Aphid saliva protein rp426 with hamp activity and use thereof

Abstract

The present application belongs to the technical field of agricultural pest prevention and control, and particularly relates to a spined cucumber bug saliva protein RP426 with HAMP activity and application thereof. The present application first identifies saliva protein RP426 secreted into soybean by spined cucumber bugs during feeding through mass spectrometry, and further researches and confirms that RP426 is a novel HAMP molecule, can activate the PTI response of plants, induce early ROS burst and up-regulated expression of defense-related genes, thereby enhancing the resistance of plants to pathogenic bacteria and phytophagous pests. The spined cucumber bug saliva protein RP426 provided by the present application makes up for the deficiency of existing Spodoptera litura prevention and control measures, reduces the use of chemical pesticides, and provides a new way for improving the resistance of plants to pathogenic bacteria and the resistance of plants to phytophagous pests.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest control technology, specifically relating to the HAMP-active salivary protein RP426 of the spotted bug and its applications. Background Technology

[0002] Spotted bee peridot ( Riptortus pedestris The tarantula (Alydidae) is a polyphagous pest belonging to the order Hemiptera. Its host range covers over 30 species of plants in 13 families, including legumes, vegetables, fruit trees, and crops. However, it shows a significant preference for soybeans. This pest is widely distributed in Asian countries such as China, South Korea, Japan, and India. Nymphs and adults often migrate into fields and congregate to cause damage during the flowering and pod-setting stages of soybeans. Affected soybean plants exhibit "green pods without fruit" symptoms, characterized by stunted green growth, failure to shed leaves, and abnormal or shriveled pods and seeds at maturity. Furthermore, the tarantula can indirectly transmit pathogenic microorganisms through its mouthparts, further leading to seed mold and significantly reducing soybean quality. In recent years, "green pods" caused by the tarantula have resulted in yield losses of up to 60% in major soybean-producing areas of my country.

[0003] Currently, the control of field spot bugs still relies primarily on chemical control, mainly depending on synthetic pyrethroids (such as deltamethrin and cypermethrin) and neonicotinoid insecticides (such as acetamiprid, thiamethoxam, and imidacloprid). Application methods often involve foliar spraying with emulsifiable concentrates, wettable powders, or water-dispersible granules. While these methods offer rapid effects, long-term use can lead to pesticide resistance in pests. More importantly, the overuse of chemical pesticides results in excessive pesticide residues, which not only damages the farmland ecosystem and harms non-target organisms such as pollinators, but also contradicts the principles of green and sustainable agricultural development. Therefore, developing environmentally friendly alternative control technologies has become a key research focus.

[0004] Herbivore-associated molecular patterns (HAMPs) are a class of effector molecules secreted into host plants via saliva during feeding by herbivore insects. These molecules are recognized by pattern recognition receptors (PRRs) located on plant cell membranes, thereby activating pattern-triggered immunity (PTI). As the first line of defense in the plant's innate immune system, PTI can rapidly initiate multi-layered defense responses, including the release of cytoplasmic calcium ions (Ca). 2+ Increased HAMP concentrations, reactive oxygen species (ROS) bursts, and activation of the mitogen-activated protein kinase (MAPK) signaling cascade can lead to broad-spectrum resistance to pests and diseases. Recent studies have found that some HAMP molecules can activate specific immune responses after being recognized by plants. Developing plant immune inducers using these molecules, or applying them to the breeding of transgenic insect-resistant crops, has become an important direction for green insect control.

[0005] However, the number of currently known HAMP molecules is limited, especially the research on hemipteran insects is less, and the related mechanism is not clear enough. Therefore, it is necessary to further discover new HAMP molecules and explore their application value in plant immunity and pest-resistant breeding. SUMMARY

[0006] In order to solve the above technical problems, the present application first identifies saliva protein RP426 secreted by Zicrona caerulea when feeding on soybean by mass spectrometry, and further research confirms that RP426 is a new type of HAMP molecule, which can activate the PTI response of plants, induce the early ROS burst and the up-regulated expression of defense-related genes, thereby enhancing the resistance of plants to pathogenic bacteria and phytophagous pests.

[0007] In one aspect, the present application provides a Zicrona caerulea saliva protein RP426 with HAMP activity, which is any one of the following (1)-(3):

[0008] (1) a protein consisting of the amino acid shown in SEQ ID NO. 1;

[0009] (2) a protein derived from (1) by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function;

[0010] (3) a protein or its derivative derived from other varieties of rice or other species and having at least 95% sequence identity and having the same function as (1).

[0011] In another aspect, the present application provides a nucleic acid encoding Zicrona caerulea saliva protein RP426 with HAMP activity, and the nucleic acid sequence is any one of the following (4)-(6):

[0012] (4) a DNA molecule with a coding region shown in SEQ ID NO. 2;

[0013] (5) a DNA molecule hybridizing to the DNA sequence defined in (4) under stringent conditions and encoding a protein with the same function;

[0014] (6) a DNA molecule having at least 95% sequence identity with the DNA sequence defined in (4) and encoding a protein with the same function.

[0015] In another aspect, the present application provides an expression cassette containing the gene.

[0016] In another aspect, the present application provides a recombinant expression vector containing the expression cassette.

[0017] In another aspect, the present application provides a plant-derived insect feeding inhibitor, wherein the active ingredient of the plant-derived insect feeding inhibitor comprises the said H. punctiventris salivary protein RP426, or the said nucleic acid, or the said recombinant expression vector.

[0018] In another aspect, the present application provides an application of the said H. punctiventris salivary protein RP426 or the said nucleic acid in improving the ability of a plant to resist pathogenic bacteria or in improving the ability of a plant to resist phytophagous pests.

[0019] In some embodiments, the application comprises overexpressing a nucleic acid encoding the H. punctiventris salivary protein RP426 in a plant to obtain a transgenic plant; or comprises directly introducing the H. punctiventris salivary protein RP426 into a plant tissue or a plant cell.

[0020] In some 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.

[0021] In some embodiments, the application specifically comprises,

[0022] (1) constructing a recombinant plant expression vector containing a nucleic acid encoding the salivary protein RP426;

[0023] (2) transforming the constructed recombinant plant expression vector into a plant tissue or a plant cell;

[0024] (3) cultivating and screening to obtain a transgenic plant with improved ability to resist pathogenic bacteria or improved ability to resist phytophagous pests; preferably, the recombinant plant expression vector is as described above.

[0025] In some embodiments, the plant is tobacco.

[0026] In some embodiments, the phytophagous pest is Helicoverpa armigera.

[0027] Compared with the prior art, the present application first identifies the salivary protein RP426 secreted by H. punctiventris when feeding on soybeans through mass spectrometry, and further studies confirm that RP426 is a new type of HAMP molecule, which can activate the PTI response of plants, induce early ROS burst and up-regulate the expression of defense-related genes, thereby enhancing the resistance of plants, especially tobacco, to pathogenic bacteria and phytophagous pests. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings required in the specific embodiment description will be briefly introduced below.

[0029] Figure 1The RP426 protein induces active oxygen burst in N. benthamiana leaf.

[0030] Figure 2 The RP426 protein up-regulates the expression of PTI marker genes in N. benthamiana; wherein, Figure A shows that the RP426 protein up-regulates the expression of Acre31 gene, Figure B shows that the RP426 protein up-regulates the expression of WRKY7 gene, Figure C shows that the RP426 protein up-regulates the expression of WRKY8 gene, and Figure D shows that the RP426 protein up-regulates the expression of PR1 gene.

[0031] Figure 3 The RP426 protein induces N. benthamiana resistance to P. capsici infection.

[0032] Figure 4 N. benthamiana leaves treated with the RP426 protein are resistant to Spodoptera litura feeding. DETAILED DESCRIPTION

[0033] Example 1 Cloning of the RP426 gene of the piezodorus guildinii

[0034] 1.1 Extraction of total RNA of the piezodorus guildinii

[0035] (1) Take one healthy adult piezodorus guildinii and place it in a sterile 2.0 mL EP tube, and add sterile grinding beads to facilitate the complete disruption of the tissue.

[0036] (2) Use liquid nitrogen to pre-cool the grinder, and set the grinding conditions to 50 Hz, 30 s, and repeat multiple times until the sample is completely ground into a fine powder to improve the efficiency of RNA extraction.

[0037] (3) Use the RNAsimple Total RNA Kit (Tiangen, China) to extract RNA. First, add 1 mL of lysis solution RZ to the ground sample, mix well using a vortex, and let it stand at room temperature for 5 min to fully lyse the cells.

[0038] (4) Centrifuge the lysed sample at 12000 rpm, 4°C for 5 min, take 800 μL of supernatant to a new centrifuge tube, and add 200 μL of pre-cooled chloroform.

[0039] (5) Shake vigorously for 15 s, then let it stand at room temperature for 3 min to promote phase separation.

[0040] (6) Continue to centrifuge at 12000 rpm, 4°C for 10 min, and 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, mix well by blowing, and then quickly transfer it to the adsorption column CR3.

[0041] (7) 12000 rpm, 4°C for 30 s, discard the flow-through and place the adsorption column back into the collection tube.

[0042] (8) Add 500 μL of RNase-free ddH2O to the adsorption column CR3, let stand at room temperature for 2 min, 12000 rpm, 4°C for 30 s, discard the flow-through and repeat this step once to enhance the purity of RNA.

[0043] (9) Add 500 μL of RNase-free ddH2O to the adsorption column CR3, let stand at room temperature for 2 min, 12000 rpm, 4°C for 30 s, discard the flow-through and repeat this step once to enhance the purity of RNA.

[0044] (10) 12000 rpm, 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.

[0045] (11) Add 30-50 μL of RNase-free ddH2O to the center of the adsorption column, incubate at room temperature for 2 min, then 12000 rpm, 4°C for 2 min to elute the RNA.

[0046] (12) Determine the concentration of RNA using a spectrophotometer, and store at -80°C for long-term preservation after quick freezing in liquid nitrogen.

[0047] 1.2 Cloning of RP426 gene

[0048] (1) Reverse transcription was performed using HiScript II Reverse Transcriptase (Novogene, China) to obtain cDNA. First, perform a genomic DNA removal reaction: in 800 ng of total RNA, add 4 μL of 4×gDNA wiper Mix, make up to 16 μL with RNase-free ddH2O, mix well by blowing, and react at 42°C for 2 min. Then configure the reverse transcription reaction system: in the above reaction system, add 4 μL of 5×HiScript II qRT SuperMix, mix well by blowing, react at 50°C for 15 min, and react at 85°C for 5 s to obtain cDNA.

[0049] (2) Using the obtained cDNA as a template, PCR amplification was performed using the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 and the super-fidelity enzyme Phanta Super-Fidelity DNA Polymerase (Novogene, China). Upstream primer: 5'-GACAAGGCCATGGCTGATATCGATTATTGCCCAAGTGACATG-3'

[0050] (SEQ ID NO. 3) Downstream primer: 5'-GGTGGTGGTGGTGGTGCTCGAGGTTTCCGTATATGTTGACCCT-3'

[0051] (SEQ ID NO. 4). The reaction system: 2 x PhantaMax Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, upstream and downstream primers (10 mM each) 2 μL each, Phanta DNA Polymerase 1 μL, template cDNA 2 μL, and ddH2O to 50 μL. According to the PCR program of 94°C pre-denaturation for 5 min, 94°C for 30 s, 56°C for 30 s, 72°C for 1.5 min, 35 cycles, and 72°C extension for 1 kb / min.

[0052] (3) The target gene fragment obtained by PCR amplification was analyzed by agarose gel electrophoresis and purified by a DNA recovery kit. After purification, the RP426 gene fragment was ligated to the cloning vector pET32a, and then transformed into E. coli DH5a competent cells and plated on LB solid medium containing carbenicillin and incubated at 37°C for 12-16 h to select single colonies containing recombinant plasmids.

[0053] (4) Positive single colonies were picked and inoculated into LB liquid medium containing carbenicillin for expansion culture, and the recombinant plasmid was extracted. Sanger sequencing was used to sequence the recombinant plasmid 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 a foundation for subsequent functional verification experiments.

[0054] Example 2 Prokaryotic expression of Rhodnius prolixus RP426 protein and purification

[0055] 1. Prokaryotic expression of RP426 protein

[0056] (1) The recombinant plasmid pET32a-RP426 was transformed into E. coli Rosetta (DE3) competent cells using the heat shock method, and plated on LB solid medium containing 50 ng / mL carbenicillin and incubated at 37°C for 12-16 h to select positive clones.

[0057] (2) Positive single colonies were picked and inoculated into 6 mL of LB liquid medium containing 50 ng / mL carbenicillin and incubated at 37°C, 200 rpm overnight to obtain a small amount of culture broth.

[0058] (3) According to the ratio of 1:100, inoculate a small amount of cultured bacteria liquid into 600 mL of LB medium, and continue to culture at 37°C with shaking, so as to gradually increase the density of the bacteria liquid.

[0059] (4) After about 2 hours of culture, measure the OD600 value every 15-20 minutes, and when the OD600 reaches 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.1 mM to induce expression. Subsequently, adjust the culture conditions to 16-18°C, and culture at 180 rpm for 12-14 h to promote the soluble expression of the recombinant protein.

[0060] (5) After the induction culture is completed, transfer the bacteria liquid to a 50 mL centrifuge tube, centrifuge at 4°C, 5000 g for 5 min, discard the supernatant, collect the bacterial precipitate, and wash it with 1×PBS buffer for 3 times to remove residual culture medium components.

[0061] (6) After resuspending the bacterial precipitate with 50 mL of 1×PBS buffer, 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 to inhibit protein degradation.

[0062] (7) Use a low-temperature ultrahigh-pressure cell disruptor (JNBIO ® mini) to break the bacteria, set the appropriate pressure, and repeat the breaking 3-4 times until the solution is clear, and obtain the cell lysate.

[0063] (8) Centrifuge the broken cell lysate at 4°C, 12000 rpm for 15 min, and take the supernatant to a pre-cooled 50 mL centrifuge tube. At this time, the crude protein of the target gene is obtained.

[0064] 2. Protein purification

[0065] (1) Add 500 μL of pretreated Ni-NTA Agarose (Qiagen) to the above 50 mL crude protein solution. Ni-NTA Agarose needs to be washed 3-5 times with His-Lysis Buffer before use. After washing, centrifuge at 4°C, 500 g for 2 min, discard the supernatant, and ensure that the Ni-NTA Agarose is fully activated.

[0066] (2) Mix the protein solution with Ni-NTA Agarose, and place it on a 4°C cold room rotary shaker for overnight incubation, so that the recombinant RP426 protein with His tag can be fully combined with Ni-NTA Agarose.

[0067] (3) After the incubation, centrifuge the solution at 500 g for 5 min at 4°C to remove unbound impurities. Then wash the Ni-NTA Agarose with His-Wash Buffer for 6 times to further remove unbound proteins. After each wash, centrifuge at 500 g for 1 min at 4°C to ensure complete removal of the wash buffer.

[0068] (4) Add 2 mL His-Elution Buffer to each sample and incubate on a cold room rotator for 30 min at 4°C to promote the elution of His-tagged proteins from the Ni-NTA Agarose.

[0069] (5) After the elution, centrifuge at 500 g for 2 min at 4°C and transfer the supernatant to a new 1.5 mL centrifuge tube. The resulting solution is the purified His-tagged RP426 recombinant protein, which has the amino acid sequence shown in SEQ ID NO. 1.

[0070] Finally, concentrate and desalt the eluted protein solution by transferring it to a Millipore 15 kDa ultrafiltration tube and centrifuging at 4000 rpm at 4°C until the protein is concentrated to approximately 500 μL. After discarding the filtrate, add 10 mL of PBS and continue centrifuging at 4000 rpm at 4°C until the remaining liquid volume is approximately 500 μL. Repeat this ultrafiltration and buffer exchange process 3 times to completely remove impurities and salt ions. Finally, centrifuge to the desired volume to obtain a high-purity RP426 protein solution.

[0071] Example 3. Measurement of active oxygen induced by N. benthamiana using the luminol method

[0072] (1) Prepare 1 μM flg22 (GenScript, China), 1 μM RP426 recombinant protein, and 1 μM GFP recombinant protein (prokaryotic expression control) for subsequent active oxygen detection experiments. Select four-week-old N. benthamiana plants and use a 0.5 cm diameter puncher to prepare leaf discs on the leaves, ensuring that the leaf front / back is facing upwards. When punching, do not rotate the puncher, but press it evenly in the up, down, left, and right directions to ensure that the leaf disc edge is smooth and complete. Gently remove the leaf disc with tweezers and place it in a 96-well plate containing 100 μL of sterilized ultrapure water, keeping the leaf front / back facing upwards to avoid experimental errors caused by leaf flipping. After treating the leaves, incubate them at room temperature in the dark overnight to eliminate mechanical stress caused by punching.

[0073] (2) The next day, sterile water was removed from the 96-well plate using 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 using ddH2O to ensure consistency of experimental systems for each group.

[0074] (3) Immediately after adding the detection buffer, the 96-well plate was placed in an ELISA reader (EnSight; PerkinElmer, USA) for detection. The experiment used a luminol-horseradish peroxidase (HRP) system for chemiluminescence detection to assess the production of reactive oxygen species (ROS) in plant cells under different treatment conditions. The detection parameters were set to Luminescence 1 mode, exposure time 0.1 s, single measurement, 60 replicates, with a one-minute interval between each replicate, continuously monitoring the dynamic changes in ROS levels.

[0075] (4) During the experiment, the room temperature was kept constant and the interference of ambient light was avoided to ensure the stability and reproducibility of the data. After the experiment, the luminescence data were collected and statistically analyzed using GraphPad Prism or Excel. The differences in luminescence intensity among different treatment groups were calculated and time-luminescence intensity change curves were plotted to evaluate the role of RP426 protein in the immune response to Nicotiana benthamiana.

[0076] like Figure 1 As shown, the luminescence signal of RP426-treated *Nicotiana benthamiana* leaves was significantly enhanced compared to GFP-treated leaves, indicating that RP426 protein can induce reactive oxygen species (ROS) production in plants. In the flg22-treated group, the level of ROS accumulation was high, indicating that flg22 successfully activated the plant's immune response. While the luminescence intensity of the RP426-treated group was slightly lower than that of flg22, it was still significantly higher than that of the control group, further supporting the role of RP426 protein in plant immune responses. Furthermore, when RP426 protein was inactivated by boiling, it could not induce ROS bursts, indicating that the immunomodulatory effect of RP426 protein depends on its native conformation, rather than simple polypeptide fragments or degradation products. These results suggest that RP426 protein may play an important role in pathogen infection and insect feeding by promoting the accumulation of reactive oxygen species and enhancing plant immune defense capabilities.

[0077] Example 4: Detection of RP426-induced PTI marker genes

[0078] 1. Extraction of total RNA from Nicotiana benthamiana

[0079] Take half of the leaf pieces treated with 1 μM RP426 / 1 μM GFP (different experimental treatment groups) protein, and extract total RNA according to the same procedure as in Example 1 to ensure consistency of experimental conditions and comparability of data. After extraction, the concentration of RNA was determined using Nanodrop, and agarose gel electrophoresis was used to detect RNA integrity to ensure the accuracy of subsequent experiments.

[0080] 2. Real-time fluorescent quantitative PCR

[0081] (1) Take 800 ng of RNA and use HiScript II Reverse Transcriptase (Aikewei, China) to perform reverse transcription according to the requirements of qPCR experiments, converting RNA to cDNA. Subsequently, the cDNA reaction mixture was diluted 2-fold using RNAase-free ddH2O to reduce the interference of residual RNA on qPCR amplification.

[0082] (2) Configure a 20 μL qPCR reaction system, with the following specific components: 10 μL 2X SYBR Green Pro Taq HSPremix (ROX Plus), 2 μL cDNA (template), 0.4 μL of 10 μM upstream and downstream primers, and RNAase-free ddH2O to make up the system to 20 μL.

[0083] (3) According to the manufacturer's instructions, use the ABI Prism 7500 rapid real-time PCR system for qPCR detection, using a two-step PCR reaction, with the following program: 95 ℃ pre-denaturation for 30 s, 95 ℃ denaturation for 5 s, 60 ℃ annealing for 30 s, and cycling 40 times.

[0084] (4) Use 2 -ΔΔCt The relative quantification method was used to calculate the expression changes of PTI marker genes in different treatment groups. The ACTIN gene was used as an internal reference to ensure the accuracy and reliability of the qPCR data. The experimental results were used to evaluate whether the RP426 protein could induce the expression of PTI-related genes and further verify its potential role in plant immune regulation.

[0085] As Figure 2As shown, the present application determines the expression of PTI marker genes Acre31, WRKY7, WRKY8 and PR1 in GFP and RP426 protein treated N. benthamiana leaves at 12 h and 24 h after treatment. The results show that compared with the GFP control, the expression level of the Acre31 gene in the RP426 protein treated leaves is up-regulated by 8 times, the expression level of the WRKY7 gene is up-regulated by 50 times, the expression level of the WRKY8 gene is up-regulated by 4 times, and the expression level of the PR1 gene is up-regulated by 100 times. The results show that RP426 protein can significantly induce the expression of PTI marker genes in N. benthamiana, indicating that RP426 protein may play an important role in plant immune response and enhance the resistance of plants to insects.

[0086] Example 5 Pathogen inoculation

[0087] (1) The P. 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 plug formation. The medium was kept moist during the culture process to avoid drying and affect the activity of the pathogen.

[0088] (2) On four-week-old N. benthamiana leaves, 1 μM RP426 recombinant protein was used for pressure injection, and expressed for 12 h to induce plant immune response. 1 μM GFP recombinant protein was used as a negative control to exclude the non-specific effects of exogenous protein expression on disease response.

[0089] (3) The treated tobacco leaves were gently cut and placed with the back facing up in a tray containing wet absorbent paper to provide a suitable humidity environment and promote pathogen infection.

[0090] (4) An equal amount of plugs from the P. capsici culture medium cultured for 2 days were directly inoculated onto the back of the leaves, and about 5 μL of sterile water was added to the surface of the plugs to improve the contact between the pathogen and the leaf tissue and promote the infection process.

[0091] (5) After inoculation, the tray was sealed with plastic wrap to maintain humidity and incubated at 25°C in the dark to simulate natural pathogen infection conditions.

[0092] (6) After 48 hours of inoculation, the leaf lesion images were taken under ultraviolet light, and the lesion area was measured using ImageJ software to assess the extent of pathogen infection. Disease symptom observation should be carried out under the same imaging conditions to ensure the comparability of the data.

[0093] (7) All experiments included at least 8 biological replicates and were performed in triplicate to ensure data stability and experimental result reproducibility.

[0094] As shown in Figure 3 Figure 6B, the lesion area of RP426-treated N. benthamiana leaves was significantly smaller than that of GFP-treated leaves in P. capsici LT263 inoculation experiment, indicating that RP426 protein played a positive role in the immune defense mechanism of N. benthamiana and improved its resistance to the pathogen. This result provides experimental evidence for further analyzing the function of RP426 in the plant immune regulation mechanism.

[0095] Example 6 Spodoptera exigua feeding preference experiment

[0096] (1) Four-week-old N. benthamiana plants were selected, and 1 μM RP426 recombinant protein was used for pressure injection (infiltration) on the front or back of the leaves, and the treatment lasted for 12 h to induce the immune response of the plant. Meanwhile, 1 μM GFP recombinant protein was injected into the leaves of the control group. Subsequently, the treated leaves were cut off and wrapped with water-absorbed defatted cotton around the petiole to prevent the leaves from wilting prematurely due to water loss. The treated leaves were placed in a circular culture dish to prepare for the insect feeding experiment.

[0097] (2) Eight second-instar larvae were selected from the laboratory-reared Spodoptera exigua population and placed in the middle of two leaves, respectively, to ensure that the larvae could freely choose different treated leaves. The culture dish was sealed with sealing film to prevent the larvae from escaping and to maintain appropriate humidity and temperature 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 larvae were allowed to feed freely for 48 h, and the feeding behavior of the insects was observed.

[0098] (3) After the experiment was completed, all the leaves were taken out and photographed, and the feeding damage was recorded under the same light conditions. ImageJ software was used to measure the damage area of the leaves after insect feeding, and data statistical analysis was performed to evaluate the effect of RP426 recombinant protein on the feeding preference of Spodoptera exigua.

[0099] As shown in Figure 4 Figure 7B, the area of Spodoptera exigua feeding on RP426-treated N. benthamiana leaves was significantly reduced compared to GFP-treated leaves, indicating that RP426 protein enhanced the resistance of N. benthamiana to Spodoptera exigua feeding, thereby affecting the feeding preference of the insects. This experiment provides experimental evidence for the potential function of RP426 protein in plant insect resistance.

[0100] Unless otherwise specifically stated, the numerical values set forth in these examples do not limit the scope of the present application. In all examples shown and described herein, unless otherwise specified, any specific value should be interpreted as merely an example, and not as a limitation, and thus, other examples of the example embodiments can have different values.

Claims

1. Use of a Piezodorus guildinii salivary protein RP426 having HAMP activity or a nucleic acid encoding the protein in increasing the ability of a plant to resist infection by a pathogenic fungus or to resist damage by a phytophagous pest, characterized in that, The amino acid sequence of the saliva protein RP426 is shown as SEQ ID NO. 1, the sequence of the encoding nucleic acid is shown as SEQ ID NO. 2, the plant is tobacco, the pathogenic bacteria is Phytophthora capsici, and the phytophagous pest is Spodoptera litura.

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