nM, 100 nM, 500 nm, and 1 μM. Select Nicotiana benthamiana of about 4 weeks old, and inject protein MLC1 into Nicotiana benthamiana leaves from the back of the leaves using a 1 ml syringe without a needle. At the same time, 1 μM EV was used as a control. After 12 hours of injection, the treated leaves were taken and placed in DAB staining solution (1 mg / ml, pH=3.8). After 8 hours of treatment at room temperature and away from light, the staining solution was removed and anhydrous ethanol was added for decolorization. After the green color of the leaves was completely removed, the leaves were taken out and photographed.

[0050] The results are as follows Figure 3 As shown in the second row: 12 hours after the leaves were treated with MLC1 recombinant protein, obvious brown deposits appeared at the injection site, and as the concentration of the injected protein increased, the area of ​​the brown deposits became larger and the color became darker, indicating that the MLC1 protein induced ROS resistance in Nicotiana benthamiana leaves.

[0051] (3) Induction of rice defense factors: For transgenic rice expressing MLC1 protein and wild-type rice, rice stems were placed in a glass cylinder containing 20 newly hatched female adults of the gray leafhopper. After 8 or 24 hours, we removed the gray leafhoppers and collected the rice stems. The samples were ground in liquid nitrogen and lysed with a labeled internal standard ( 2 D4-SA, 2 D6-JA and 2 D6-JA-Ile) were extracted with ethyl acetate and analyzed for salicylic acid, jasmonic acid, and jasmonate-isoleucine using high-performance liquid chromatography-tandem mass spectrometry. Hydrogen peroxide concentrations were quantified using the Amplex Red Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen).

[0052] The results are as follows Figure 4 As shown: 4A is the statistics of hydrogen peroxide content, 4B is the statistics of jasmonic acid-isoleucine content, 4C is the statistics of jasmonic acid content, and 4D is the statistics of salicylic acid content. The results of quantitative analysis of hormone concentration showed that protein MLC1 can significantly induce the increase of hydrogen peroxide, jasmonic acid and jasmonic acid-isoleucine content.

[0053] Example 3 Protein MLC1 induces insect resistance in rice

[0054] (1) Inducing resistance of rice to brown planthoppers: One MLC1 transgenic rice plant (oe4 strain or oe7 strain) and one wild-type rice plant were enclosed in a glass cylinder (8 cm in diameter and 8 cm in height), and 15 fourth-instar nymphs were released. The number of brown planthoppers / brown planthopper nymphs on each plant was counted at 1, 2, 4, 8, 24 and 48 hours after release; for the fecundity test, one female brown planthopper / brown planthopper and two male brown planthoppers / brown planthoppers were placed on the rice stem covered with the glass cylinder (0-8 cm above the ground). After seven days, the number of eggs laid by each female insect was counted under an optical microscope.

[0055] The results are as follows Figure 5 As shown, 5A is the number of gray planthoppers on each oe4 rice plant, 5B is the number of gray planthoppers on each oe7 rice plant, 5C is the egg-laying amount of gray planthoppers on different rice plants, 5D is the number of brown planthoppers on each oe4 rice plant, 5E is the number of brown planthoppers on each oe7 rice plant, and 5F is the egg-laying amount of brown planthoppers on different rice plants: After the MLC1 gene was transferred into the rice plants, the number of gray planthoppers / brown planthoppers staying on the rice plants was significantly reduced, and the egg-laying amount of gray planthoppers / brown planthoppers was significantly reduced, indicating that MLC1 can induce rice resistance to gray planthoppers.

[0056] (2) Inducing resistance of rice to Chilo suppressalis: A protein MLC1 transgenic rice plant (oe4 line or oe7 line) and a wild-type rice plant were sealed in a glass cylinder (8 cm in diameter and 8 cm in height). After weighing, five second-instar Chilo suppressalis nymphs were placed in the glass cylinder and attached to the basal stem. After feeding for 7 days, each insect was re-weighed to assess weight gain.

[0057] The results are as follows Figure 6 As shown: After the MLC1 gene was transferred into rice plants, the feeding of the Chilo suppressalis was significantly reduced, indicating that MLC1 can induce rice resistance to the Chilo suppressalis.

[0058] Example 4 Preparation of short peptide MP41

[0059] (1) Discovery of the elicitor peptide MP41

[0060] The applicant found that the protein MLC1 sequence has a conserved domain through sequence comparison, and obtained the short peptide MP41 by truncating the protein. The amino acid sequence of the short peptide MP41 is shown in the sequence listing SEQ ID NO:3, and the nucleotide sequence encoding the short peptide MP41 is shown in the sequence listing SEQ ID NO:4.

[0061] The amino acid sequence of the short peptide MP41 is shown in the sequence listing SEQ ID NO: 3:

[0062] GTKKKGEKTISFEEFLPVFSQARKDKDCGVYEDFIECLKLY.

[0063] The nucleotide sequence encoding the short peptide MP41 is shown in the sequence listing SEQ ID NO:4:

[0064] GGCACCAAGAAGAAGGGTGAGAAGACAATCTCATTCGAAGAGTTCCTGCCAGTGTTCAGCCAGGCGAGGAAGGACAAGGACTGCGGTGTCTACGAGGACTTCATTGAGTGCTTGAAGCTCTAC.

[0065] (2) Synthesis of the stimulatory peptide MP41: The stimulatory peptide MP41 was directly synthesized by GenScript Corporation.

[0066] Example 5 Short peptide MP41 induces plant defense response

[0067] (1) Inducing cell death and reactive oxygen species (ROS) production in Nicotiana benthamiana leaves: The concentration of the short peptide MP41 was adjusted to 1 nM, 20 nM, 50 nM, 100 nM, 500 nM, and 1 μM. Select Nicotiana benthamiana leaves of about 4 weeks old, and inject the MP41 short peptide into the Nicotiana benthamiana leaves from the back of the leaves using a 1 ml syringe without a needle. At the same time, pure water was used as a control. After 48 hours of injection, the allergic necrosis reaction was observed. 12 hours after the injection, the treated leaves were taken and placed in DAB staining solution (1 mg / ml, pH=3.8). After 8 hours of treatment at room temperature and away from light, the staining solution was removed and anhydrous ethanol was added for decolorization. After the green color of the leaves was completely removed, the leaves were taken out and photographed.

[0068] The results are as follows Figure 7 MP41 at a concentration of more than 50 nM can induce allergic cell death visible to the naked eye in Nicotiana benthamiana leaves. 12 hours after the leaves were treated with MP41, obvious brown deposits appeared at the injection site. As the concentration of the injected short peptide increased, the area of ​​the brown deposits became larger and the color became darker, indicating that MP41 induced the production of ROS in Nicotiana benthamiana leaves.

[0069] (2) Inducing the expression of defense-related genes in Nicotiana benthamiana leaves: After the short peptide MP41 and pure water were injected into Nicotiana benthamiana leaves, samples were taken at 24h and 48h, respectively, and RNA was extracted using a plant RNA extraction kit, and genomic DNA was removed to obtain high-purity RNA. The first-strand cDNA was synthesized using a reverse transcription kit. According to the instructions of the quantitative kit, 2μL of the reverse transcription product was taken as a template, and then the real-time fluorescence quantitative PCR method was used to quantify the RNA. EF-1α as internal reference genes, and jasmonic acid signaling-related genes for Nicotiana benthamiana resistance-related genes NbPR and NbPR The expression level was measured. The primers used are as follows:

[0070] NbEF1a-QF: 5′-AGAGGCCCTCAGACAAAC-3′, as shown in SEQ ID NO: 7;

[0071] NbEF1a-QR: 5′-TAGGTCCAAAGGTCACAA-3′, as shown in SEQ ID NO: 8;

[0072] NbPR3-QF: 5'-TGGGGTTATTGCTGGCTTAG-3', as shown in SEQ ID NO:9;

[0073] NbPR3-QR: 5′- GGGTCATCCAAAACCAGAGA-3′, as shown in SEQ ID NO: 10;

[0074] NbPR4-QF: 5′-GGCCAAGATTCCTGTGGTAGAT-3′, as shown in SEQ ID NO: 11;

[0075] NbPR4-QR: 5'-CACTGTTGTTTGAGTTCCTGTTCCT-3', as shown in SEQ ID NO:12.

[0076] The results are as follows Figure 8 As shown, 8A is NbPR The expression level of 8B is NbPR Expression of jasmonic acid signaling-related genes: Fluorescence quantitative PCR results showed that MP41 short peptide could significantly induce jasmonic acid signaling-related genes 1d and 2d after injection of Nicotiana benthamiana NbPR and NbPR expression.

[0077] (3) Inducing rice defense factors: After the rice stems were treated with short peptide MP41 and water, the rice stems were collected, the samples were ground in liquid nitrogen, and the samples were diluted with a labeled internal standard ( 2 D4-SA, 2 D6-JA and 2 D6-JA-Ile) were extracted with ethyl acetate and analyzed for salicylic acid, jasmonic acid, and jasmonate-isoleucine using high-performance liquid chromatography-tandem mass spectrometry. Hydrogen peroxide concentrations were quantified using the Amplex Red Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen).

[0078] The results are as follows Fig. 9As shown, 9A is the content of hydrogen peroxide, 9B is the content of jasmonic acid-isoleucine, 9C is the content of jasmonic acid, and 9D is the content of salicylic acid: the results of quantitative analysis of hormone concentrations showed that MP41 could significantly induce an increase in the contents of hydrogen peroxide, jasmonic acid and jasmonic acid-isoleucine.

[0079] Example 6 Short peptide MP41 induces insect resistance in tobacco and rice

[0080] (1) Inducing tobacco resistance to whitefly: 15 whitefly adults were placed in the center of each pair of leaves treated with MLC1 recombinant protein or water. The number of insects on each leaf was recorded at 1, 2, 4, 8, 12, 24 and 48 hours.

[0081] The results are as follows Fig.10 As shown: After the leaves of Nicotiana benthamiana were treated with MLC1 recombinant protein, the number of insects was significantly reduced, indicating that MLC1 recombinant protein can induce the resistance of Nicotiana benthamiana to whitefly.

[0082] (2) Inducing resistance of rice to Laodelphax striatellus:

[0083] One rice plant with stems painted with short peptide MP41 and one rice plant with stems painted with pure water were sealed in a glass cylinder (8 cm in diameter; 8 cm in height), and 15 fourth-instar nymphs were released. The number of LAPH / NBPH nymphs on each plant was counted 1, 2, 4, 8, 24, and 48 hours after release;

[0084] The results are as follows Fig.11 As shown, 11A is the number of gray planthoppers on each rice plant, 11B is the egg-laying amount of gray planthoppers, 11C is the number of brown planthoppers on each rice plant, and 11D is the egg-laying amount of brown planthoppers: after applying the short peptide MP41, the number of gray planthoppers / brown planthoppers staying on rice can be significantly reduced, indicating that MP41 can induce rice resistance to gray planthoppers / brown planthoppers.

[0085] (3) Inducing resistance of rice to Chilo suppressalis: A rice plant with stems coated with short peptide MP41 and a rice plant with stems coated with pure water were sealed in a glass cylinder (8 cm in diameter and 8 cm in height). After weighing, five second-instar nymphs were placed in the glass cylinder and attached to the base of the stem. After feeding for 7 days, each insect was re-weighed to assess weight gain.

[0086] The results are as follows Fig.12 As shown: After the short peptide MP41 was treated with rice, the feeding of the Chilo suppressalis was significantly reduced, indicating that MP41 can induce rice resistance to the Chilo suppressalis.

[0087] Example 7 MP41 homologous short peptides induce plant defense responses

[0088] (1) Cell death of different plants: The leaves of different plants (tomato, eggplant, pepper, cotton) were treated with MP41 short peptide. The concentration of the short peptide was adjusted to 0.5 μM and then infiltrated into the leaves of different species using a needle-free syringe.

[0089] The results are as follows Fig.13 As shown: MP41 homologous short peptides can effectively induce cell death in cotton, eggplant, pepper and wheat leaves.

[0090] (2) Inducing tobacco cell death and reactive oxygen species: MLC1 homologous proteins of 8 representative arthropods were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the MLC1 homologous gene sequences were aligned using Geneious 7 to find sequences homologous to the MP41 short peptide. The MP41 homologous short peptides were constructed into the plant transient expression vector pBINPLUS, transformed into Agrobacterium and injected into tobacco. After 4 days, the leaf cell necrosis was observed and photographed. The leaves 4 days after injection were placed in DAB staining solution (1 mg / ml, pH=3.8), kept in the dark at room temperature for 8 hours, then the staining solution was removed and anhydrous ethanol was added for decolorization. After the green color of the leaves was completely removed, the leaves were taken out, photographed, and the production of reactive oxygen species was evaluated.

[0091] The results are as follows Fig.14 As shown: From the Hemiptera insect brown planthopper (Nl, N. lugens ) and Bemisia tabaci (Bt, B. tabaci ) of MP41 homologous short peptides, derived from the lepidopteran insect fall armyworm (Sf, S. frugiperda ) and Spodoptera exigua (Se, S. exigua ) of MP41 homologous short peptides, derived from the dipteran insect Drosophila (Dm, D. melanogaster ) and Anopheles stephensi (As, A. stephensi ) of MP41 homologous short peptides, derived from the spider mite (Tur, T. urticae ) and Haemaphysalis longicornis (Hl, H. longicornis ) MP41 homologous short peptides can induce tobacco cell death and reactive oxygen production.

[0092] Example 8 Protein MLC1 induces rice resistance to three planthoppers in the field

[0093] Twenty-four MLC1 transgenic rice plants (oe4 and oe7 lines) and 24 wild-type rice plants were planted in the field, the rice was covered with a mesh net, and about 500 small brown planthoppers, 500 brown planthoppers, and 500 white-backed planthoppers were released into the field. The total number of all rice planthoppers (including nymphs and adults) on each rice plant was counted 40, 54, and 68 days after planting the rice.

[0094] The results are as follows Fig.15 As shown: After the MLC1 gene was transferred into rice plants, the number of three planthoppers staying on rice was significantly reduced, indicating that MLC1 can induce field resistance of rice to three planthoppers.

[0095] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. Use of a short peptide MP41 having an amino acid sequence as shown in SEQ ID NO: 3 in improving plant insect resistance, wherein: The plant is rice or tobacco, and the insect resistance specifically refers to resistance to gray planthopper, brown planthopper, whitefly, whiteback planthopper or striped stem borer.

2. The use according to claim 1, characterized in that: The nucleotide sequence encoding the short peptide MP41 is shown in SEQ ID NO:

4.

3. Application of the salivary sheath protein MLC1 of Laodelphax striatellus in improving the insect resistance of plants, characterized in that: The amino acid sequence of the protein MLC1 is shown in SEQ ID NO: 1; the plant is rice or tobacco, and the insect resistance specifically refers to resistance to Laodelphax striatellus, Nilaparvata lugens, Bemisia tabaci, Whiteback Planthopper or Chilo suppressalis.

4. The use according to claim 3, characterized in that: The nucleotide sequence encoding protein MLC1 is shown in SEQ ID NO:

2.

5. A method for improving insect resistance of plants, characterized in that: The short peptide MP41 or the protein MLC1 is sprayed or injected on plant tissues; wherein the plants are rice and tobacco, and the insect resistance specifically refers to resistance to gray leafhopper, brown leafhopper, whitefly, whiteback planthopper or striped stem borer; The amino acid sequence of the short peptide MP41 is shown in SEQ ID NO:3, and the amino acid sequence of the protein MLC1 is shown in SEQ ID NO:1.

Images