A short peptide NP19, a polypeptide Nlsp5 and their applications

Abstract

The short peptide NP19, polypeptide Nlsp5 provided by the present invention and their applications belong to the technical field of bioengineering. The short peptide NP19 and polypeptide Nlsp5 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 polypeptide Nlsp5 of the present invention provide a new way for improving the insect resistance of plants, provide resources for the development and application of future biological pesticides, can be used as a new type of microbial protein pesticide to defend against insect pest attacks, and also provide a basis for future transgenic plants of this gene, having broad application prospects in agricultural production.

Description

Technical Field

[0001] The present invention relates to a short peptide NP19, a polypeptide Nlsp5 and their applications, belonging to the field of bioengineering technology. Background Art

[0002] In recent years, biological control methods have been increasingly widely used in pest control. Biological control utilizes natural enemies or microorganisms in nature to control pests. For example, natural enemies such as parasitic wasps, predatory insects, fungi, bacteria, etc. are used to suppress the number of pests. In addition, the insect resistance of plants themselves has also received increasing attention. Through the study of the natural insect resistance mechanism of plants, scientists have found that many plants can resist pests by secreting specific chemical substances 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 technologies face some challenges. First, the effect of biological control is usually greatly affected by environmental conditions, and some natural enemies or microorganisms may not achieve the best effect under different climate and soil conditions. Second, although some natural insect resistance elicitors have been discovered, the mechanisms of these substances are not yet fully understood, and the stability and persistence of their applications are still difficult points in research. Therefore, screening for highly efficient and stable insect resistance elicitors has become an important research direction at present.

[0004] In recent years, screening and identifying insect resistance elicitors has become a research hotspot in the field of biological control. Through in-depth research on plant immune responses, scientists have gradually revealed the roles of various natural insect resistance elicitors (such as certain proteins, carbohydrates, and small molecule compounds) in the insect resistance mechanism. In particular, the immune signaling pathway mediated by pattern recognition receptors (PRRs) has been proven to play an important role in the defense of plants against pests. Based on these findings, researchers are exploring how to improve the insect resistance of plants by exogenously applying these elicitors or by genetic engineering means.

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

[0006] Therefore, developing new insect resistance elicitors or improving the plant's own immune system through genetic engineering to enhance its defense ability against pests is an important direction for the development of current biological control technologies. Summary of the Invention

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

[0008] To solve the above technical problems:

[0009] The first object of the present invention is to provide a short peptide NP19 derived from the secreted protein of the salivary sheath of Nilaparvata lugens, and the amino acid sequence of the short peptide NP19 is shown in SEQ ID NO: 3.

[0010] Furthermore, the nucleotide sequence encoding the short peptide NP19 is shown in SEQ ID NO: 4.

[0011] The second object of the present invention is to provide a polypeptide Nlsp5 secreted by the salivary sheath of Nilaparvata lugens, which includes the above-mentioned short peptide NP19.

[0012] Furthermore, the amino acid sequence of the polypeptide Nlsp5 is shown in SEQ ID NO: 1.

[0013] Furthermore, the nucleotide sequence encoding the polypeptide Nlsp5 is shown in SEQ ID NO: 2.

[0014] The third object of the present invention is to provide the application of the above-mentioned short peptide NP19 or the above-mentioned polypeptide Nlsp5 in improving the insect resistance of tobacco or rice.

[0015] Furthermore, the short peptide NP19 or the polypeptide Nlsp5 is used as an elicitor to stimulate the defense response or hypersensitive response of tobacco or rice.

[0016] Furthermore, the short peptide NP19 or the polypeptide Nlsp5 is sprayed on tobacco or rice as a pesticide or as the main component of a pesticide.

[0017] The fourth object of the present invention is to provide the application of the above-mentioned short peptide NP19 and the above-mentioned polypeptide Nlsp5 in the preparation of pesticides.

[0018] The beneficial technical effects achieved by the present invention: 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 of 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 future transgenic plants of this gene, and have broad application prospects in agricultural production. Brief Description of the Drawings

[0019] Figure 1 Results of Nlsp5-induced cell death and reactive oxygen species production in Nicotiana benthamiana

[0020] Figure 2 Results of Nlsp5 expression levels in transgenic rice

[0021] Figure 3 Results of Nlsp5-induced rice defense factors

[0022] Figure 4 Results of Nlsp5-induced resistance of rice to Nilaparvata lugens

[0023] Figure 5 Results of Nlsp5-induced resistance of rice to Laodelphax striatellus

[0024] Figure 6 Results of Nlsp5-induced field resistance of rice to three species of planthoppers

[0025] Figure 7 Results of Nlsp5-induced resistance of rice to Chilo suppressalis

[0026] Figure 8 Results of NP19-induced cell death and reactive oxygen species production in Nicotiana benthamiana

[0027] Figure 9 Results of NP19-induced expression of defense-related genes in Nicotiana benthamiana

[0028] Figure 10 Results of NP19-induced rice defense factors

[0029] Figure 11 Results of NP19-induced resistance of Nicotiana benthamiana to Bemisia tabaci

[0030] Figure 12 Results of NP19-induced resistance of rice to Nilaparvata lugens and Laodelphax striatellus

[0031] Figure 13 Results of NP19-induced resistance of rice to Chilo suppressalis

[0032] Figure 14 Results of NP19-induced cell death in different plants

[0033] Figure 15 Agrobacterium gel electrophoresis detection band diagram of the present invention

[0034] Figure 16 Results of the short peptide NP19-induced insect resistance in cotton of the present invention Detailed implementation manners

[0035] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0036] The present invention patent will be further described below in conjunction with the accompanying drawings and embodiments.

[0037] The polypeptides Nlsp5 and the 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 polypeptides Nlsp5 and the short peptide NP19 in the present application, some plant species such as rice, tobacco, and cotton are selected for detailed description, but this does not limit the protection scope of the present application. At the same time, in terms of the types of insects, the following embodiments select Nilaparvata lugens, Laodelphax striatellus, Sogatella furcifera, Bemisia tabaci, Chilo suppressalis, etc. for detailed description, but it does not mean that the short peptides and polypeptides in the present application have no resistance to other insects.

[0038] Example 1 Construction of transgenic plants expressing polypeptide Nlsp5

[0039] 1. Preparation of target protein: Collect the salivary sheath secreted proteins of Nilaparvata lugens, perform protein sequencing to obtain the nucleotide sequences of the proteins, respectively construct the nucleotide sequences of different proteins onto the transient expression vector pBINPLUS, and then transform the expression vector into Agrobacterium and inject it into Nicotiana benthamiana. Screen out the proteins that can induce the hypersensitive necrosis of Nicotiana benthamiana cells, and name them Nlsp5. Its amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence encoding polypeptide Nlsp5 is shown in SEQ ID NO:2.

[0040] SEQ ID NO:1:

[0041] MASFTSLLSALVLVAGSQGVAVNPMGMADAGLGMVDAGVSAGMGLASTGMDAGKGLANAGMGAASSMSGRMTEEAKKLNGNIQDMLGAQMKGGANILNNMLKMDAAVLTEMGTMISSIAHLTGATSQQMLQILQEVMTKGPLAGAQMLQRLLAYIAQKKIAAGDKIVSKTSSFADGMKQKSFIPLNMLDNMYRTLGSVNDITSALTGTPSLAASKAATALTPPVV.

[0042] SEQ ID NO:2:

[0043] ATGGCTTCATTCACATCTCTATTATCCGCTCTTGTGTTGGTCGCTGGTTCACAGGGAGTGGCTGTGAACCCAATGGGAATGGCGGATGCAGGACTGGGAATGGTGGATGCAGGAGTTTCAGCGGGAATGGGACTGGCAAGTACTGGAATGGATGCAGGAAAGGGCTTGGCGAATGCAGGAATGGGTGCTGCTAGTTCCATGTCTGGTCGCATGACGGAGGAAGCTAAAAAATTAAATGGAAACATTCAGGACATGTTAGGAGCGCAGATGAAGGGAGGTGCAAACATACTGAATAATATGTTGAAGATGGACGCTGCCGTTCTCACTGAAATGGGGACAATGATCAGTTCTATTGCACACTTAACTGGAGCAACATCACAACAGATGTTGCAAATACTACAAGAAGTCATGACGAAAGGACCACTCGCAGGTGCTCAAATGTTACAGAGGTTATTAGCATACATTGCACAAAAGAAGATAGCAGCAGGTGATAAGATTGTTTCCAAGACCAGCAGCTTCGCTGATGGCATGAAGCAAAAATCGTTTATACCATTAAACATGTTGGATAATATGTACAGAACGCTTGGATCAGTTAACGATATAACATCAGCATTGACTGGCACTCCTTCGCTTGCCGCATCAAAAGCAGCTACAGCCCTCACTCCACCCGTAGTTTGA。

[0044] 2. Construction of plasmid: Under the control of the CaMV35S promoter, the above nucleotide sequence encoding polypeptide Nlsp5 was ligated into the vector pCAMBIA1301 to obtain plasmid pCAMBIA1301-Nlsp5.

[0045] Processes 1 and 2 were sent to Wuhan Boyuan Biotechnology Co., Ltd. for completion.

[0046] 3. Plasmid transformation: Take 1 μl of plasmid pCAMBIA1301-Nlsp5 and add it to 50 μl of Agrobacterium tumefaciens EHA105 competent cells. After thorough mixing, transfer the mixture to an electroporation cuvette. After electroporation, add 1 ml of LB liquid medium, mix well, and transfer it to a 1.5 ml centrifuge tube. Incubate it on a shaker at 30 °C and 180 rpm for 30 min. Then, take 50 μl of the activated Agrobacterium liquid and inoculate it onto an LB solid medium, and incubate it in the dark at 30 °C for 48 h.

[0047] 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, and detect it by gel electrophoresis. Prepare a 1% agarose gel and load the samples. The results are as Figure 15 shown: When the electrophoresis bands of the positive control and the sample are clear and of the correct size, and there is no band in the negative control, it indicates that the sample can proceed to the next step.

[0048] 5. Induce transformation to prepare callus:

[0049] Preparation work: Prepare the required experimental equipment and reagents, including rice seeds without mildew spots and with normal germ pores, alcohol lamp, alcohol cotton (75% pure alcohol), lighter, culture dish, disposable rubber gloves, large forceps, small forceps, filter paper (after sterilization), timer, 4.5% sodium hypochlorite, empty triangular flask (after sterilization), sterile water, sealing film, MS induction medium (containing 3 mg / L 2,4-D), agar, sucrose, waste liquid tank, etc.

[0050] Sterilize the laminar flow hood: Wipe the laminar flow hood with alcohol cotton, and place the experimental supplies in the laminar flow hood and irradiate them with ultraviolet light for more than half an hour and blow them for more than 10 minutes.

[0051] Seed disinfection: Place the seeds in an empty triangular flask, disinfect them with 75% alcohol for 1 min, wash them with sterile water, once a minute for 1 min, pour in sodium hypochlorite and disinfect for 15 minutes, shake several times every minute. Pour out the sodium hypochlorite and add sterile water to wash 5 times, 2 minutes each time. Pour out the sterile water, and pour the seeds into a culture dish with filter paper to dry.

[0052] Inoculation: Heat the small forceps until it turns red on the alcohol lamp. After it cools down, put the dry seeds into the induction medium (1 L of NB medium, 300 mg / L of casein hydrolysate, 500 mg / L of glutamine, 500 mg / L of proline, 100 mg / L of inositol, 30 g / L of sucrose, 2.6 mg / L of PHytagel, NB + 2,4-D 2 mg / L), about 15 seeds per petri dish. Seal the petri dish with sealing film for 2 - 3 layers.

[0053] Cultivation: Put the petri dish into the incubator for cultivation and wait for the formation of callus.

[0054] 6. Agrobacterium infection: Pick Agrobacterium into the infection solution (500 μL of 1 M MgCl2, 1 mL of 0.5 M MES pH 5.7, 50 μL of 100 mM Acetosyringone, made up to 50 mL with ddH2O, freshly prepared). Prepare an Agrobacterium resuspension with OD600 = 0.2. Pick the callus into a triangular flask, add the Agrobacterium resuspension, after infecting for 10 - 15 minutes, discard the bacterial liquid, and inoculate the callus onto the co-culture medium (NB + 2,4-D 2 mg / L + AS 100 μM / L), and co-culture at 20 °C for 48 - 72 h.

[0055] 7. Callus screening: Inoculate the callus in step 6 onto the screening medium (NB + 2,4-D 2 mg / L, Car 250 mg / L, Hyg 30 mg / L), and culture it in the dark at 26 °C for 20 - 30 days; inoculate the positive callus onto the secondary screening medium (NB + 2,4-D 2 mg / L, Hyg 50 mg / L). When picking the callus, be sure to pick monoclonal callus, and culture it in the dark at 26 °C for 7 - 10 days.

[0056] 8. Differentiation and rooting: Inoculate the positive callus onto the differentiation medium (NB, KT 10 mg / L, NAA 0.4 mg / L), and culture it under light at 25 - 27 °C for 15 - 20 days. After the buds grow to 2 - 5 cm, inoculate them onto the rooting medium (1 / 2 MS inorganic salts, MS organic components, Hyg 30 mg / L), and culture it under light at 30 °C for 7 - 10 days.

[0057] 9. Detection of positive seedlings: Extract rice genomic DNA by the CTAB method, screen for homozygous transgenic plants by hygromycin resistance, GUS staining and PCR. Verify the expression of the Nlsp5 gene in rice by qRT-PCR. The primers for PCR screening of the strain are:

[0058] Nlsp5-oe-F: 5‘-GTTAGGAGCGCAGATGAAGG-3‘, as shown in SEQ ID NO:5;

[0059] Nlsp5-oe-R: 5'-ACATTTGAGCACCTGCGAGT-3', as shown in SEQ ID NO:6;

[0060] Primers for qRT-PCR verification of the expression of Nlsp5 gene in rice:

[0061] RT-Nlsp5-oe-F: 5'-AGATTGTTTCCAAGACCAGCA-3', as shown in SEQ ID NO:7;

[0062] RT-Nlsp5-oe-R: 5'-CGTTAACTGATCCAAGCGTTC-3', as shown in SEQ ID NO:8.

[0063] Results: Verified by qRT-PCR, 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, as Figure 2 shown.

[0064] Example 2 Polypeptide Nlsp5 Induces Defense Factors in Transgenic Rice

[0065] Select the transgenic rice expressing Nlsp5 and wild-type rice in Example 1. Put the rice stems into a glass cylinder, which contains 20 newly hatched female adults of Nilaparvata lugens. After 8 and 24 hours, take out the Nilaparvata lugens and collect the rice stems. Grind the samples in liquid nitrogen, extract with ethyl acetate containing labeled internal standards ( 2 D 4 -SA, 2 D 6 -JA and 2 D 6 -JA-Ile), and analyze salicylic acid, jasmonic acid and jasmonic acid-isoleucine by high performance liquid chromatography tandem mass spectrometry. Quantitatively analyze the hydrogen peroxide concentration using AmplexRed Hydrogen Peroxide / Peroxidase Assay Kit (Invitrogen).

[0066] Results: The results of quantitative analysis of hormone concentrations showed that the transfer of Nlsp5 gene in rice could significantly induce the increase in the contents of hydrogen peroxide, jasmonic acid and jasmonic acid-isoleucine, as Figure 3 shown, where 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.

[0067] Example 3 Polypeptide Nlsp5 Induces Defense Factors in Tobacco

[0068] The constructed pBINPLUS-Nlsp5 vector was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. Agrobacterium tumefaciens strain GV3101 was cultured in LB liquid medium containing kanamycin and rifampicin at a culture temperature of 28 °C for 24 hours. The recombinant strain was washed three times with infiltration buffer (1M MgCl 2 , 100 mM MES, 150 mM acetosyringone) and resuspended to an OD 600 of 0.4. Then, the suspension of Agrobacterium tumefaciens cells was injected into the leaves of Nicotiana benthamiana using a needleless syringe. The results are as shown in Figure 1 : The polypeptide Nlsp5 can induce a visible hypersensitive cell death phenomenon on Nicotiana benthamiana. Among them, 1A shows cell death and 1B shows ROS burst.

[0069] Example 4 Insect Resistance Induced by Polypeptide Nlsp5 in Transgenic Rice

[0070] (1) Inducing Resistance of Rice to Brown Planthopper

[0071] The Nlsp5 transgenic rice plants (oe4 line or oe5 line) obtained by the method of Example 1 and wild-type rice plants were enclosed in a glass cylinder (diameter 8 cm; height 8 cm), and 15 fourth-instar nymphs were released. The number of 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 and two male brown planthoppers were placed on the rice stem (0 - 8 cm above the ground) covered with a glass cylinder. Seven days later, the number of eggs laid by each female was counted under an optical microscope.

[0072] Results: After transferring the Nlsp5 gene into rice plants, the number of brown planthoppers staying on the rice can be significantly reduced, and the egg-laying amount of brown planthoppers can be significantly decreased, indicating that Nlsp5 can induce the resistance of rice to brown planthoppers, as shown in Figure 4 : Among them, 4A shows the number of brown planthoppers on each oe4-line rice plant, 4B shows the number of brown planthoppers on each oe5-line rice plant, and 4C shows the number of eggs laid by brown planthoppers on different plants.

[0073] (2) Inducing Resistance of Rice to Small Brown Planthopper

[0074] The Nlsp5 transgenic rice plants (oe4 line or oe5 line) obtained by the method of Example 1 and wild-type rice plants were enclosed in a glass cylinder (8 cm in diameter; 8 cm in height), and 15 fourth-instar nymphs were released. The Laodelphax striatellus nymphs on each plant were counted at 1, 2, 4, 8, 24, and 48 hours after release; for the fecundity test, one female Laodelphax striatellus and two male Laodelphax striatellus were placed on the rice stem (0 - 8 cm above the ground) covered with a glass cylinder. Seven days later, the number of eggs laid by each female was counted under an optical microscope.

[0075] Results: After transferring the Nlsp5 gene into rice plants, the number of Laodelphax striatellus staying on the rice can be significantly reduced, and the egg-laying amount of Laodelphax striatellus can be significantly reduced, indicating that Nlsp5 can induce rice resistance to Laodelphax striatellus, as Figure 5 shown. Among them, 5A is the number of Laodelphax striatellus on each oe4-line rice plant, 5B is the number of Laodelphax striatellus on each oe5-line rice plant, and 5C is the number of eggs laid by Laodelphax striatellus on different plants.

[0076] (3) Inducing field resistance of rice to three kinds of planthoppers

[0077] Twenty-four Nlsp5 transgenic rice plants (oe4 line and oe5 line) obtained by the method of Example 1 and 24 wild-type rice plants were planted in the field, the rice was covered with a perforated net, and about 500 brown planthoppers, 500 brown planthoppers, and 500 white-backed planthoppers were released into the field. At 40, 54, and 68 days after planting the rice, the total number of all planthoppers (including nymphs and adults) on each rice plant was counted.

[0078] Results: After transferring the Nlsp5 gene into rice plants, the number of three kinds of planthoppers staying on the rice can be significantly reduced, indicating that Nlsp5 can induce field resistance of rice to three kinds of planthoppers, as Figure 6 shown.

[0079] (4) Inducing resistance of rice to Chilo suppressalis

[0080] The Nlsp5 transgenic rice plants (oe4 line or oe5 line) obtained by the method of Example 1 and wild-type rice plants were enclosed in a glass cylinder (8 cm in diameter; 8 cm in height). After weighing, 5 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 reweighed to evaluate the weight gain.

[0081] Results: After transferring the Nlsp5 gene into rice plants, the feeding of Chilo suppressalis can be significantly reduced, indicating that Nlsp5 can induce rice resistance to Chilo suppressalis, as Figure 7 shown.

[0082] Example 5: Short peptide NP19 induces plant defense response

[0083] 1. Synthesis of short peptide NP19: According to sequence alignment in the laboratory, it was found that the polypeptide Nlsp5 sequence has a conserved domain. The protein was truncated to obtain the short peptide NP19. The amino acid sequence of the short peptide NP19 is shown in SEQ ID NO:3 in the sequence listing, and the nucleotide sequence encoding the short peptide NP19 is shown in SEQ ID NO:4 in the sequence listing. The nucleotide sequence of the short peptide NP19 was sent to GenScript for synthesis.

[0084] SEQ ID NO:3: KGPLAGAQMLQRLLAYIAQ;

[0085] SEQ ID NO:4:

[0086] AAAGGACCACTCGCAGGTGCTCAAATGTTACAGAGGTTATTAGCATACATTGCACAA.

[0087] 2. Inducing cell death and reactive oxygen species (ROS) production in Nicotiana benthamiana leaves: Adjust the concentration of NP19 to 1 nM, 20 nM, 50 nM, 100 nM, 500 nM, 1 μM. Select Nicotiana benthamiana plants about 4 weeks old, and use a 1 ml syringe without a needle to inject the short peptide NP19 into the Nicotiana benthamiana leaves from the back of the leaves. At the same time, pure water was used as a control. After 48 hours of injection, observe the hypersensitive necrosis reaction. After 12 hours of injection, take the treated leaves and put them into DAB staining solution (1 mg / ml, pH = 3.8), and treat them in the dark at room temperature for 8 h. Then remove the staining solution, add absolute ethanol for decolorization. After all the green color of the leaves has faded, take out the leaves and take pictures.

[0088] Results: NP19 at a concentration of 50 nM or above can induce visible hypersensitive cell death on Nicotiana benthamiana. After 12 h of treatment with NP19 on the leaves, obvious brown deposits appeared at the injection site. Moreover, 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 induces ROS production in Nicotiana benthamiana leaves, as Figure 8 shown.

[0089] 3. Inducing the expression of defense-related genes in Nicotiana benthamiana: After injecting the short peptide NP19 and pure water into Nicotiana benthamiana leaves, samples were taken at 24 h and 48 h 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. According to the instructions of the quantitative kit, take 2 μL of the reverse transcription product as a template, and then by real-time fluorescence quantitative PCR method, using EF-1α as the internal reference gene, for the jasmonic acid signal-related genes related to the resistance of Nicotiana benthamianaNbPR3 and NbPR4 the expression levels were measured. The primers used were as follows:

[0090] NbEF1a-QF: 5'-AGAGGCCCTCAGACAAAC-3', as shown in SEQ ID NO:9;

[0091] NbEF1a-QR: 5'-TAGGTCCAAAGGTCACAA-3', as shown in SEQ ID NO:10;

[0092] NbPR3-QF: 5'-TGGGGTTATTGCTGGCTTAG-3', as shown in SEQ ID NO:11;

[0093] NbPR3-QR: 5'-GGGTCATCCAAAACCAGAGA-3', as shown in SEQ ID NO:12;

[0094] NbPR4-QF: 5'-GGCCAAGATTCCTGTGGTAGAT-3', as shown in SEQ ID NO:13;

[0095] NbPR4-QR: 5'-CACTGTTGTTTGAGTTCCTGTTCCT-3', as shown in SEQ ID NO:14.

[0096] Results: The results of fluorescence quantitative PCR showed that the NP19 peptide could significantly induce the expression of jasmonic acid signal-related genes NbPR3 and NbPR4 after injection into Nicotiana benthamiana for 1 day and 2 days, as Figure 9 shown, where 9A is the expression level of the related gene NbPR3 and 9B is the expression level of the related gene NbPR4 .

[0097] 4. Inducing rice defense factors: After treating rice stems with the NP19 peptide and water, the rice stems were collected, ground in liquid nitrogen, extracted with ethyl acetate containing labeled internal standards ( 2 D 4 -SA, 2 D 6 -JA and 2 D 6 -JA-Ile), and salicylic acid, jasmonic acid, and jasmonic acid-isoleucine were analyzed using high-performance liquid chromatography tandem mass spectrometry. The hydrogen peroxide concentration was quantitatively analyzed using an AmplexRed hydrogen peroxide / peroxidase assay kit (Invitrogen).

[0098] Results: The results of quantitative analysis of hormone concentrations showed that NP19 could significantly induce an increase in the contents of hydrogen peroxide, jasmonic acid, and jasmonoyl-isoleucine, as Figure 10 shown, where 10A is the content of hydrogen peroxide, 10B is the content of jasmonoyl-isoleucine, 10C is the content of jasmonic acid, and 10D is the content of salicylic acid.

[0099] Example 6 Induction of Insect Resistance in Tobacco and Rice by Short Peptide NP19

[0100] (1) Induction of Resistance to Bemisia tabaci in Tobacco

[0101] Fifteen adult Bemisia tabaci were placed in the center of each pair of leaves treated with short peptide NP19 or water. The number of insects on each leaf was recorded at 1, 2, 4, 8, 12, 24, and 48 hours.

[0102] Results: After treating Nicotiana benthamiana leaves with short peptide NP19, the number of insects could be significantly reduced, indicating that short peptide NP19 could induce resistance to Bemisia tabaci in Nicotiana benthamiana, as Figure 11 shown.

[0103] (2) Induction of Resistance to Nilaparvata lugens / Laodelphax striatellus in Rice

[0104] A rice plant treated with short peptide NP19 by smearing the stem and a rice plant treated with pure water by smearing the stem were enclosed in a glass cylinder (diameter 8 cm; height 8 cm), and 15 fourth-instar nymphs were released. The nymphs of Nilaparvata lugens / Laodelphax striatellus on each plant were counted at 1, 2, 4, 8, 24, and 48 hours after release;

[0105] Results: After smearing with short peptide NP19, the number of Nilaparvata lugens / Laodelphax striatellus staying on the rice could be significantly reduced, and the egg-laying amounts of both Nilaparvata lugens and Laodelphax striatellus also decreased significantly, indicating that NP19 could induce resistance to Nilaparvata lugens / Laodelphax striatellus in rice, as Figure 12 shown, where 12A is the number of Laodelphax striatellus on each rice plant, 12B is the egg-laying amount of Laodelphax striatellus, 12C is the number of Nilaparvata lugens on each rice plant, and 12D is the egg-laying amount of Nilaparvata lugens.

[0106] (3) Induction of Resistance to Chilo suppressalis in Rice

[0107] A rice plant treated with short peptide NP19 by smearing the stem and a rice plant treated with pure water by smearing the stem were enclosed in a glass cylinder (diameter 8 cm; height 8 cm). After weighing, 5 second-instar nymphs were placed in the glass cylinder and attached to the basal stem. After 7 days of feeding, each insect was reweighed to evaluate weight gain.

[0108] Results: After treating rice with short peptide NP19, the feeding of Chilo suppressalis could be significantly reduced, indicating that NP19 could induce resistance to Chilo suppressalis in rice, as Figure 13 shown.

[0109] Example 7: Short peptide NP19 induces cell death in different plant cells

[0110] Treat plant leaves with short peptide NP19. For dicotyledonous plants (cotton, eggplant), adjust the short peptide to a concentration of 0.5 μM and then use a needleless syringe to infiltrate it into the leaves of different species. For monocotyledonous plants (rice, corn), use quartz sand to create micro-wounds and then infiltrate the peptide solution into the plants.

[0111] Results: Short peptide NP19 can induce cell death in the leaves of cotton, eggplant, rice, and corn, as Figure 14 shown.

[0112] Example 8: Short peptide NP19 induces insect resistance in cotton

[0113] Selectivity test: Introduce 8 second-instar cotton bollworm larvae or 15 adult female cotton aphids into the center of each pair of cotton leaves treated with 0.5 μM NP19 short peptide or water. Record the number of insects on each leaf at 1, 2, 4, 8, 12, 24, and 48 hours.

[0114] Place each pair of cotton leaves treated with 0.5 μM NP19 short peptide or water in a petri dish. Place four adult cotton aphids on each leaf. After 3 days, count the newly born offspring under a microscope.

[0115] Results: Compared with the cotton leaves treated with short peptide NP19, cotton bollworm larvae or cotton aphids tend to feed on the cotton leaves treated with water; compared with the cotton leaves treated with short peptide NP19, the reproductive ability of cotton aphids on the cotton leaves treated with water is significantly stronger. It shows that NP19 can induce cotton to produce resistance to cotton bollworms or cotton aphids, as Figure 16 shown, where 16A is the growth picture of cotton aphids or cotton bollworms on cotton leaves, 16B is the statistical curve of the number of cotton bollworms, 16C is the statistical curve of the number of cotton aphids, and 16D is the statistical curve of the number of offspring of cotton aphids.

[0116] The present invention has been disclosed in the above preferred embodiments, but it is not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation shall 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. A nucleotide sequence encoding a short peptide NP19, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

4.

3. Use of the short peptide NP19 according to any one of claims 1 to 2, or the polypeptide Nlsp5 comprising the short peptide NP19 in the preparation of a plant insecticide for controlling gray planthopper, brown planthopper, whitefly, cotton bollworm, whiteback planthopper or striped stem borer, wherein the amino acid sequence of the polypeptide Nlsp5 is shown in SEQ ID NO:

1.

4. The use according to claim 3, 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.

5. The use according to claim 3, characterized in that: The short peptide NP19 is sprayed on tobacco or rice as a pesticide or as a main component of a pesticide.

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