Short peptide NP19, polypeptide Nlsp5 and application of short peptide NP19 and polypeptide Nlsp5

Abstract

The invention provides a short peptide NP19, a polypeptide Nlsp5 and application thereof, and belongs to the technical field of bioengineering. The oligopeptide NP19 and the polypeptide Nlsp5 can obviously activate the immune system of the plant, improve the insect resistance of the plant, enhance the insect resistance of the plant, and effectively prevent or reduce the occurrence of insect pests. The oligopeptide NP19 and the polypeptide Nlsp5 provide a new way for improving the insect resistance of plants, provide resources for development and application of biological pesticides in the future, can be used as a novel microbial protein pesticide to defend attack of insect pests, also provide a basis for transgenic plants with the gene in the future, and have wide application prospects in agricultural production.

Description

Technical Field

[0001] The invention relates to a short peptide NP19, a polypeptide Nlsp5 and applications thereof, and belongs to the technical field of bioengineering. Background Art

[0002] In recent years, biological control methods have been increasingly used in pest control. Biological control uses natural enemies or microorganisms in nature to control pests, such as using natural enemies such as parasitic wasps, predatory insects, fungi, bacteria, etc. to suppress the number of pests. In addition, the insect resistance of plants themselves has also received more and more attention. Through the study of the natural insect resistance mechanism of plants, scientists have found that many plants can resist pests by secreting specific chemicals or activating immune responses. When these plants are attacked by pests, they can respond quickly, activate the plant's immune system, and release some natural insect resistance elicitors to inhibit the growth and reproduction of pests.

[0003] However, current biological control technology faces some challenges. First, the effectiveness of biological control is usually greatly affected by environmental conditions, and some natural enemies or microorganisms may not perform optimally under different climate and soil conditions. Second, although some natural insecticide elicitors have been discovered, the mechanisms of these substances are not yet fully understood, and their application stability and duration of effectiveness remain difficult research issues. Therefore, screening out highly efficient and stable insecticide elicitors has become an important direction of current research.

[0004] In recent years, screening and identification of insect resistance elicitors has become a research hotspot in the field of biological control. Through in-depth studies of plant immune responses, scientists have gradually revealed the role of various natural insect resistance elicitors (such as certain proteins, sugars, and small molecules) in insect resistance mechanisms. In particular, the immune signaling pathway mediated by receptor kinases (PRRs) has been shown to play an important role in plant defense against insect pests. Based on these findings, researchers are exploring how to improve plant resistance to insect pests by exogenous application of these elicitors or through genetic engineering.

[0005] However, although some anti-insect elicitors have been discovered and initially applied, there are still many challenges in improving their application in actual agricultural production. For example, how to improve the absorption efficiency of elicitors in plants, prolong their action time in plants, and enhance their broad spectrum and stability against different types of insect pests are still key technical problems that need to be solved urgently.

[0006] Therefore, developing new insect-resistant elicitors or improving the plant's own immune system through genetic engineering to enhance its ability to defend against insect pests is an important direction for the development of biological control technology. Summary of the invention

[0007] In order to solve the above-mentioned defects and shortcomings in the prior art, the present invention provides a short peptide NP19, a polypeptide Nlsp5 and applications thereof, which can effectively improve the insect resistance of tobacco or rice.

[0008] To solve the above technical problems: The first object of the present invention is to provide a short peptide NP19 derived from a salivary sheath secretion protein of brown planthopper, wherein the amino acid sequence of the short peptide NP19 is shown in SEQ ID NO:3.

[0009] Furthermore, the nucleotide sequence encoding the short peptide NP19 is shown in SEQ ID NO:4.

[0010] The second object of the present invention is to provide a brown planthopper salivary sheath secretion polypeptide Nlsp5, including the above-mentioned short peptide NP19.

[0011] Furthermore, the amino acid sequence of the polypeptide Nlsp5 is shown in SEQ ID NO:1.

[0012] Furthermore, the nucleotide sequence encoding the polypeptide Nlsp5 is shown in SEQ ID NO:2.

[0013] The third object of the present invention is to provide the use of the above-mentioned short peptide NP19 or the above-mentioned polypeptide Nlsp5 in improving the insect resistance of tobacco or rice.

[0014] Furthermore, the short peptide NP19 or polypeptide Nlsp5 is used as an elicitor to stimulate the defense response or allergic response of tobacco or rice.

[0015] Furthermore, the short peptide NP19 or the polypeptide Nlsp5 is used as a pesticide or as a main component of a pesticide and sprayed on tobacco or rice.

[0016] The fourth purpose of the present invention is to provide an application of the above-mentioned short peptide NP19 and the above-mentioned polypeptide Nlsp5 in the preparation of pesticides.

[0017] The beneficial technical effects achieved by the present invention are as follows: the short peptide NP19, the polypeptide Nlsp5 and their applications provided by the present invention can significantly activate the plant immune system, improve the insect resistance of plants, enhance the ability of plants to resist insects, and effectively prevent or reduce the occurrence of insect pests. The short peptide NP19 and the polypeptide Nlsp5 described in the present invention provide a new way to improve the insect resistance of plants, provide resources for the development and application of biological pesticides in the future, can be used as a new type of microbial protein pesticide to defend against insect pests, and also provide a basis for the future transgenic plants of the gene, and have broad application prospects in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of Nlsp5 inducing Nicotiana benthamiana cell death and reactive oxygen production are shown in Figure 2. Figure 2 The result diagram of Nlsp5 expression level in transgenic rice; Figure 3 This is the result diagram of Nlsp5 inducing rice defense factors; Figure 4 This is the result of Nlsp5 inducing rice resistance to brown planthoppers; Figure 5 This is the result of Nlsp5 inducing rice resistance to Laodelphax striatellus; Figure 6 This is the result of Nlsp5-induced rice resistance to three planthoppers in the field; Figure 7 This is the result of Nlsp5 inducing rice resistance to Chilo suppressalis; Figure 8 The results of NP19-induced cell death and reactive oxygen production in Nicotiana benthamiana cells; Fig. 9 This is the result of NP19 inducing the expression of defense-related genes in Nicotiana benthamiana; Fig.10 This is the result diagram of NP19 inducing rice defense factors; Fig.11 This is the result of NP19 inducing resistance of Nicotiana benthamiana to Bemisia tabaci; Fig.12 This is the result of NP19 inducing rice resistance to brown planthopper and gray planthopper; Fig.13 This is the result of NP19 inducing rice resistance to Chilo suppressalis; Fig.14 This is a diagram showing the results of NP19 inducing cell death in different plants; Fig.15 This is the band diagram of the Agrobacterium gel electrophoresis detection of the present invention; Fig.16 This is a graph showing the results of the short peptide NP19 of the present invention inducing insect resistance in cotton. DETAILED DESCRIPTION

[0019] 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.

[0020] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0021] The polypeptide Nlsp5 and short peptide NP19 disclosed in the present application can improve the resistance of different plants to different insects. In order to more clearly illustrate the insect resistance of the polypeptide Nlsp5 and short peptide NP19 of the present application, some plant species such as rice, tobacco, and cotton are selected for detailed description, but this is not a limitation on the scope of protection of the present application. At the same time, in terms of the types of insects, the following examples select brown planthoppers, gray planthoppers, whiteback planthoppers, whiteflies, and striped stem borers for detailed description, but it does not mean that the short peptides and polypeptides of the present application have no resistance to other insects.

[0022] Example 1 Construction of polypeptide Nlsp5 transgenic plants 1. Preparation of target protein: The salivary sheath secretion proteins of brown planthopper were collected and sequenced to obtain the nucleotide sequences of the proteins. The nucleotide sequences of different proteins were respectively constructed into the transient expression vector pBINPLUS, and then the expression vectors were transformed into Agrobacterium and injected into Nicotiana benthamiana. The protein that can induce allergic necrosis of Nicotiana benthamiana cells was screened out and named Nlsp5. Its amino acid sequence is shown in SEQID NO:1, and the nucleotide sequence encoding the polypeptide Nlsp5 is shown in SEQID NO:2.

[0023] SEQ ID NO: 1: MASFTSLLSALVLVAGSQGVAVNPMGMADAGLGMVDAGVSAGMGLASTGMDAGKGLANAGMGAASSMSGRMTEEAKKLNGNIQDMLGAQMKGGANILNNMLKMDAAVLTEMGT MISSIAHLTGATSQQMLQILQEVMTKGPLAGAQMLQRLLAYIAQKKIAAGDKIVSKTSSFADGMKQKSFIPLNMLDNMYRTLGSVNDITSALTGTPSLAASKAATALTPPVV.

[0024] SEQ ID NO: 2: .

[0025] 2. Construction of plasmid: Under the control of CaMV35S promoter, the nucleotide sequence encoding polypeptide Nlsp5 was ligated into vector pCAMBIA1301 to obtain plasmid pCAMBIA1301-Nlsp5.

[0026] Processes 1 and 2 were sent to Wuhan Boyuan Biotechnology Co., Ltd. for completion.

[0027] 3. Plasmid transformation: Take 1ul plasmid pCAMBIA1301-Nlsp5 and add it to 50ul EHA105 Agrobacterium competent cells, mix thoroughly and then transfer to an electroporation cup. After electroporation, add 1ml LB liquid culture medium, mix thoroughly and then transfer to a 1.5ml centrifuge tube. Incubate on a shaker at 30℃ and 180rpm for 30min. Take 50ul of the activated Agrobacterium liquid and inoculate it on LB solid culture medium. Incubate in the dark at 30℃ for 48h.

[0028] 4. Agrobacterium detection: Synthesize the corresponding detection primers (the base sequence of the forward primer F is shown in SEQ ID NO: 15: TACTGGAATGGATGCAGGA; the base sequence of the reverse primer R is shown in SEQ ID NO: 16: ACATTTGAGCACCTGCGAGT), configure the PCR system, perform gel electrophoresis detection, prepare 1% agarose gel, and spot the sample. The results are as follows Fig.15 As shown: When the electrophoresis bands of the positive control and sample are clear and of the correct size, and the negative control has no bands, it indicates that the sample can proceed to the next step.

[0029] 5. Inducing transformation and preparing callus tissue: Preparation: Prepare the necessary experimental equipment and reagents, including rice seeds without mold spots and normal buds, alcohol lamp, alcohol cotton (75% pure alcohol), lighter, culture dish, disposable rubber gloves, large tweezers, small tweezers, filter paper (sterilized), timer, 4.5% sodium hypochlorite, empty conical flask (sterilized), sterile water, sealing film, MS induction medium (containing 3 mg / L2,4-D), agar, sucrose, waste liquid tank, etc.

[0030] Disinfection of the clean bench: Wipe the clean bench with alcohol cotton, and place the experimental supplies in the clean bench under ultraviolet light for more than half an hour, and blow air for more than 10 minutes.

[0031] Seed disinfection: Place the seeds in an empty triangular bottle, disinfect with 75% alcohol for 1 minute, wash with sterile water for 1 minute, pour in sodium hypochlorite for disinfection for 15 minutes, and shake several times every minute. Pour out the sodium hypochlorite and wash with sterile water 5 times, 2 minutes each time. Pour out the sterile water and pour the seeds into a petri dish with filter paper to dry.

[0032] Inoculation: Heat the tweezers on an alcohol lamp, and after cooling, place the dried seeds in the induction medium (NB medium 1L, hydrolyzed casein 300 mg / L, glutamine 500 mg / L, proline 500 mg / L, inositol 100mg / L, sucrose 30 g / L, PHytagel 2.6 mg / L, NB+2,4-D 2mg / L), about 15 seeds per dish. Seal the dish with 2~3 layers of sealing film.

[0033] Cultivation: Place the culture dish in an incubator and wait for the formation of callus tissue.

[0034] 6. Agrobacterium infection: Pick up Agrobacterium and put it in the infection solution (1 M MgCl2 500 μL, 0.5 M MES pH 5.7 1mL, 100 mM Acetosyringone 50 μL, ddH2O to 50 mL, prepare it before use.), prepare Agrobacterium resuspension with OD600=0.2, pick up callus in a triangular flask, add Agrobacterium resuspension, infect for 10-15 minutes and discard the bacterial solution, inoculate the callus into co-cultivation medium (NB+2,4-D 2mg / L+AS 100uM / L), and co-cultivate at 20℃ for 48-72h.

[0035] 7. Callus screening: inoculate the callus tissue in step 6 into the screening medium (NB+2, 4-D 2 mg / L, Car 250 mg / L, Hyg 30 mg / L), and culture in the dark at 26°C for 20-30 days; inoculate the positive callus into the secondary screening medium (NB+2, 4-D 2 mg / L, Hyg 50 mg / L), be sure to pick the single clone callus during the callus tissue picking process, and culture in the dark at 26°C for 7-10 days.

[0036] 8. Differentiation and rooting: Inoculate the positive callus into differentiation medium (NB, KT 10mg / L, NAA 0.4mg / L), culture at 25-27℃ light for 15-20 days, and after 2-5cm buds are differentiated, inoculate into rooting medium (1 / 2MS inorganic salts, MS organic components, Hyg30 mg / L), and culture at 30℃ light for 7-10 days.

[0037] 9. Positive seedling detection: CTAB method was used to extract rice genomic DNA, hygromycin resistance, GUS staining and PCR were used to screen homozygous transgenic plants, and qRT-PCR was used to verify the expression of Nlsp5 gene in rice. The PCR screening primers were: Nlsp5-oe-F: 5′-GTTAGGAGCGCAGATGAAGG-3′, as shown in SEQ ID NO: 5; Nlsp5-oe-R: 5′-ACATTTGAGCACCTGCGAGT-3′, as shown in SEQ ID NO: 6; qRT-PCR verification of the expression of Nlsp5 gene in rice Primers: RT-Nlsp5-oe-F: 5′-AGATTGTTTCCAAGACCAGCA-3′, as shown in SEQ ID NO: 7; RT-Nlsp5-oe-R: 5′-CGTTAACTGATCCAAGCGTTC-3′, as shown in SEQ ID NO:8.

[0038] Results: qRT-PCR verification showed that the expression of Nlsp5 gene was not detected in wild-type rice (WT), while the expression of Nlsp5 gene was detected in homozygous transgenic plants oe4 and oe5. Figure 2 shown.

[0039] Example 2 Polypeptide Nlsp5 induces transgenic rice defense factors Transgenic rice expressing Nlsp5 and wild-type rice from Example 1 were selected, and rice stems were placed in a glass cylinder containing 20 newly hatched female adults of brown planthoppers. After 8 and 24 hours, the brown planthoppers were removed and the rice stems were collected. 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 AmplexRed Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen).

[0040] Results: The quantitative analysis of hormone concentrations showed that the introduction of the Nlsp5 gene into rice could significantly induce an increase in the contents of hydrogen peroxide, jasmonic acid, and jasmonic acid-isoleucine. Figure 3 As shown, 3A is the content of hydrogen peroxide, 3B is the content of jasmonic acid-isoleucine, 3C is the content of jasmonic acid, and 3D is the content of salicylic acid.

[0041] Example 3: Polypeptide Nlsp5 induces tobacco defense factors The constructed pBINPLUS-Nlsp5 vector was introduced into the Agrobacterium tumefaciens GV3101 strain by electroporation. The Agrobacterium tumefaciens GV3101 strain was cultured in LB liquid medium containing kanamycin and rifampicin at 28°C for 24 hours. The recombinant strain was washed three times with infiltration buffer (1 M MgCl2, 100 mM MES, 150 mM acetosyringone) and resuspended to OD 600 The Agrobacterium tumefaciens cell suspension was then injected into the leaves of Nicotiana benthamiana using a needle-free syringe. Figure 1 As shown: The peptide Nlsp5 can induce visible allergic cell death in Nicotiana benthamiana, where 1A is cell death and 1B is ROS burst.

[0042] Example 4: Polypeptide Nlsp5 induces insect resistance in transgenic rice (1) Inducing resistance of rice to brown planthopper The Nlsp5 transgenic rice plants (oe4 strain or oe5 strain) and wild-type rice plants obtained according to the method of Example 1 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 nymphs on each plant was counted 1, 2, 4, 8, 24 and 48 hours after release; for the fecundity test, one female brown planthopper and two male brown planthoppers were placed on the rice stalks covered with glass cylinders (0-8 cm above the ground). After seven days, the number of eggs laid by each female insect was counted under an optical microscope.

[0043] Results: After the Nlsp5 gene was transferred into rice plants, the number of brown planthoppers staying on rice and the number of brown planthoppers laying eggs were significantly reduced, indicating that Nlsp5 can induce rice resistance to brown planthoppers. Figure 4 As shown, 4A is the number of brown planthoppers on each rice plant of oe4 strain, 4B is the number of brown planthoppers on each rice plant of oe5 strain, and 4C is the number of eggs laid by brown planthoppers on different plants.

[0044] (2) Inducing resistance of rice to Laodelphax striatellus The Nlsp5 transgenic rice plants (oe4 strain or oe5 strain) obtained according to the method of Example 1 and wild-type rice plants were enclosed in glass cylinders (8 cm in diameter and 8 cm in height), and 15 fourth-instar nymphs were released. The nymphs of gray leafhoppers on each plant were counted 1, 2, 4, 8, 24 and 48 hours after release; for the fecundity test, one female gray leafhopper and two male gray leafhoppers were placed on the rice stalks covered with glass cylinders (0-8 cm above the ground). After seven days, the number of eggs laid by each female insect was counted under an optical microscope.

[0045] Results: After the Nlsp5 gene was transferred into rice plants, the number of gray leafhoppers staying on rice and the egg laying of gray leafhoppers were significantly reduced, indicating that Nlsp5 can induce rice resistance to gray leafhoppers. Figure 5 Among them, 5A is the number of gray leafhoppers on each rice plant of oe4 strain, 5B is the number of gray leafhoppers on each rice plant of oe5 strain, and 5C is the number of eggs laid by gray leafhoppers on different plants.

[0046] (3) Inducing field resistance of rice to three planthoppers 24 Nlsp5 transgenic rice plants (oe4 strain and oe5 strain) obtained according to the method of Example 1 and 24 wild-type rice plants were planted in a field, the rice was covered with a mesh net, and about 500 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.

[0047] Results: After the Nlsp5 gene was transferred into rice plants, the number of three planthoppers staying on rice was significantly reduced, indicating that Nlsp5 can induce field resistance of rice to three planthoppers. Figure 6 shown.

[0048] (4) Inducing resistance of rice to Chilo suppressalis The Nlsp5 transgenic rice plants (oe4 strain or oe5 strain) and wild-type rice plants obtained according to the method of Example 1 were sealed in glass cylinders (8 cm in diameter and 8 cm in height), and after weighing, five second-instar nymphs of Chilo suppressalis were placed in the glass cylinders and attached to the base stem. After feeding for 7 days, each insect was re-weighed to evaluate weight gain.

[0049] Results: After the Nlsp5 gene was transferred into rice plants, the feeding of the Chilo suppressalis was significantly reduced, indicating that Nlsp5 can induce resistance of rice to the Chilo suppressalis. Figure 7 shown.

[0050] Example 5 Short peptide NP19 induces plant defense response 1. Synthesis of short peptide NP19: The laboratory found that the polypeptide Nlsp5 sequence has a conserved domain based on sequence comparison, and the short peptide NP19 was obtained by truncating the protein. The amino acid sequence of the short peptide NP19 is shown in the sequence list SEQ ID NO:3, and the nucleotide sequence encoding the short peptide NP19 is shown in the sequence list SEQ ID NO:4. The nucleotide sequence of the short peptide NP19 was sent to GenScript for synthesis.

[0051] SEQ ID NO:3: KGPLAGAQMLQRLLAYIAQ; SEQ ID NO:4: AAAGGACCACTCGCAGGGTGCTCAAATGTTACAGAGGTTATTAGCATACATTGCACAA.

[0052] 2. Induce cell death and reactive oxygen species (ROS) production in Nicotiana benthamiana leaves: adjust the concentration of NP19 to 1nM, 20nM, 50nM, 100nM, 500nM, and 1μM. Select Nicotiana benthamiana of about 4 weeks old, and inject the NP19 short peptide into the Nicotiana benthamiana leaves from the back of the leaves with a 1ml syringe without a needle. At the same time, pure water was used as a control. After 48 hours of injection, observe the allergic necrosis reaction. 12 hours after the injection, take the treated leaves and put them in DAB staining solution (1mg / ml, pH=3.8). After 8 hours of treatment at room temperature and away from light, remove the staining solution, add anhydrous ethanol for decolorization, and take out the leaves after the green color of the leaves is completely removed and take pictures.

[0053] Results: NP19 at a concentration of 50 nM or more could induce visible allergic cell death in N. benthamiana. After 12 h of NP19 treatment, 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 NP19 induced ROS production in N. benthamiana leaves. Figure 8 shown.

[0054] 3. Induce the expression of defense-related genes in Nicotiana benthamiana: After the NP19 short peptide 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: NbEF1a-QF: 5′-AGAGGCCCTCAGACAAAC-3′, as shown in SEQ ID NO: 9; NbEF1a-QR: 5′-TAGGTCCAAAGGTCACAA-3′, as shown in SEQ ID NO: 10; NbPR3-QF: 5′-TGGGGTTATTGCTGGCTTAG-3′, as shown in SEQ ID NO: 11; NbPR3-QR: 5′-GGGTCATCCAAAACCAGAGA-3′, as shown in SEQ ID NO: 12; NbPR4-QF: 5′-GGCCAAGATTCCTGTGGTAGAT-3′, as shown in SEQ ID NO: 13; NbPR4-QR: 5′-CACTGTTGTTTGAGTTCCTGTTCCT-3′, as shown in SEQ ID NO:14.

[0055] Results: Fluorescence quantitative PCR results showed that NP19 short peptide could significantly induce jasmonic acid signaling-related genes after 1d and 2d injection of N. benthamiana NbPR and NbPR Expressions such as Fig. 9 As shown, among which 9A is the related gene NbPR The expression level of 9B is related gene NbPR The amount of expression.

[0056] 4. Inducing rice defense factors: After treating rice stems with NP19 short peptide 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 AmplexRed Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen).

[0057] Results: The quantitative analysis results of hormone concentrations showed that NP19 could significantly induce the increase of hydrogen peroxide, jasmonic acid and jasmonic acid-isoleucine contents. Fig.10 As shown, 10A is the content of hydrogen peroxide, 10B is the content of jasmonic acid-isoleucine, 10C is the content of jasmonic acid, and 10D is the content of salicylic acid.

[0058] Example 6 Short peptide NP19 induces insect resistance in tobacco and rice (1) Inducing tobacco resistance to whitefly Fifteen adult whiteflies were placed in the center of each pair of leaves treated with NP19 short peptide or water. The number of insects on each leaf was recorded at 1, 2, 4, 8, 12, 24 and 48 hours.

[0059] Results: After the leaves of N. benthamiana were treated with NP19 short peptide, the number of insects was significantly reduced, indicating that NP19 short peptide could induce the resistance of N. benthamiana to whitefly. Fig.11 shown.

[0060] (2) Inducing resistance of rice to brown and gray planthoppers One rice plant with stems painted with short peptide NP19 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 brown / grass planthopper nymphs on each plant were counted 1, 2, 4, 8, 24, and 48 hours after release; Results: After applying the short peptide NP19, the number of brown / grass planthoppers staying on rice was significantly reduced, and the egg laying of brown / grass planthoppers and gray planthoppers also decreased significantly, indicating that NP19 can induce rice resistance to brown / grass planthoppers. Fig.12 As shown, 12A is the number of gray planthoppers on each rice plant, 12B is the egg-laying amount of gray planthoppers, 12C is the number of brown planthoppers on each rice plant, and 12D is the egg-laying amount of brown planthoppers.

[0061] (3) Inducing resistance of rice to Chilo suppressalis One rice plant with stems painted with short peptide NP19 and one rice plant with stems painted with pure water were sealed in glass cylinders (8 cm in diameter; 8 cm in height) and weighed. Five second-instar nymphs were placed in the glass cylinders and attached to the base stem. After feeding for 7 days, each insect was re-weighed to assess weight gain.

[0062] Results: The short peptide NP19 treated rice significantly reduced the feeding of the Chilo suppressalis, indicating that NP19 can induce resistance of rice to the Chilo suppressalis. Fig.13 shown.

[0063] Example 7 Short peptide NP19 induces cell death in different plants Treat plant leaves with the short peptide NP19. For dicots (cotton, eggplant), the short peptide was adjusted to a concentration of 0.5uM and then infiltrated into the leaves of different species using a needle-free syringe. For monocots (rice, corn), micro wounds were made using quartz sand and then the peptide solution was infiltrated into the plants.

[0064] Results: The short peptide NP19 could induce cell death in cotton, eggplant, rice and corn leaves. Fig.14 shown.

[0065] Example 8 Short peptide NP19 induces insect resistance in cotton Selective test: 8 second-instar larvae of cotton bollworm or 15 female adults of cotton aphid were introduced into the center of each pair of cotton leaves treated with 0.5 μM NP19 short peptide or water. The number of insects on each leaf was recorded at 1, 2, 4, 8, 12, 24 and 48 hours.

[0066] Each pair of cotton leaves treated with 0.5 μM NP19 short peptide or water was placed in a petri dish. Four cotton aphid adults were placed on each leaf, and after 3 days, the newborn offspring were counted under a microscope.

[0067] Results: Compared with cotton leaves treated with short peptide NP19, cotton bollworm larvae or cotton aphids tended to feed on cotton leaves treated with water; compared with cotton leaves treated with short peptide NP19, cotton aphids had significantly stronger reproductive capacity on cotton leaves treated with water. This indicates that NP19 can induce cotton to develop resistance to cotton bollworm or cotton aphids, such as Fig.16 As shown, 16A is a growth picture of cotton aphids or cotton bollworms on cotton leaves, 16B is a statistical curve of cotton bollworm numbers, 16C is a statistical curve of cotton aphid numbers, and 16D is a statistical curve of cotton aphid offspring numbers.

[0068] 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. A short peptide NP19 derived from a salivary sheath secretory protein of brown planthopper, characterized in that: The amino acid sequence of the short peptide NP19 is shown in SEQ ID NO:

3.

2. The short peptide NP19 derived from the salivary sheath secretion protein of brown planthopper according to claim 1, characterized in that: The nucleotide sequence encoding the short peptide NP19 is shown in SEQ ID NO:

4.

3. A brown planthopper salivary sheath secretion polypeptide Nlsp5, characterized in that: The invention comprises the short peptide NP19 as described in any one of claims 1 to 2.

4. The brown planthopper salivary sheath secretion polypeptide Nlsp5 according to claim 3, characterized in that: The amino acid sequence of the polypeptide Nlsp5 is shown in SEQ ID NO:

1.

5. The brown planthopper salivary sheath secretion polypeptide Nlsp5 according to claim 4, characterized in that: The nucleotide sequence encoding the polypeptide Nlsp5 is shown in SEQ ID NO:

2.

6. Use of the short peptide NP19 according to any one of claims 1 to 2 or the polypeptide Nlsp5 according to any one of claims 3 to 5 in improving insect resistance of plants.

7. The use according to claim 6, characterized in that: The short peptide NP19 or polypeptide Nlsp5 is used as an elicitor to stimulate the defense response or allergic response of the plant.

8. The use according to claim 6, characterized in that: The short peptide NP19 is sprayed on tobacco or rice as a pesticide or as a main component of a pesticide.

9. Use of the short peptide NP19 according to any one of claims 1 to 2 in the preparation of pesticides.

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