Brown planthopper elicitor polypeptide NP30, protein Nlsp7 and application thereof

Abstract

The invention provides brown planthopper elicitor polypeptide NP30 and protein Nlsp7 and application thereof, and the polypeptide NP30 or protein Nlsp7 provided by the invention can obviously activate a plant immune system, improve the insect resistance of plants, enhance the insect resistance of the plants, and effectively prevent or reduce the occurrence of insect pests. The invention provides a new way for improving the insect resistance of plants, provides resources for the development and application of biological pesticides in the future, can be used as a novel microbial protein pesticide for defending the attack of insect pests, also provides a basis for transgenic plants with the gene in the future, and has a wide application prospect in agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a brown planthopper elicitor polypeptide NP30, a protein Nlsp7, and their applications. Background Technology

[0002] Currently, the main measures for controlling crop pests rely on chemical control and the breeding of insect-resistant varieties. However, chemical control primarily depends on pesticides, and excessive pesticide use leads to environmental pollution and pesticide residues. Furthermore, issues such as insect gene mutations leading to pesticide resistance and the need to overcome resistance genes reduce the control effectiveness of insect-resistant varieties. The effectiveness of both pest control strategies is severely impacted, necessitating the development of environmentally friendly, safe, economical, and efficient pest control strategies.

[0003] Recently, biological control methods have been increasingly widely used in pest management. Biological control utilizes natural enemies or microorganisms in nature to control pests, such as parasitic wasps, predatory insects, fungi, and bacteria, to suppress pest populations. In addition, the insect resistance capabilities of plants themselves have received increasing attention. Through research on the natural insect resistance mechanisms of plants, researchers have discovered that many plants can resist pests by secreting specific chemicals or activating immune responses. When attacked by pests, these plants can respond rapidly, activating their immune system and releasing natural insect-resistant factors to inhibit the growth and reproduction of pests.

[0004] Insects and host plants have long co-evolved, with plants developing complex and diverse defense mechanisms to resist insect feeding and attacks. The most important of these defenses is the activation of herbivorous insect-associated molecular patterns (HAMPs) / elicitors released by insects through oral secretions or oviposition via pattern recognition receptors (PRRs) located on the cell membrane. In recent years, significant progress has been made in the study of HAMPs in chewing insects. Reported HAMPs include inceptin, volicitin, caeliferin, glucose oxidase, lipase, β-glucosidase, phospholipase C, and β-galactofuranose from the insect oral cavity OS. Furthermore, with the publication of studies on NlMLP in the brown planthopper and CathB3 in the peach aphid, breakthroughs have been achieved in the study of elicitors from piercing-sucking insect pests.

[0005] However, despite the discovery and development of several HAMPs / elicitors into novel biopesticides, improving their effectiveness in actual agricultural production remains a significant challenge. For example, extending the duration of action of HAMPs / elicitors within plants, increasing their absorption efficiency, and enhancing their broad-spectrum and stability against different pests are still critical technical problems that urgently need to be solved. Therefore, developing novel insect-resistant elicitors or improving the plant's immune system through genetic engineering to enhance its defense against pests is an important direction in the development of biological control technology. Summary of the Invention

[0006] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a brown planthopper elicitor polypeptide NP30, protein Nlsp7 and their applications, which can effectively improve the insect resistance of tobacco or rice.

[0007] To solve the above technical problems: The first objective of this invention is to provide a brown planthopper elicitor polypeptide NP30, the amino acid sequence of which is shown in SEQ ID NO:3; and the nucleotide sequence encoding the polypeptide NP30 is shown in SEQ ID NO:4.

[0008] A second objective of this invention is to provide a brown planthopper elicitor protein Nlsp7, the amino acid sequence of which is shown in SEQ ID NO:1; and the nucleotide sequence encoding which is shown in SEQ ID NO:2.

[0009] A third objective of this invention is to provide an application of the aforementioned polypeptide NP30 and protein Nlsp7 in improving the insect resistance of rice or tobacco.

[0010] Furthermore, the polypeptide NP30 or protein Nlsp7 is used as an elicitor to stimulate the plant's defense or allergic response.

[0011] Furthermore, the polypeptide NP30 or protein Nlsp7 is sprayed onto tobacco or rice as a pesticide or as the main component of a pesticide.

[0012] The fourth objective of this invention is to provide an application of the above-mentioned polypeptide NP30 and protein Nlsp7 in the preparation of pesticides.

[0013] The beneficial technical effects achieved by this invention are as follows: The polypeptide NP30 or protein Nlsp7 provided by this invention can significantly activate the plant immune system, improve plant insect resistance, enhance the plant's ability to resist insects, and effectively prevent or reduce the occurrence of insect pests. This provides a new approach to improving plant insect resistance, provides resources for the future development and application of biological pesticides, can be used as a novel microbial protein pesticide to defend against insect infestations, and also provides a basis for future transgenic plants based on this gene, showing broad application prospects in agricultural production. Attached Figure Description

[0014] Figure 1 Gel electrophoresis image of crude Nlsp7 prokaryotic expression extract; Figure 2 Here is a gel electrophoresis image of Nlsp7 purified protein; Figure 3 Figure showing the results of Nlsp7-induced cell death and reactive oxygen species production in Nicotiana benthamiana cells; Figure 4 The result of Nlsp7-induced Benedict's tobacco MAPK signaling pathway is shown in the figure. Figure 5 Figure showing the results of Nlsp7 inducing resistance in rice to brown planthopper and gray planthopper; Figure 6 Figure showing the results of Nlsp7-induced resistance to rice stem borer; Figure 7 Figure showing the results of NP30-induced cell death and reactive oxygen species production in Nicotiana benthamiana cells. Figure 8 Figure showing the results of NP30-induced expression of genes related to defense in Nicotiana benthamiana; Figure 9 The results of NP30-induced Benedict's tobacco MAPK signaling pathway are shown in the figure. Figure 10 Figure showing the results of NP30-induced resistance of *Nicotiana benthamiana* to whiteflies; Figure 11 Figure showing the results of NP30-induced resistance in rice to brown planthopper and gray planthopper; Figure 12 Figure showing the results of NP30-induced resistance to rice stem borer; Figure 13 Figure 1 shows the results of NP30-induced cell death in different plants. Figure 14 Figure showing the field resistance results of rice to three planthoppers induced by Nlsp7. Detailed Implementation

[0015] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0016] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all obtainable through conventional commercial channels.

[0017] Example 1: Prokaryotic expression and purification of protein Nlsp7 The elicitor protein Nlsp7 disclosed in this embodiment is derived from the salivary sheath secretion protein of the brown planthopper. The nucleotide sequence of the protein was obtained by protein sequencing. The nucleotide sequences of different proteins were constructed into the transient expression vector pBINPLUS, and the expression vector was transformed into Agrobacterium and injected into Nicotiana benthamiana. The protein that can induce allergic necrosis of Nicotiana benthamiana cells was screened and named Nlsp7. Its amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence encoding the protein Nlsp7 is shown in SEQ ID NO:2.

[0018] SEQID NO:1: MRAALILLIVSAIIIDSAMAGPKSKKGKSKRSRERIVYAQPPPTPVIIQGAAPYNYDNRGYYDNRPYPGDGRGYYDANGVWINGGYNGPYPNNGPVVVYPNNGPYVQPTYGPQVVY.

[0019] SEQID NO:2: ATGAGGGCTGCCCTGATTCTTCTCATCGTATCTGCCATCATTATTGATTCGGCCATGGCAGGCCCCAAATCGAAGAAAGGCAAGAGCAAGAGGCGATCAAGGGAGAGAATCGTGTATGCACAGCCTCCTCCAACCCCAGTCATCATCCAAGGTGCTGCTCCATACAACTATGACAAC AGAGGCTACTATGACAACAGGCCTTACCCTGGAGATGGTCGCGGATATTATGACGCCAACGGTGTCTGGATCAATGGAGGCTACAATGGACCTTACCCCAATAATGGTCCAGTGGTTGTCTACCCCAATAATGGTCCTTATGTGCAGCCTACCTATGGACCACAGGTTGTCTACTAG.

[0020] (1) Identification of protein Nlsp7 Based on the principle that insect-secreted proteins can be recognized by plants and induce an immune response, our laboratory constructed the Nlsp7 protein coding sequence into the plant transient expression vector pBINPLUS, transformed Agrobacterium, and injected it into tobacco, confirming its ability to induce hypersensitive necrosis in Nicotiana benthamiana cells. Specifically, the constructed pBINPLUS-Nlsp7 vector was introduced into Agrobacterium tumefaciens strain GV3101 using an electroporation method. Agrobacterium tumefaciens strain GV3101 was cultured in LB broth containing kanamycin and rifampin at 28°C for 24 hours. The recombinant strain was washed three times with osmotic buffer (1 mmol / L MgCl2, 100 mM MISS, 150 mM acetylsyl eugenol) and resuspended to OD200. 600 The value was 0.4. Then, using a needle-free injector, a suspension of Agrobacterium tumefaciens cells was injected into the leaves of Nicotiana benthamiana, resulting in anaphylactic necrosis of the tobacco, which was the target protein.

[0021] (2) Construction of prokaryotic expression vectors Specific primers encoding the Nlsp7 gene sequence were designed: forward primer: 5'-ctgtattttcagggcgaattcatgagggctgccctgattct-3', as shown in SEQ ID NO:5; reverse primer: 5'-caggtcgactctagaggatccctagtagacaacctgtggtc-3', as shown in SEQ ID NO:6. Using brown planthopper cDNA as a template, the full-length Nlsp7 gene was amplified (94 °C 5 min; 94 °C 30 s, 58 °C 30 s, 72 °C 30 s, 34 cycles; 72 °C 5 min). The pHMTc vector was digested with EcoRI and BamHI, and the Nlsp7 fragment was cloned into the pHMTc vector using recombinant cloning. The recombinant vector was transformed into *Escherichia coli* DH5α strain, plated on agar plates containing ampicillin, and positive clones were screened by colony PCR. The plasmids were extracted from the screened positive clones to obtain the recombinant vector.

[0022] Results: The prokaryotic expression vector for protein Nlsp7 was obtained through colony PCR and sequencing verification.

[0023] (3) Inducing the expression of protein Nlsp7 1. Recombinant plasmid transformed into Escherichia coli Rosetta I. Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 min.

[0024] II. Heat shock at 42℃ for 90 seconds, then immediately place on ice for 5 minutes, and add 600 μL of LB culture medium.

[0025] III. Shake at 37℃ and 220 r / min for 1 h, then centrifuge and spread the entire sample onto LB plates containing 50 μg / mL Amp or Kan, and incubate upside down at 37℃ overnight.

[0026] 2. IPTG-induced expression of recombinant bacterial fusion protein Ⅰ. Pick a single clone from the transformation plate and inoculate it into a test tube containing 3 mL LB medium with 50 μg / mL Amp or Kan, and incubate overnight at 37°C with shaking at 220 r / min.

[0027] II. The next day, inoculate at a ratio of 1:100 into 30 mL of LB culture medium containing 50 μg / mL Amp or Kan, and shake at 37℃ and 220 r / min until the bacterial OD600 is 0.6-0.8.

[0028] III. Take out 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer.

[0029] IV. a. Add IPTG to the remaining culture to a final concentration of 0.5 mM, and incubate at 37°C with shaking at 220 r / min for 4 h to induce fusion protein expression. b. Add IPTG to the remaining culture to a final concentration of 0.5 mM, and incubate at 11°C with shaking at 220 r / min overnight to induce fusion protein expression.

[0030] V. Take 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in PBS; after sonicating the resuspended solution, take the supernatant and pellet respectively and resuspend them in loading buffer.

[0031] VI. 12% SDS-PAGE analysis was performed, with Coomassie brilliant blue staining for banding. Results: SDS-PAGE analysis showed that the Nlsp7 fusion protein, with a molecular weight of approximately 14 KD, was present in both the supernatant and the precipitate. Figure 1 As shown.

[0032] (4) Purification of recombinant protein After overnight induction, the bacterial cells were centrifuged in 50 ml centrifuge tubes at 8000 rpm for 10 min at 4 °C, and the cells were collected. The cells were then suspended in 10-20 ml of lysis buffer (NaH2PO4 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8.0), and 20 μL of lysozyme was added. The cells were incubated on ice for 30 min. After sonication for 1 s, 3 s, and 30 min, the cells were centrifuged at 10000 rpm for 70 min at 4 °C, and the supernatant was collected. A 400 μL nickel column was washed twice with 10 ml of lysis buffer by centrifugation at 3100 rpm for 5 min at 4 °C, and the supernatant was added. The column was then incubated on a shaker at 4 °C for 2 h. After incubation at 4°C, 3210 rpm, 5 min, remove the supernatant, resuspend in 10 ml of 20 mM wash buffer (NaH2PO4 50 mM, NaCl 300 mM, Imidazole 20 mM, pH=8.0), and centrifuge three times at 4°C, 3100 rpm, 5 min. Add 10 ml of 50 mM wash buffer (NaH2PO4 50 mM, NaCl 300 mM, Imidazole 50 mM, pH=8.0) to the centrifuge tube, wash three times, then add 250 μL of elution buffer (NaH2PO4 50 mM, NaCl 300 mM, Imidazole 250 mM, pH=8.0) and elute 10 times. Add 10 μL of protein solution to 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 protein purification. Replace the purified protein with imidazole-free Tris buffer using a Millipore Amicon Ultra-15c ultrafiltration tube for subsequent assays.

[0033] Results: Purified single Nlsp7 recombinant protein was obtained. SDS-PAGE analysis showed that the purified Nlsp7 fusion protein was consistent with the predicted molecular weight and exhibited a single band, as shown in the image. Figure 2 As shown.

[0034] Example 2: Protein Nlsp7 induces plant defense response (1) Inducing tobacco allergic reactions The concentrations of protein Nlsp7 were adjusted to 1 nM, 20 nM, 50 nM, 100 nM, 500 nm, and 1 μM. Approximately 4-week-old *Nicotiana benthamiana* plants were selected, and the recombinant Nlsp7 protein was injected into the leaves from the back using a 1 ml syringe without a needle. Simultaneously, 1 μM of EV was used as a control. Allergic necrosis was observed 48 hours after injection.

[0035] The results are as follows Figure 3 As shown, protein concentrations above 100 nM can induce visible allergic cell death in *Fumiganthus benthamianus*.

[0036] (2) Inducing ROS production in tobacco leaves The concentrations of protein Nlsp7 were adjusted to 1 nM, 20 nM, 50 nM, 100 nM, 500 nm, and 1 μM. Approximately 4-week-old *Nicotiana benthamiana* plants were selected, and protein Nlsp7 was injected into the leaves from the back using a 1 ml syringe without a needle. Simultaneously, 1 μM of EV was used as a control. Twelve hours after injection, the treated leaves were collected and placed in DAB staining solution (1 mg / ml, pH=3.8). After 8 hours of treatment at room temperature in the dark, the staining solution was removed, and anhydrous ethanol was added for decolorization. Once all the green color had disappeared from the leaves, they were removed and photographed.

[0037] The results are as follows Figure 3 As shown: 12 h after treatment of leaves with protein Nlsp7, obvious brown deposits appeared at the injection site. As the concentration of injected protein increased, the area of ​​brown deposits also increased and the color became darker, indicating that Nlsp7 recombinant protein induced ROS production in tobacco leaves.

[0038] (3) Inducing the tobacco MAPK signaling pathway Four-week-old Nicotiana benthamiana leaves were treated with 0.5 μM flg22 and Nlsp7rec / EV, and total protein was extracted. The isolated proteins were separated by 12% (v / v) SDS-PAGE gel chromatography and analyzed by immunoblotting. Immunoblotting was performed using antiphosphorylated p44 / 42 MAPK antibody (1:5,000, Cell Signaling Technology, USA) and horseradish peroxidase-labeled anti-rabbit IgG (1:10,000, Sigma, USA) as secondary antibodies to determine the phosphorylation of MAPK proteins, including MPK3, MPK4, and MPK6.

[0039] The results are as follows Figure 4 As shown: The MAPK signaling pathway analysis results show that the Nlsp7 protein can significantly induce MAPK signaling.

[0040] Example 3: Nlsp7 protein induces insect resistance in rice (1) Inducing resistance in rice to brown planthopper / gray planthopper One transgenic Nlsp7 rice plant (either the oe1 or oe3 line) 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 planthopper / gray planthopper nymphs on each plant was counted at 1, 2, 4, 8, 24, and 48 hours after release. For reproductive capacity testing, one female brown planthopper / gray planthopper and two male brown planthoppers / gray planthoppers were placed on the rice stalks enclosed in the glass cylinder (0-8 cm above the ground). Seven days later, the number of eggs laid by each female was counted under an optical microscope.

[0041] The results are as follows Figure 5 As shown, the introduction of the Nlsp7 gene into rice plants significantly reduced the number of brown planthoppers / gray planthoppers remaining on the rice plants and significantly reduced the number of eggs laid by brown planthoppers / gray planthoppers, indicating that Nlsp7 can induce resistance in rice to brown planthoppers.

[0042] (2) Inducing resistance in rice to rice stem borer One Nlsp7 transgenic rice plant (either the oe1 or oe3 line) and one wild-type rice plant were enclosed in a glass cylinder (8 cm in diameter and 8 cm in height). After weighing, five second-instar rice stem borer nymphs were placed in the glass cylinder and attached to the basal stem. Seven days after feeding, each insect was weighed again to assess weight gain.

[0043] The results are as follows Figure 6 As shown, the introduction of the Nlsp7 gene into rice plants significantly reduced the feeding of rice stem borers, indicating that Nlsp7 can induce resistance in rice to rice stem borers.

[0044] Example 4: Preparation of short peptide NP30 (1) Discovery of the elicitor peptide NP30 Based on sequence alignment, our laboratory discovered that the protein elicitor Nlsp7 sequence possesses a conserved domain. Our laboratory truncated the protein to obtain the short peptide NP30, the amino acid sequence of which is shown in SEQ ID NO:3 of the sequence listing, and the nucleotide sequence encoding the short peptide NP30 is shown in SEQ ID NO:4 of the sequence listing.

[0045] SEQ ID NO:3: GPKSKKGKSKRRSRERIVYAQPPPTPVIIQ; SEQ ID NO:4: GGCCCCAAATCGAAGAAAGGCAAGAGCAAGAGGCGATCAAGGGAGAGAATCGTGTATGCACAGCCTCCTCCAACCCCAGTCATCATCCAA.

[0046] (2) Synthesis of the elicitor peptide NP30 The elicitor peptide NP30 was directly synthesized by GenScript.

[0047] Example 5: Induction of plant defense response by the elicitor peptide NP30 Induces cell death and reactive oxygen species (ROS) production in tobacco leaves. The NP30 concentration was adjusted to 1 nM, 20 nM, 50 nM, 100 nM, 500 nM, and 1 μM. Approximately 4-week-old *Nicotiana benthamiana* leaves were selected, and the NP30 short peptide was injected into the leaves from the back using a 1 ml syringe without a needle. Pure water was used as a control. Allergic necrosis was observed 48 hours after injection. Twelve hours after injection, the treated leaves were collected and placed in DAB staining solution (1 mg / ml, pH=3.8). After 8 hours of treatment at room temperature in the dark, the staining solution was removed, and anhydrous ethanol was added for decolorization. Once the green color had completely disappeared from the leaves, they were removed and photographed.

[0048] Results: NP30 concentrations above 50 nM induced visible hypersensitive cell death in *Nicotiana benthamiana*. Twelve hours after NP30 treatment, obvious brown deposits appeared at the injection sites, and the area and color of the brown deposits increased with increasing short peptide concentration, indicating that NP30 induced ROS production in *Nicotiana benthamiana* leaves. Figure 7 As shown.

[0049] (2) Inducing the expression of genes related to the defense of Smoke Benedict's tobacco After injecting NP30 short peptide and pure water into tobacco leaves, samples were taken at 24h and 48h, respectively. RNA was extracted using a plant RNA extraction kit, and genomic DNA was removed to obtain high-purity RNA. First-strand cDNA was synthesized using a reverse transcription kit. Following the instructions of the quantitative PCR kit, 2μL of the reverse transcription product was used as a template, and then real-time quantitative PCR was performed. EF-1α This is an internal reference gene, a gene related to resistance to Nicotiana benthamiana, and a jasmonic acid signaling gene. NbPR3 and NbPR4 Expression levels were measured. The primers used are as follows: NbEF1a-QF: 5'-AGAGGCCCTCAGACAAAC-3', as shown in SEQ ID NO: 7; NbEF1a-QR: 5'-TAGGTCCAAAGGTCACAA-3', as shown in SEQ ID NO:8; NbPR3-QF: 5'-TGGGGTTATTGCTGGCTTAG-3', as shown in SEQ ID NO:9; NbPR3-QR: 5'-GGGTCATCCAAAACCAGAGA-3', as shown in SEQ ID NO:10; NbPR4-QF: 5'- GGCCAAGATTCCTGTGGTAGAT-3', as shown in SEQ ID NO: 11; NbPR4-QR: 5'- CACTGTTGTTTGAGTTCCTGTTCCT-3', as shown in SEQ ID NO:12; Results: Quantitative real-time PCR results showed that the NP30 short peptide significantly induced jasmonic acid signaling-related genes 1 and 2 days after injection of Nicotiana benthamiana. NbPR3 and NbPR4 Expressions, such as Figure 8 As shown.

[0050] (3) Inducing the tobacco MAPK signaling pathway Four-week-old *Nicotiana benthamiana* leaves were treated with 0.5 μM flg22 and NP30 / Water, and total protein was extracted. The isolated proteins were separated by 12% (v / v) SDS-PAGE gel chromatography and analyzed by Western blotting. Immunoblotting was performed using anti-phosphorylated p44 / 42 MAPK antibody (1:5,000, Cell Signaling Technology, USA) and horseradish peroxidase-labeled anti-rabbit IgG (1:10,000, Sigma, USA) as secondary antibodies to determine the phosphorylation of MAPK proteins, including MPK3, MPK4, and MPK6.

[0051] Results: Analysis of the MAPK signaling pathway showed that the NP30 peptide significantly induced MAPK signaling, such as Figure 9 As shown.

[0052] Example 6: Induction of insect resistance in tobacco and rice by the elicitor peptide NP30 (1) Inducing tobacco resistance to whiteflies Fifteen adult whiteflies were placed in the center of each pair of leaves treated with Nlsp7 recombinant protein or water. The number of insects on each leaf was recorded at 1, 2, 4, 8, 12, 24, and 48 hours.

[0053] Results: Treatment of *N. benthamiana* leaves with the Nlsp7 recombinant protein significantly reduced insect populations, indicating that the Nlsp7 recombinant protein can induce resistance in *N. benthamiana* to whiteflies. Figure 10 As shown.

[0054] (2) Inducing resistance in rice to planthoppers One rice plant with stems coated with the short peptide NP30 and another with stems coated with pure water were enclosed in glass cylinders (8 cm in diameter and 8 cm in height), and 15 fourth-instar nymphs were released. The number of brown planthopper / gray planthopper nymphs on each plant was counted at 1, 2, 4, 8, 24, and 48 hours after release. For reproductive capacity testing, one female brown planthopper / gray planthopper and two male brown planthoppers / gray planthoppers were placed on the rice stems covered with the glass cylinders (0-8 cm above the ground). Seven days later, the number of eggs laid by each female was counted under an optical microscope.

[0055] Results: Applying the short peptide NP30 significantly reduced the number of brown planthoppers / gray planthoppers remaining on rice plants and significantly reduced their egg production, indicating that NP30 can induce resistance in rice to brown planthoppers / gray planthoppers. Figure 11 As shown.

[0056] (3) Inducing resistance in rice to rice stem borer One rice plant whose stem was coated with the short peptide NP30 and another rice plant whose stem was 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 basal stem. Seven days after feeding, each insect was weighed again to assess weight gain.

[0057] Results: Treatment of rice with the short peptide NP30 significantly reduced rice stem borer feeding, indicating that NP30 can induce resistance in rice to the rice stem borer. Figure 12 As shown.

[0058] Example 7: Results of NP30-induced cell death in different plants (images) Plant leaves were treated with NP30 short peptides. For dicotyledonous plants (peppers, cotton), the short peptides were adjusted to appropriate concentrations and then infiltrated into the leaves of different species using a needle-free injector. For monocotyledonous plants (corn), micro-wounds were created using quartz sand, and then the peptide solution was infiltrated into the plants.

[0059] Results: NP30 can induce cell death in the leaves of pepper, cotton, and maize, such as Figure 13 As shown.

[0060] Example 8: Field resistance results of rice induced by Nlsp7 against three planthoppers (Figure 8) Twenty-four Nlsp7 transgenic rice plants (oe1 and oe3 lines) and 24 wild-type rice plants were planted in the field, covered with perforated netting, and approximately 500 brown planthoppers, 500 gray planthoppers, and 500 white-backed planthoppers were released into the field. The total number of planthoppers (including nymphs and adults) on each rice plant was counted at 70 and 80 days after planting.

[0061] Results: Transplantation of the Nlsp7 gene into rice plants significantly reduced the number of the three planthopper species remaining on the rice plants, indicating that Nlsp7 can induce field resistance in rice to the three planthopper species.

[0062] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. A brown planthopper elicitor polypeptide NP30, characterized in that: The amino acid sequence of the polypeptide NP30 is shown in SEQ ID NO:

3.

2. The brown planthopper elicitor polypeptide NP30 according to claim 1, characterized in that: The nucleotide sequence encoding polypeptide NP30 is shown in SEQ ID NO:

4.

3. A brown planthopper elicitor protein Nlsp7, characterized in that: The amino acid sequence of protein Nlsp7 is shown in SEQ ID NO:

1.

4. The brown planthopper elicitor protein Nlsp7 according to claim 1, characterized in that: The nucleotide sequence encoding the protein Nlsp7 is shown in SEQ ID NO:

2.

5. The application of the polypeptide NP30 according to any one of claims 1-2, or the protein Nlsp7 according to any one of claims 3-4, in improving the insect resistance of rice or tobacco.

6. The application according to claim 5, characterized in that: The polypeptide NP30 or protein Nlsp7 is used as an elicitor to stimulate the plant's defense or allergic response.

7. The application according to claim 5, characterized in that: The polypeptide NP30 or protein Nlsp7 is sprayed onto tobacco or rice as a pesticide or as the main component of a pesticide.

8. The use of the polypeptide NP30 according to any one of claims 1-2, or the protein Nlsp7 according to any one of claims 3-4, in the preparation of pesticides.

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