Emblycoris pallidiflora sialoprotein RP426 with HAMP activity and application thereof

Abstract

The invention belongs to the technical field of prevention and control of agricultural pests, and particularly relates to an apis cerana sialoprotein RP426 with HAMP activity and application of the apis cerana sialoprotein RP426. The sialoprotein RP426 secreted into the soybean during feeding of the bee bugs is identified through a mass spectrometry technology for the first time, and further research proves that the RP426 is a novel HAMP molecule and can activate PTI reaction of plants, induce early ROS outbreak of the plants and defend up-regulated expression of related genes, so that the resistance of the plants to pathogenic bacteria and phytophagous pests is enhanced. According to the present invention, with the application of the Emblycoris punctata sialoprotein RP426, the defects of the existing prodenia litura prevention and control measures are made up, the use of the chemical pesticide is reduced, and the new approach is provided for improving the resistance of the plant to the pathogenic bacteria and the resistance of the plant to the phytophagous pests.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural pest control, and particularly relates to a salivary protein RP426 of the genus Psoralea corymbosa with HAMP activity and an application thereof. Background Art

[0002] Bee stink bug ( Riptortus pedestris ) belongs to the family Alydidae of the order Hemiptera. It is a polyphagous pest whose host range covers more than 30 plant species in 13 families, including legumes, vegetables, fruit trees and crops. However, it shows a significant feeding preference for soybeans. The pest is widely distributed in Asian countries such as China, South Korea, Japan and India. Nymphs and adults often migrate into the fields during the flowering and podding period of soybeans to cause damage in clusters. The affected soybean plants show symptoms of "green symptomatic" of "pods but no fruits", that is, the soybean plants remain green and do not shed leaves during the mature stage, and the pods and grains develop abnormally or shriveled. In addition, the bee-eared stink bug can indirectly transmit pathogenic microorganisms through its mouthparts, further causing grain mildew and significantly reducing the quality of soybeans. In recent years, the "green symptomatic" caused by the bee-eared stink bug in my country's main soybean producing areas has caused yield losses of up to 60%.

[0003] Currently, the control of bee-edge bugs in fields still relies primarily on chemical control, relying primarily on synthetic pyrethroids (cypermethrin and deltamethrin) and neonicotinoid insecticides (acetamiprid, thiamethoxam, and imidacloprid). Foliar application of emulsifiable concentrates, wettable powders, or water-dispersible granules is common. While fast-acting, long-term use can lead to the development of pesticide resistance in pests. Of greater concern is the excessive use of chemical pesticides, which leads to excessive pesticide residues, damaging farmland ecosystems and harming non-target organisms such as pollinators. It also contradicts the concept of sustainable, green agriculture. Therefore, the development of environmentally friendly alternative control technologies has become a current research priority.

[0004] Herbivore-associated molecular patterns (HAMPs) are a class of effector molecules secreted into the host plant through saliva during feeding by herbivorous insects. These molecules are recognized by pattern recognition receptors (PRRs) located on the plant cell membrane, thereby activating pattern-triggered immunity (PTI). As the first line of defense of the plant innate immune system, PTI can rapidly initiate a multi-layered defense response, including the activation of cytosolic calcium ions (Ca 2+ ) concentrations, reactive oxygen species (ROS) bursts, and activation of the mitogen-activated protein kinase (MAPK) signaling cascade, thereby conferring broad-spectrum resistance to pests and diseases. Recent research has revealed that some HAMP molecules, when recognized by plants, can trigger specific immune responses. Utilizing these molecules to develop plant immune elicitors or to breed transgenic insect-resistant crops has become an important direction for green insect control.

[0005] However, the number of known HAMP molecules is limited, especially those from piercing-sucking insects, and their mechanisms of action are not yet fully understood. Therefore, it is necessary to further discover new HAMP molecules and explore their application in plant immunity and insect-resistant breeding. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention identified for the first time through mass spectrometry technology the salivary protein RP426 secreted into soybeans by the bee-margin stink bug when feeding, and further studies confirmed that RP426 is a new type of HAMP molecule that can activate the plant PTI response, induce early ROS burst in plants and upregulate the expression of defense-related genes, thereby enhancing the plant's resistance to pathogens and herbivorous pests.

[0007] In one aspect, the present invention provides a salivary protein RP426 of the bee stink bug having HAMP activity, wherein the salivary protein RP426 is as shown in any one of the following (1)-(3): (1) A protein consisting of the amino acids shown in SEQ ID NO. 1; (2) A protein derived from (1) having the same function as the amino acid represented by SEQ ID NO. 1, wherein one or more amino acid residues are substituted and / or deleted and / or added; (3) Proteins or their derivatives derived from other varieties of rice or other species that have at least 95% sequence identity and have the same function as (1).

[0008] On the other hand, the present invention provides a nucleic acid encoding a salivary protein RP426 of the bee stink bug having HAMP activity, wherein the nucleic acid sequence is shown in any one of the following (4)-(6): (4) A DNA molecule whose coding region is the sequence shown in SEQ ID NO. 2; (5) A DNA molecule that hybridizes with the DNA sequence defined in (4) under stringent conditions and encodes a protein with the same function; (6) A DNA molecule that has at least 95% sequence identity with the DNA sequence defined in (4) and encodes a protein with the same function.

[0009] In another aspect, the present invention provides an expression cassette containing the gene.

[0010] In another aspect, the present invention provides a recombinant expression vector containing the expression cassette.

[0011] In another aspect, the present invention provides a plant-derived insect feeding inhibitor, wherein the active ingredient of the plant-derived insect feeding inhibitor comprises the salivary protein RP426 of the schizont, the nucleic acid, or the recombinant expression vector.

[0012] On the other hand, the present invention provides the use of the salivary protein RP426 of the schizont or the nucleic acid in improving the ability of plants to resist infection by pathogens or to resist damage by herbivorous pests.

[0013] In certain embodiments, the use comprises overexpressing the nucleic acid encoding the saliva protein RP426 of the bee stink bug in a plant to obtain a transgenic plant; or comprises directly introducing the saliva protein RP426 of the bee stink bug into plant tissues or plant cells.

[0014] In certain embodiments, the amino acid sequence of the salivary protein RP426 is as described above, and the nucleic acid encoding the salivary protein RP426 is as described above.

[0015] In some embodiments, the application is specifically, (1) Construct a recombinant plant expression vector containing the nucleic acid encoding salivary protein RP426; (2) Transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) Cultivating and screening transgenic plants with improved resistance to pathogen infection or herbivorous pest damage; preferably, the recombinant plant expression vector is as described above.

[0016] In certain embodiments, the plant is tobacco.

[0017] In certain embodiments, the herbivorous pest is Spodoptera litura.

[0018] Compared with the existing technology, the present invention identified for the first time through mass spectrometry technology the salivary protein RP426 secreted into soybeans by the bee-margin stink bug when feeding, and further research confirmed that RP426 is a new type of HAMP molecule that can activate the plant PTI response, induce early ROS burst in plants and upregulate the expression of defense-related genes, thereby enhancing the resistance of plants, especially tobacco, to pathogens and herbivorous pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the specific embodiments.

[0020] Figure 1 RP426 protein induces reactive oxygen species burst in Nicotiana benthamiana leaves.

[0021] Figure 2The RP426 gene upregulates the expression of PTI marker genes in Nicotiana benthamiana. Figure A shows that the RP426 protein upregulates the expression of the Acre31 gene, Figure B shows that the RP426 protein upregulates the expression of the WRKY7 gene, Figure C shows that the RP426 protein upregulates the expression of the WRKY8 gene, and Figure D shows that the RP426 protein upregulates the expression of the PR1 gene.

[0022] Figure 3 RP426 protein induces Nicotiana benthamiana to resist infection by Phytophthora capsici.

[0023] Figure 4 The RP426 protein-treated Nicotiana benthamiana leaves resisted feeding by Spodoptera litura. DETAILED DESCRIPTION

[0024] Example 1 Cloning of the RP426 gene of the bee stink bug

[0025] 1.1 Extraction of total RNA from the bee-margined stink bug (1) Place a healthy adult of the bee-edge bug in a sterile 2.0 mL EP tube and add sterile grinding beads to promote sufficient tissue disruption.

[0026] (2) Use a liquid nitrogen pre-cooled grinder to grind the sample of the bee-margin stink bug. Set the grinding conditions to 50 Hz, 30 s, and repeat several times until the sample is completely ground into a fine powder to improve the efficiency of RNA extraction.

[0027] (3) RNA was extracted using the RNAsimple Total RNA Kit (Tiangen, China). First, 1 mL of lysis buffer RZ was quickly added to the ground sample, mixed thoroughly using a vortexer, and allowed to stand at room temperature for 5 min to fully lyse the cells.

[0028] (4) Centrifuge the lysed sample at 12000 rpm and 4°C for 5 min, take 800 μL of supernatant and transfer it to a new centrifuge tube, and add 200 μL of pre-cooled chloroform.

[0029] (5) Shake vigorously for 15 seconds and then let it stand at room temperature for 3 minutes to promote phase separation.

[0030] (6) Continue centrifugation at 12000 rpm and 4°C for 10 min. After centrifugation, three layers are formed. Take about 400 μL of the upper aqueous phase and transfer it to a new centrifuge tube. Then add 200 μL of pre-cooled anhydrous ethanol, blow and mix well, and then quickly transfer it to the adsorption column CR3.

[0031] (7) Centrifuge at 12000 rpm and 4°C for 30 seconds, discard the effluent, and return the adsorption column to the collection tube.

[0032] (8) Add 500 μL of rinse solution RW to the adsorption column CR3, place at room temperature for 2 minutes, centrifuge at 12000 rpm and 4°C for 30 seconds, discard the effluent, and repeat this step once to enhance RNA purity.

[0033] (9) Add 500 μL of rinse solution RW to the adsorption column CR3, place at room temperature for 2 minutes, centrifuge at 12000 rpm and 4°C for 30 seconds, discard the effluent, and repeat this step once to enhance RNA purity.

[0034] (10) Centrifuge at 12000 rpm and 4°C for 2 min. After removing the residual liquid, transfer the adsorption column CR3 to a new centrifuge tube and blow dry on ice in a clean bench for 8 min.

[0035] (11) Add 30-50 μL RNase-Free ddH2O to the center of the adsorption column, incubate at room temperature for 2 min, and then centrifuge at 12,000 rpm and 4°C for 2 min to elute the RNA.

[0036] (12) The RNA concentration was measured using a spectrophotometer, and after quick freezing in liquid nitrogen, it was stored at -80°C for long-term storage.

[0037] 1.2 RP426 gene cloning (1) Reverse transcription was performed using HiScript II Reverse Transcriptase (Novozymes, China) to obtain cDNA. First, genomic DNA removal reaction was performed: 4 μL 4× gDNA wiper mix was added to 800 ng of total RNA, and the volume was made up to 16 μL with RNase-free ddH2O. The mixture was pipetted and mixed, and the reaction was incubated at 42°C for 2 min. Then, a reverse transcription reaction system was prepared. 4 μL 5× HiScript II qRT Super Mix was added to the above reaction system. The mixture was gently pipetted and mixed, and the reaction was incubated at 50°C for 15 min and 85°C for 5 s to obtain cDNA.

[0038] (2) The obtained cDNA was used as a template to perform PCR amplification using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 and the super-fidelity enzyme Phanta Super-Fidelity DNA Polymerase (Nuoweizan, China). Upstream primer: 5'-GACAAGGCCATGGCTGATATCGATTATTGCCCAAGTGACATG-3' (SEQ ID NO. 3) Downstream primer: 5'-GGTGGTGGTGGTGGTGCTCGAGGTTTCCGTATATGTTGACCCT-3' (SEQ ID NO. 4). Reaction system: 25 μL of 2× PhantaMax Buffer, 1 μL of dNTP Mix (10 mM each), 2 μL of upstream and downstream primers (10 mM each), 1 μL of Phanta DNA Polymerase, 2 μL of template cDNA, and ddH₂O to 50 μL. Amplify using the PCR protocol: initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1.5 min, followed by extension at 72°C at 1 kb / min.

[0039] (3) The target gene fragment obtained by PCR amplification was analyzed by agarose gel electrophoresis and purified using a DNA recovery kit. After purification, the RP426 gene fragment was ligated into the cloning vector pET32a, then transformed into Escherichia coli DH5α competent cells and plated on LB solid medium containing carbenicillin. The cells were cultured at 37°C for 12-16 hours, and single clones containing the recombinant plasmid were screened.

[0040] (4) Positive monoclonal colonies were picked and inoculated into LB liquid medium containing carbenicillin for expansion culture, and the recombinant plasmid was extracted. The recombinant plasmid was sequenced using Sanger sequencing to confirm whether the RP426 target gene was successfully inserted. Finally, the recombinant plasmid pET32a-RP426 containing the sequence shown in SEQ ID NO. 2 was obtained, laying the foundation for subsequent functional verification experiments.

[0041] Example 2 Prokaryotic expression and purification of the protein of the RP426 stink bug

[0042] 1. Prokaryotic expression of RP426 protein (1) Transform the recombinant plasmid pET32a-RP426 into Escherichia coli Rosetta (DE3) competent cells using the heat shock method. Plate the cells on LB solid medium containing 50 ng / mL carbenicillin and incubate at 37°C for 12-16 h to screen for positive clones.

[0043] (2) Pick a positive monoclonal colony and inoculate it into 6 mL of LB liquid culture medium containing 50 ng / mL carbenicillin. Incubate the culture at 37°C and 200 rpm with shaking overnight to obtain a small amount of culture solution.

[0044] (3) Inoculate a small amount of cultured bacteria into 600 mL of LB medium at a ratio of 1:100 and continue shaking culture at 37°C to gradually increase the density of the culture.

[0045] (4) After culturing for about 2 hours, measure the OD600 value every 15-20 minutes. When the OD600 reaches 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.1 mM to induce expression. Subsequently, adjust the culture conditions to 16-18°C and shake at 180 rpm for 12-14 hours to promote soluble expression of the recombinant protein.

[0046] (5) After the induction culture is completed, transfer the bacterial solution to a 50 mL centrifuge tube and centrifuge at 5000 g for 5 min at 4°C. Discard the supernatant, collect the bacterial pellet, and wash it three times with 1× PBS buffer to remove residual culture medium components.

[0047] (6) Add 50 mL of 1× PBS buffer to the bacterial pellet and resuspend it. Then add 200 μL of lysozyme (4 μL per 1 mL) and 500 μL of protease inhibitor PMSF (10 μL per 1 mL). Mix thoroughly and place on ice until ready to use to inhibit protein degradation.

[0048] (7) Use low temperature ultra-high pressure cell disruptor (JNBIO ® Use a microbead microcentrifuge (microbead microcentrifuge) to disrupt the bacteria, set an appropriate pressure, and repeat the disruption 3 to 4 times until the solution is clear to obtain the cell lysis product.

[0049] (8) Centrifuge the cell lysate at 4°C, 12,000 rpm for 15 min, and transfer the supernatant to a pre-cooled 50 mL centrifuge tube to obtain the crude protein of the target gene.

[0050] 2. Protein purification (1) Add 500 μL of pretreated Ni-NTA Agarose (Qiagen) to the above 50 mL crude protein solution. Before use, the Ni-NTA Agarose should be washed 3-5 times with His-Lysis Buffer. After washing, centrifuge at 500 g for 2 minutes at 4°C and discard the supernatant to ensure that the Ni-NTA Agarose is fully activated.

[0051] (2) Mix the protein solution with Ni-NTA Agarose, place it on a rotating shaker in a cold room at 4°C, and incubate overnight to allow the recombinant RP426 protein with the His tag to fully bind to the Ni-NTA Agarose.

[0052] (3) After incubation, centrifuge the solution at 4°C, 500 g for 5 min and discard the supernatant to remove unbound proteins. Wash the Ni-NTA Agarose six times with His-Wash Buffer to further remove unbound proteins. After each wash, centrifuge at 4°C, 500 g for 1 min to ensure complete removal of the eluate.

[0053] (4) Add 2 mL of His-Elution Buffer to each sample and incubate on a rotating shaker in a cold room at 4°C for 30 min to promote the competitive elution of His-tagged proteins from Ni-NTA Agarose.

[0054] (5) After elution, centrifuge at 4°C, 500 g for 2 min and transfer the supernatant to a new 1.5 mL centrifuge tube. This is the purified His-tagged RP426 recombinant protein, whose amino acid sequence is shown in SEQ ID NO.1.

[0055] Finally, the protein was concentrated and desalted. The eluted protein solution was transferred to a Millipore 15 kDa ultrafiltration tube and centrifuged at 4°C, 4000 rpm until the protein was concentrated to approximately 500 μL. After discarding the filtrate, 10 mL of PBS was added and centrifuged again at 4°C, 4000 rpm until the residual volume was approximately 500 μL. This ultrafiltration and liquid exchange process was repeated three times to completely remove impurities and salt ions. A final centrifugation was performed to the desired experimental volume to obtain a highly pure RP426 protein solution.

[0056] Example 3 Luminol method for measuring reactive oxygen species induced by Nicotiana benthamiana

[0057] (1) Prepare 1 μM flg22 (GenScript, China), 1 μM RP426 recombinant protein, and 1 μM GFP recombinant protein (prokaryotic expression control) for subsequent reactive oxygen species detection experiments. Select Nicotiana benthamiana plants of about four weeks old and use a 0.5 cm diameter punch to prepare leaf discs on the leaves, ensuring that the front / back of the leaves are facing upward. Do not rotate the punch during punching, but press evenly in the four directions of up, down, left, and right to ensure that the edges of the leaf discs are smooth and complete. Gently remove the leaf disc with tweezers and place it in a 96-well plate containing 100 μL sterile ultrapure water, still keeping the front / back of the leaves facing upward to avoid experimental errors caused by leaf flipping. After the leaves are treated, they are left to stand at room temperature in the dark overnight to eliminate the mechanical stress caused by punching.

[0058] (2) The next day, the sterile water in the 96-well plate was removed with a pipette and 100 μL of detection buffer was added to each well. The buffer consisted of 100 μM LO12 (Fujifilm, Japan), 20 μg / mL horseradish peroxidase (Sigma-Aldrich, USA), and 1 μM flg22 / 1 μM RP426 / 1 μM GFP (different experimental treatment groups). The remaining volume was made up to a final volume of 100 μL with ddH2O to ensure consistency of the experimental system in each group.

[0059] (3) After adding the assay buffer, the 96-well plate was immediately placed in a microplate reader (EnSight; PerkinElmer, USA) for detection. Luminol-horseradish peroxidase (HRP) system was used for chemiluminescence detection to assess the production of reactive oxygen species (ROS) in plant cells under different treatment conditions. The assay parameters were set to Luminescence 1 mode, 0.1 s exposure time, single measurement, 60 replicates, and a one-minute interval between replicates to continuously monitor the dynamic changes in ROS levels.

[0060] (4) Keep the room temperature constant during the experiment to avoid interference from ambient light to ensure data stability and reproducibility. After the experiment, collect luminescence data and perform statistical analysis using GraphPad Prism or Excel to calculate the differences in luminescence intensity between different treatment groups and draw a time-luminescence intensity change curve to evaluate the role of RP426 protein in the immune response of Nicotiana benthamiana.

[0061] like Figure 1 As shown, RP426-treated Nicotiana benthamiana leaves showed significantly enhanced luminescence compared to GFP-treated leaves, indicating that RP426 can induce the production of reactive oxygen species (ROS). High levels of ROS accumulation were observed in the flg22-treated group, indicating that flg22 successfully activated the plant's immune response. While the luminescence intensity in the RP426-treated group was slightly lower than that in the flg22-treated group, it was still significantly higher than that in the control group, further supporting the role of RP426 in plant immune responses. Furthermore, when RP426 was inactivated by boiling, it failed to induce ROS bursts, indicating that its immune-inducing effect relies on its native conformation rather than being a simple peptide fragment or degradation product. These results suggest that RP426 may play an important role in pathogen infection and insect feeding by promoting the accumulation of ROS and enhancing plant immune defenses.

[0062] Example 4 Detection of PTI marker genes induced by RP426

[0063] 1. Extraction of Total RNA from Nicotiana benthamiana Total RNA was extracted from half a Nicotiana benthamiana leaf treated with 1 μM RP426 / 1 μM GFP (different experimental treatment groups) following the same RNA extraction procedures as in Example 1 to ensure consistency of experimental conditions and comparability of data. The extracted RNA concentration was determined using a Nanodrop, and RNA integrity was assessed by agarose gel electrophoresis to ensure accuracy in subsequent experiments.

[0064] 2. Real-time fluorescence quantitative PCR (1) 800 ng of RNA was taken and reverse transcribed into cDNA using HiScript II Reverse Transcriptase (Aicore, China) according to the qPCR experimental requirements. Subsequently, the cDNA reaction mixture was diluted 2-fold using RNAase-free ddH2O to reduce the interference of residual RNA on qPCR amplification.

[0065] (2) Prepare a 20 μL qPCR reaction system, the specific components are as follows: 10 μL 2X SYBR Green Pro Taq HS Premix (ROX Plus), 2 μL cDNA (template), 0.4 μL each of 10 μM upstream and downstream primers, and RNAase-free ddH2O to fill the system to 20 μL.

[0066] (3) qPCR detection was performed using the ABI Prism 7500 Fast Real-Time PCR System according to the manufacturer's instructions. A two-step PCR reaction was performed with the following procedure: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and annealing at 60°C for 30 s, for 40 cycles.

[0067] (4) Adopt 2 -ΔΔCt Relative quantification was used to calculate changes in the expression of PTI marker genes across treatment groups. The ACTIN gene was used as an internal control to ensure the accuracy and reliability of the qPCR data. These results were used to evaluate whether RP426 protein can induce the expression of PTI-related genes and further validate its potential role in plant immune regulation.

[0068] like Figure 2As shown, the present invention measured the expression of the PTI marker genes Acre31, WRKY7, WRKY8, and PR1 in leaves of Nicotiana benthamiana treated with GFP and RP426 protein 12 and 24 hours after treatment. The results showed that compared to the GFP control, the expression level of the Acre31 gene in leaves treated with RP426 protein was upregulated by 8-fold, the expression level of the WRKY7 gene was upregulated by 50-fold, the expression level of the WRKY8 gene was upregulated by 4-fold, and the expression level of the PR1 gene was upregulated by 100-fold. These results demonstrate that RP426 protein can significantly induce the expression of PTI marker genes in Nicotiana benthamiana, suggesting that RP426 protein may play an important role in plant immune responses, enhancing plant resistance to insects.

[0069] Example 5 Pathogen Inoculation

[0070] (1) The capsici Phytophthora capsici strain LT263 used in this study was cultured on 10% (v / v) V8 juice medium at 25°C in the dark for 2 days to promote mycelial growth and fungus formation. The medium was kept moist during the culture process to avoid drying out, which would affect the activity of the pathogen.

[0071] (2) 1 μM RP426 recombinant protein was pressure-injected into four-week-old Nicotiana benthamiana leaves for 12 h to induce plant immune responses. 1 μM GFP recombinant protein was used as a negative control to exclude nonspecific effects of exogenous protein expression on disease responses.

[0072] (3) Take the treated tobacco leaves, cut them gently, and place them with the back side facing up on a tray containing moistened absorbent paper to provide a suitable humidity environment to promote pathogen infection.

[0073] (4) Cut an equal amount of fungus blocks from the 2-day-old culture medium of pepper phytophthora and inoculate them directly onto the back of the leaves. At the same time, add about 5 μL of sterile water to the surface of the fungus blocks to increase the contact between the pathogen and the leaf tissue and promote the infection process.

[0074] (5) After inoculation, seal the tray with plastic wrap to maintain humidity and culture it in a dark environment at 25°C to simulate natural pathogen infection conditions.

[0075] (6) 48 hours after inoculation, take images of leaf lesions under UV light and measure the lesion area using ImageJ software to assess the degree of pathogen infection. Observation of disease symptoms should be performed under the same imaging conditions to ensure data comparability.

[0076] (7) All experiments included at least 8 biological replicates and were performed three times independently to ensure the stability of the data and the reproducibility of the experimental results.

[0077] like Figure 3 As shown in the results, in an experiment inoculated with Phytophthora capsici LT263, the lesion area of ​​leaves in N. benthamiana treated with RP426 was significantly smaller than that in the GFP-treated group, indicating that the RP426 protein plays a positive role in the immune defense mechanism of N. benthamiana, improving its resistance to pathogens. This result provides experimental evidence for further analysis of the function of RP426 in plant immune regulation.

[0078] Example 6 Feeding preference experiment of Spodoptera litura

[0079] (1) Four-week-old Nicotiana benthamiana plants were selected and pressure-injected with 1 μM RP426 recombinant protein on the front or back of leaves for 12 hours to induce an immune response. Simultaneously, 1 μM GFP recombinant protein was injected into control leaves. The treated leaves were then cut and the petioles were wrapped with soaked cotton wool to prevent premature wilting due to water loss. The treated leaves were placed in circular Petri dishes in preparation for insect feeding experiments.

[0080] (2) Eight second-instar larvae were selected from a laboratory-reared population of Spodoptera litura and placed in the middle of two leaves, ensuring that the larvae could freely choose the leaves with different treatments. The culture dish was sealed with parafilm to prevent the larvae from escaping, and the appropriate humidity and temperature were maintained to ensure the stability of the feeding experiment. The culture dish was placed in a growth environment of 25°C, 16 h light / 8 h dark, and the insects were allowed to feed freely for 48 h. The feeding behavior of the insects was observed.

[0081] (3) After the experiment, all leaves were removed and photographed, and the feeding damage was recorded under the same lighting conditions. ImageJ software was used to measure the leaf damage area after insect feeding, and statistical analysis was performed to evaluate the effect of RP426 recombinant protein on the feeding preference of Spodoptera litura.

[0082] like Figure 4 As shown, compared to GFP-treated leaves, leaves of N. benthamiana treated with RP426 showed significantly less feeding area by Spodoptera litura, indicating that RP426 enhances resistance to Spodoptera litura feeding in N. benthamiana, thereby affecting the insect's feeding preference. This experiment provides experimental evidence for the potential function of RP426 in plant insect resistance.

[0083] Unless otherwise specified, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments can have different values.

Claims

1. Use of the salivary protein RP426 of the staphylococcus punctatus having HAMP activity or a nucleic acid encoding the protein in improving the ability of plants to resist infection by pathogens or to resist damage by herbivorous pests, characterized in that: The amino acid sequence of the salivary protein RP426 is shown as SEQ ID NO. 1, the sequence of the encoding nucleic acid is shown as SEQ ID NO. 2, and the plant is tobacco.

2. The use according to claim 1, characterized in that The application comprises over-expressing the encoding nucleic acid of the salivary protein RP426 of the schizont in plants to obtain transgenic plants.

3. The use according to claim 2, characterized in that The application is specifically the following steps: (1) Construct a recombinant plant expression vector containing the nucleic acid encoding salivary protein RP426; (2) Transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) Cultivate and screen transgenic plants with improved resistance to pathogen infection or damage from herbivorous pests.

4. The use according to claim 3, characterized in that The plant is tobacco, the pathogen is Phytophthora capsici, and the herbivorous pest is Spodoptera litura.

5. The use according to claim 3, characterized in that The herbivorous pest is Spodoptera litura.

6. Use of a biological material containing the salivary protein RP426 of the staph stink bug or a nucleic acid encoding the protein for improving the ability of plants to resist infection by pathogens or to resist damage by herbivorous pests, characterized in that: The biological material is any of the following, 1) An expression cassette containing the encoding nucleic acid according to claim 1; 2) an expression vector containing the encoding nucleic acid according to claim 1; 3) an expression vector containing the expression cassette described in 1); 4) a recombinant microorganism containing the expression vector described in 2); 5) A recombinant microorganism containing the expression vector described in 3).

7. The use according to claim 6, characterized in that The plant is tobacco, the pathogen is Phytophthora capsici, and the herbivorous pest is Spodoptera litura.

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