Brown planthopper G-protein coupled receptor Methuselah-like 2 and application thereof

Abstract

The invention discloses a brown planthopper G-protein coupled receptor Methuselah-like 2 and application of the brown planthopper G-protein coupled receptor Methuselah-like 2. The amino acid sequence of the G-protein coupled receptor Methuselah-like 2 is as shown in SEQ ID NO. 2. The dsRNA (dsMthl2) of the brown planthopper G-protein coupled receptor Methuselah-like 2 disclosed by the invention has better gene silencing efficiency, so that the ecdysis rate of brown planthopper nymphs can be obviously reduced, epidermis dysplasia can be caused, an ordered and uniform endodermis structure cannot be formed, the outer epidermis is thickened, and the structure is loose. The brown planthopper G-protein coupled receptor Methuselah-like 2 disclosed by the invention can be used as an important molecular target for preventing and controlling brown planthopper, is expected to fully play the function of ecological prevention and control while inhibiting pests, and can be widely applied to crop breeding, biopesticide research and development and biological prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a brown planthopper G-protein coupled receptor Methuselah-like 2 and its applications. Background Technology

[0002] Brown planthopper Nilaparvata lugens The brown planthopper, belonging to the order Hemiptera and family Planthopperidae, is a significant pest in rice-growing regions of my country and Asia, threatening my country's food security. It is an insect with incomplete metamorphosis, consisting of three life stages: egg, nymph, and adult, with the nymph having five instars. Female adult brown planthoppers lay their eggs inside rice stems using their slender reproductive organs. On average, each female can lay over 300 eggs during its entire oviposition period, and this can even reach over 600 eggs in susceptible rice varieties. The adult lifespan is over 15 days. The brown planthopper is a monophagous pest with piercing-sucking mouthparts, feeding on the sap of the rice phloem. Severe infestations can lead to malnutrition and lodging in rice plants, ultimately resulting in reduced yields or even total crop failure. Furthermore, brown planthoppers can cause various rice viral diseases, such as rice tooth-leaf dwarf virus (RRSV) and rice black-streaked dwarf virus (RBSDV). Currently, brown planthopper control in agricultural production generally employs a combination of agricultural, biological, and chemical control methods, with chemical pesticides as the primary approach, supplemented by the breeding of resistant rice and biological control. However, long-term use of chemical pesticides easily leads to the "3R" problems—environmental pollution from pesticide residues, increased pesticide resistance in pests, and resurgence—seriously impacting people's living environment and food safety. Therefore, finding a sustainable, safe, and green control method is crucial for brown planthopper control.

[0003] RNA interference (RNAi) is a naturally occurring post-transcriptional gene silencing mechanism within organisms. When the sense and antisense strands of a target gene are introduced into an organism, they inhibit the transcriptional expression of the target gene. RNA biopesticides utilize the principle of RNA interference to suppress the expression of important functional genes in organisms, causing developmental arrest or death of harmful organisms, thereby achieving the purpose of pest and disease control. This technology does not alter the genome of harmful organisms and does not have adverse effects on the ecosystem. Due to its advantages such as precision, high efficiency, and being environmentally friendly and pollution-free, RNA biopesticides have attracted the attention of plant protection experts.

[0004] G-protein-coupled receptors (GPCRs) are currently considered the largest and most widely distributed family of cell surface receptors, consisting of seven transmembrane helical structures of cell membrane proteins. They can be activated by extracellular ligands, transmitting extracellular signals into the cell and then regulating functions such as growth, development, behavior, reproduction, stress resistance, and lifespan. Therefore, GPCRs are currently considered important potential targets for developing next-generation insecticides. The only receptor in the GPCR-B3 family, Methuselah / Methuselah-like (Mth / Mthl), was first discovered in Drosophila melanogaster and can affect various physiological functions of insects, including lifespan, stress resistance, molting, and reproduction. The Mthl1-5 subtypes exist in brown planthoppers. Currently, the function of Mthl2 in brown planthoppers is unclear, and whether it can serve as a gene target for effective control of brown planthoppers has not been reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a brown planthopper G-protein coupled receptor Methuselah-like2 and its application, and inhibiting its expression can effectively control the population size of brown planthoppers.

[0006] This invention is achieved through the following technical solution:

[0007] Methuselah-like 2, a G-protein coupled receptor for brown planthopper, has the amino acid sequence shown in SEQ ID NO.2.

[0008] A gene encoding the aforementioned brown planthopper G-protein-coupled receptor Methuselah-like 2, the gene being a full-length ORF sequence, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] A primer pair for obtaining the above-mentioned gene, the primer pair comprising upstream and downstream primers, the nucleotide sequence of the upstream primer being shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer being shown in SEQ ID NO.4.

[0010] Application of inhibitors that suppress the expression of the brown planthopper G-protein coupled receptor Methuselah-like 2 in the control of brown planthoppers.

[0011] Application of inhibitors that suppress the expression of the brown planthopper G-protein coupled receptor Methuselah-like 2 in the preparation of insecticides.

[0012] Preferably, the inhibitor is a dsRNA synthesized based on the brown planthopper G-protein coupled receptor Methuselah-like 2.

[0013] Preferably, the dsRNA is prepared using a method comprising the following steps:

[0014] Step 1) Design upstream and downstream primers based on the brown planthopper G-protein coupled receptor Methuselah-like 2. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.6, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.7.

[0015] Step 2) Obtain a DNA fragment by PCR amplification, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0016] Step 3) The PCR amplification product was purified and then transcribed in vitro to synthesize the dsRNA.

[0017] The beneficial effects of this invention are as follows:

[0018] The dsRNA (dsMthl2) of the brown planthopper G-protein-coupled receptor Methuselah-like 2 of this invention exhibits good gene silencing efficiency, significantly reducing the molting rate of brown planthopper nymphs, resulting in poor epidermal development, failure to form an orderly and uniform inner epidermal structure, and thickened and loosely structured outer epidermis. Compared with the control group treated with dsEGFP, the molting process of brown planthopper nymphs treated with dsMthl2 is slow and the molting success rate is low. In addition, after injection of dsMthl2, the ovaries of female brown planthopper adults are incompletely developed, failing to exhibit a plump and cottony appearance, resulting in a significant decrease in egg production and offspring numbers. The brown planthopper G-protein-coupled receptor Methuselah-like 2 of this invention can serve as an important molecular target for the control of brown planthoppers, and is expected to fully exert the function of ecological control while suppressing pests, and can be widely used in crop breeding, biopesticide development, and biological control. Attached Figure Description

[0019] Figure 1 The agarose gel electrophoresis diagram of dsMthl2 synthesized in vitro in Example 2 is shown below: M is the marker, 1 is the synthesized dsRNA (dsMthl2), and 2 is the control group dsEGFP.

[0020] Figure 2 To illustrate the interference efficiency of different doses of dsMthl2 on the Methuselah-like 2 target gene of brown planthopper nymphs in Example 3, dsEGFP served as the control group (left column): A represents the silencing efficiency of different concentrations of dsMthl2 on the target gene; B represents the silencing efficiency of 60 ng dsMthl2 injected at different times on the target gene.

[0021] Figure 3The effect of dsMthl2 injection on molting of brown planthoppers in Example 3, with dsEGFP as the control group: A shows the effect of dsMthl2 injection on molting rate of brown planthoppers; B shows the effect of dsMthl2 injection on epidermal thickness of brown planthopper nymphs (TEM image); C shows the effect of dsMthl2 injection on growth and development of brown planthopper nymphs.

[0022] Figure 4 The effect of dsMthl2 injection on ovarian development in female brown planthoppers in Example 3 is shown below: A: 2 days after dsEGFP injection; B: 2 days after dsMthl2 injection; C: 4 days after dsEGFP injection; D: 4 days after dsMthl2 injection; E: 6 days after dsEGFP injection; F: 6 days after dsMthl2 injection; G: 8 days after dsEGFP injection; H: 8 days after dsMthl2 injection.

[0023] Figure 5 The effects of dsMthl2 injection on reproductive parameters and progeny populations of female brown planthoppers in Example 3 are as follows: A shows the effect of dsEGFP and dsMthl2 injection on oviposition rate of female brown planthoppers; B shows the effect of dsEGFP (left) and dsMthl2 (right) injection on pre-oviposition and oviposition duration of female brown planthoppers; C shows the effect of dsEGFP and dsMthl2 injection on the population growth index of brown planthoppers; D shows the effect of dsEGFP and dsMthl2 injection on the population size of F1 progeny of brown planthoppers. The effects of injection of dsEGFP and dsMthl2 on the hatching rate of F1 progeny of brown planthopper; the effects of injection of dsEGFP and dsMthl2 on the sex ratio of F1 progeny of brown planthopper; the effects of injection of dsEGFP and dsMthl2 on the population size of F2 progeny of brown planthopper; the effects of injection of dsEGFP and dsMthl2 on the hatching rate of F2 progeny of brown planthopper; and the effects of injection of dsEGFP and dsMthl2 on the sex ratio of F2 progeny of brown planthopper. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0026] Based on transcriptome analysis of female brown planthoppers that fed on rice treated with jinggangmycin (JGM) for 2 days, our research group found that compared with untreated rice, the expression level of Mthl2 in female brown planthoppers that fed on JGM-treated rice for 2 days was significantly upregulated, while the expression levels of Mthl1, Mthl3, Mthl4, and Mthl5 showed no significant difference. Based on this, the following experiment was designed.

[0027] Example 1: Cloning of the Methuselah-like 2 encoding gene in the brown planthopper

[0028] 1. Rearing of brown planthoppers

[0029] The tested rice variety was Yangdao 6 (indica rice), provided by the Jiangsu Lixiahe Research Institute. After soaking and germination, the seeds were sown in standard outdoor cement ponds (60cm×100cm×200cm). When the seedlings reached the 6-leaf stage, they were transplanted into 16cm diameter plastic containers, with 3 holes per container and 4 seedlings per hole. The experiment continued until the rice tillering stage. Throughout the experiment, the ponds were covered with 80-mesh insect-proof netting to prevent pest intrusion. The tested brown planthopper population came from the China National Rice Research Institute (Hangzhou, China) and had been continuously reared in the laboratory for many years without exposure to any pesticides. Brown planthoppers were reared using indoor-cultured rice seedlings that had not been exposed to pesticides. Seedlings were replaced promptly according to the planthopper population. The rearing conditions were 26±2℃, relative humidity 65%–75%, and a photoperiod of L:D = 16h:8h.

[0030] 2. RNA extraction from brown planthopper nymphs

[0031] Total RNA was extracted from brown planthoppers using the TRIzol method (Thermo Fisher Scientific, USA). The extraction steps are as follows:

[0032] (1) Place the collected samples into a 1.5 mL EP tube without RNase, add 500 μL TRIzol, and grind the samples thoroughly with a grinding stick;

[0033] (2) After thorough grinding, add 500 μL of TRIzol to the EP tube again and let it stand at room temperature for 5 min;

[0034] (3) After centrifuging at 12,000 rpm for 10 min at 4℃, transfer the supernatant into a new 1.5 mL EP tube without RNase;

[0035] (4) Add 200 μL of chloroform to the new EP tube, seal the centrifuge tube tightly, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0036] (5) Centrifuge at 4℃ and 12000rpm for 15min. At this time, the mixture will be divided into three layers. Use a pipette to take small amounts of the colorless aqueous phase (RNA is dissolved in the aqueous phase) from the top layer and transfer it to a new RNase-free EP tube.

[0037] (6) Add an equal volume of isopropanol to the colorless water obtained in step (5), mix well, and let stand at room temperature for 30 minutes to fully precipitate RNA.

[0038] (7) After centrifuging at 12,000×g for 10 min at 4℃, remove the supernatant;

[0039] (8) Add 1 mL of 75% ethanol to the obtained RNA precipitate to wash the precipitate, centrifuge at 5,000×g for 5 min at 4°C, and carefully remove the supernatant;

[0040] (9) Then repeat step (8) three times;

[0041] (10) After air-drying at room temperature for 5-10 minutes, add 20 μL RNase Free ddH2O to fully dissolve the precipitate to obtain the RNA solution, which is then stored at -80℃ for later use.

[0042] (11) The concentration and purity of RNA samples were detected using NanoDrop 2000, OD 260 / 280 The values ​​range from 1.8 to 2.1; 1.5% agarose gel electrophoresis is used to detect the presence of genomic residues or protein contamination, as well as the integrity of RNA bands.

[0043] 3. Cloning of the Methuselah-like 2 coding gene

[0044] (1) The ORF of the Methuselah-like 2 coding gene of the brown planthopper was analyzed based on the Open Reading Frames (ORF) prediction URL (http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html). Specific primers for the Methuselah-like 2 coding gene were designed using Primer Premier 5.0 software to amplify its ORF. The cloning primers are as follows:

[0045] F (SEQ ID NO.3): 5'-GTGGAAATAGGTGACATGTAG-3';

[0046] R (SEQ ID NO. 4): 5'-TGGCAGATGTTGAAGATGTCGG-3'.

[0047] (2) The reaction system is shown in Table 1 below:

[0048] Table 1 Reaction System

[0049]

[0050]

[0051] (3) After gently tapping to mix, briefly centrifuge at low speed to collect the reaction solution to the bottom of the tube. The reaction procedure is shown in Table 2 below:

[0052] Table 2 Reaction Procedure

[0053]

[0054] (4) After the reaction, agarose gel electrophoresis (2%) was used to detect whether the bands of the PCR products were the same size as the target gene.

[0055] (5) The target band was cut and recovered using a gel recovery kit. The steps are as follows:

[0056] ① Place the agarose gel under ultraviolet light and cut out a portion containing the target fragment, keeping the amount as small as possible;

[0057] ② Transfer the gel block into a 2mL EP tube, add 400μL Binding Solution, and heat the gel block in the EP tube at 60℃ in a metal bath until the gel block is completely dissolved;

[0058] ③ Transfer the mixture into an adsorption tube with a filter membrane, let it stand at room temperature for 2 minutes, and centrifuge at 6,000×g for 1 minute;

[0059] ④ Repeat step ③ to remove residual liquid from the collection tube;

[0060] ⑤ Add 500 μL of WA to the adsorption tube, centrifuge at 12,000 × g for 1 min, and remove the residual liquid in the collection tube;

[0061] ⑥ Add 500 μL Wash Solution to the adsorption tube, centrifuge at 12,000 × g for 1 min, and remove the residual liquid in the collection tube;

[0062] ⑦ Repeat step ⑥ to remove residual liquid from the collection tube;

[0063] ⑧Then centrifuge at 12,000×g for 2 min, transfer the adsorption tube to a new 1.5 mL EP tube, and place at room temperature for 10 min;

[0064] ⑨ Add 20 μL of RNase-free ddH2O to the filter membrane in the middle of the adsorption tube, let it stand at room temperature for 3 min, and centrifuge at 12,000×g for 2 min to obtain the product of gel recovery.

[0065] ⑩ The purity and concentration of the recovered product were determined by agarose gel electrophoresis (2%) and Nano Drop 2000 assay.

[0066] (6) The product after gel extraction, recovery, and purification was linked to the TA / Blunt-Zero support. The linkage reaction system is shown in Table 3 below:

[0067] Table 3 Connection Reaction System

[0068]

[0069] (7) The recombinant plasmid obtained in step (6) was introduced into competent DH5α cells, and cloned, screened and purified. The specific steps are as follows:

[0070] ① Thaw competent DH5α cells in an ice-water mixture;

[0071] ② When the cells are completely thawed, dispense them into 50 μL tubes of competent cells, add 5 μL of the recombinant plasmid obtained in step (6) to each tube, gently tap the bottom of the tube to mix them, and let them stand in an ice-water mixture for 30 min.

[0072] ③ After the settling period, remove the centrifuge tube and heat shock it at 42℃ for 45 seconds. Then quickly remove it and transfer it to an ice-water mixture and let it stand for 2 minutes.

[0073] ④ Add 900 μL of liquid LB medium to the mixture after the reaction in step ③, and place it in a shaker at 37°C and 200 rpm for 1 h to revive competent cells;

[0074] ⑤ After shaking and incubating for 1 hour, centrifuge at 2500 rpm for 3 minutes, take out 900 μL of supernatant with a pipette and remove it, resuspend the bacterial cells and spread them evenly on LB plates containing ampicillin resistance.

[0075] ⑥ Transfer the plate to a 37°C incubator for a few minutes until the bacterial culture is dry, then invert the plate and incubate overnight;

[0076] ⑦ Select 3 single clones and transfer them into 1 mL of LB liquid medium. Incubate on a shaker for 4 h. Then streak the bacterial culture onto an LB plate and incubate again. Select 6 single clones and incubate them in the same way for 4 h.

[0077] ⑧ The purified monoclonal bacterial culture was verified by PCR using the M13 primer. Six positive clones were selected and sequenced to obtain the sequences and verify their accuracy.

[0078] The sequencing results were compared with the transcriptome search results to obtain the full-length nucleotide sequence of the Methuselah-like 2 encoding gene, as shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0079] Example 2: Synthesis of Methuselah-like 2-specific dsRNA from the brown planthopper G-protein-coupled receptor

[0080] 1. Rearing of brown planthoppers

[0081] The experimental methods and procedures are the same as in Example 1.

[0082] 2. Total RNA extraction and cDNA synthesis

[0083] The experimental methods and procedures are the same as in Example 1.

[0084] 3. Synthesis of Methuselah-like 2-specific dsRNA

[0085] (1) The full-length sequence of the target gene was cloned, and primers for synthesizing dsRNA were designed using Primer Premier 5.0 software. The dsRNA template sequence was 300-500 bp in length. Primers were designed and synthesized (Nanjing Qingke Biotechnology Co., Ltd., Nanjing). The target fragment was obtained by PCR amplification using 2×Rapid Taq Master Mix (Nanjing Novizan Biotechnology Co., Ltd., Nanjing): 95℃, 3 min (pre-denaturation); 95℃, 15 sec (denaturation); 57℃, 15 sec (annealing); 72℃ extension for 30 sec, for a total of 30 cycles; and finally, 72℃ extension for 10 min to obtain the target fragment (SEQ ID NO.5).

[0086] The primer sequences are as follows:

[0087] F (SEQ ID NO.6): 5'-TAATACGACTCACTATAGGGGTACCGCGGGACCATTTCC-3';

[0088] R (SEQ ID NO. 7): 5'-TAATACGACTCACTATAGGGTTTGCTCAGGGCGGA CTGG-3'.

[0089] (2) The size of the amplified product was determined by 2% agarose gel electrophoresis. The target band was cut off and recovered under UV light and purified using FastPure Gel DNA Extraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd., Nanjing).

[0090] (3) Using T7 RiboMAX TMThe dsRNA was synthesized using the Express RNAi system kit (Promega, USA), and the reaction system was prepared in 1.5 mL Axygen centrifuge tubes as shown in Table 4 below:

[0091] Table 4 Reaction System

[0092]

[0093] (4) Place the mixed liquid in the centrifuge tube in a preheated water bath at 37°C for 30 min; then at 70°C for 15 min. Remove the centrifuge tube and allow it to cool at room temperature for 20 min.

[0094] (5) Take out a 0.2 mL Axygen centrifuge tube and add 199 μL RNase-Free ddH2O and 1 μL RNaseA Solution, and shake to mix.

[0095] (6) After step (4) is completed, add 1 μL of the mixed liquid from step (5) and 1 μL of RQ1 RNase-Free DNase, shake to mix, place in a preheated water bath, 37°C, 30 min;

[0096] (7) After the reaction is complete, add 50 μL of 95% ethanol and 2 μL of sodium acetate, 3.0 M (pH = 5.2), shake to mix, and let stand on ice for 5 min; centrifuge at 12,000 × g, 4 °C for 10 min, and discard the supernatant.

[0097] (8) Add 750 μL of 75% ethanol to wash the RNA precipitate; centrifuge at 7,500×g, 4℃ for 5 min, and discard the supernatant;

[0098] (9) Repeat step (8);

[0099] (10) After the dsRNA precipitate is dried, add an appropriate amount of nuclease-free water to dissolve it for 5 min, and use 3% agarose gel electrophoresis to determine the integrity of the dsRNA. Use NanoDrop2000 (Thermo Fisher Scientific, USA) to detect the dsRNA concentration and store it at -80℃ for later use.

[0100] like Figure 1 As shown, 1 is the dsRNA (dsMthl2) synthesized in this embodiment; 2 is the control group dsEGFP.

[0101] Example 3: Effects of Methuselah-like 2-specific dsRNA on molting and reproduction of brown planthoppers.

[0102] 1. Injection of Methuselah-like 2-specific dsRNA from brown planthopper G-protein-coupled receptor

[0103] Fourth instar brown planthopper nymphs of uniform growth were selected and anesthetized with CO2 gas. Different doses (20 ng, 40 ng, 60 ng, 80 ng, 100 ng) of dsMthl2 synthesized in Example 2 were injected into the mesothorax of the nymphs. Different doses of dsEGFP were used as control groups. After injection, the nymphs were transferred to fresh 14-day-old rice seedlings for rearing. The rice seedlings were replaced as needed based on the nymph population. The rearing conditions were 26±2℃, relative humidity 65%–75%, and photoperiod L:D = 16 h:8 h. Each treatment group and control group had three independent biological replicates, with 15 nymphs per replicate.

[0104] After determining the optimal dsRNA injection concentration, brown planthopper nymph samples were collected at 12h, 24h, 48h, 72h and 96h after injection to further clarify the duration of the dsMthl2 silencing effect.

[0105] 2. Detection of the silencing efficiency of the Methuselah-like 2 G-protein-coupled receptor gene in brown planthopper.

[0106] Brown planthopper nymphs treated as described above were injected with dsMthl2 and dsEGFP. RNA was extracted from nymphs injected with different doses and at different time points using the optimal dsRNAs, and cDNA was synthesized (RNA extraction and cDNA synthesis were the same as in Example 1). Gene silencing efficiency was detected using quantitative real-time PCR (qRT-PCR). Three independent biological replicates were performed for each treatment group and the control group, with 15 nymphs per replicate. The qRT-PCR reaction system is shown in Table 5 below.

[0107] Table 5 qRT-PCR reaction system

[0108]

[0109] After a brief, low-speed centrifugation, collect the reaction solution to the bottom of the tube. The qRT-PCR reaction procedure is shown in Table 6 below:

[0110] Table 6 qRT-PCR reaction procedure

[0111]

[0112] Experimental results are as follows Figure 2As shown, different doses of dsMthl2 all exhibited good silencing effects on the Methuselah-like 2 target gene, and the silencing efficiency gradually increased with increasing injection dose. At a dsRNA injection concentration of 60 ng, the silencing effect stabilized and did not continue to increase with further increases in dsRNA injection concentration. The silencing effect on the target gene remained above 80%. Figure 2 (A), and the silencing effect of the target gene can remain above 75% after 48 hours. Figure 2 (B)

[0113] 3. Effects of dsMthl2 injection on molting of brown planthopper nymphs

[0114] (1) Experimental steps

[0115] Fifth-instar early-stage brown planthopper nymphs were injected with dsMthl2, with dsEGFP injection serving as the control group. Each treatment group and control group had three independent biological replicates, with 50 nymphs per replicate. After injection, the nymphs were transferred to fresh rice seedlings. The survival of the nymphs was observed and recorded daily. The phenotype of the nymphs was observed using a stereomicroscope (OLYMPUS CX23, Japan), and photographs were taken using a DS-Fi2 digital camera (Nikon, Japan). Finally, the molting rate of the fifth-instar nymphs was calculated.

[0116] To further clarify the effect of dsMthl2 on the epidermis of brown planthopper nymphs, early 5th instar brown planthopper nymphs were injected with dsMthl2, with dsEGFP injection serving as the control group. After injection, the nymphs were transferred to fresh rice seedlings for rearing. After 72 hours, surviving nymphs were selected, and the 4th to 8th segments of the dorsal and ventral epidermis of the brown planthopper were dissected under a stereomicroscope. The sections were immediately immersed in 2.5% glutaraldehyde fixative to prepare ultrathin sections (70 nm) of the epidermal tissue. The samples were observed and photographed using transmission electron microscopy (TEM) at an accelerating voltage of 80 kV.

[0117] (2) Experimental Results

[0118] Experimental results are as follows Figure 3 As shown, the molting ability of 5th instar brown planthoppers was downregulated after injection of dsMthl2. Compared with the control group treated with dsEGFP, the molting rate of 5th instar early nymphs of brown planthoppers treated with dsMthl2 was reduced by 29%. Figure 3 The levels of dsMthl2 in the 5th instar early-stage brown planthopper nymphs were significantly lower than those in the control group. Transmission electron microscopy results showed that after 4 days of treatment with dsMthl2, the inner epidermis of the brown planthopper was poorly developed, failing to form an orderly and uniform inner epidermal structure, while the outer epidermis was thickened and loosely structured. Figure 3(B). Compared to dsEGFP treatment of 5th instar early brown planthopper nymphs, which resulted in molting on day 5, dsMthl2 treatment of 5th instar early brown planthopper nymphs resulted in molting on day 6, with a slow molting process and low molting success rate. Figure 3 (C)

[0119] 4. Effects of dsMthl2 injection on the reproduction of female adult brown planthoppers

[0120] (1) Experimental steps

[0121] Fifth-instar brown planthopper nymphs injected with dsMthl2 (with dsEGFP as a control) were reared until initial emergence. Short-winged female brown planthoppers were harvested at 2, 4, 6, and 8 days after emergence. Their ovaries were dissected, and ovarian development morphology was photographed. The ovarian tubules were also dissected separately, and their development morphology was photographed. The ovarian grading index was recorded (n=33, N=3). The ovarian area was calculated using ImageJ software under a DS-Fi2 digital camera (Nikon, Japan) (n=15, N=3).

[0122] Population parameters of female brown planthoppers were determined by pairing unmated females with untreated males. Each pair was then transferred to rice stalks in the tillering stage (4 holes per bucket, 3 plants per hole), and sealed with nylon netting. Once the F1 generation nymphs reached the third instar, the number of offspring was counted, and they were transferred to new rice stalks placed in glass containers. After emergence, the sex ratio was recorded, and unhatched brown planthopper eggs were counted by dissecting the rice stalks. The hatching rate and population growth index (n=15, N=3) were calculated. The F1 generation nymphs were then raised to emergence. Unmated females were paired with untreated males, and each pair was transferred to rice stalks in the tillering stage (4 holes per bucket, 3 plants per hole), and sealed with nylon netting. After the brown planthopper offspring (F2 generation) nymphs reached the 3rd instar, the number of offspring was counted and transferred to new rice stalks, placed in glass cups, and fed until they emerged. The sex ratio was recorded. At the same time, the unhatched brown planthopper eggs were counted by dissecting the rice stalks, and the hatching rate and population growth index (n=15, N=3) were calculated.

[0123] (2) Experimental Results

[0124] Experimental results are as follows Figure 4 , 5 As shown, after silencing the brown planthopper Methuselah-like 2 using RNAi, the ovaries of females emerging two days prior were not fully developed and did not exhibit a plump, cottony appearance. Figure 4 (A, B); After treatment with dsEGFP, arched eggs appeared in the ovarian tubules of female brown planthoppers 4 days after emergence; after treatment with dsMthl2, arched eggs appeared 6 days after emergence. Figure 4(CF); Compared with the control group treated with dsEGFP, the female brown planthoppers that emerged 8 days after injection of dsMthl2 had incomplete ovarian development, with only a few mature eggs (CF); Figure 4 (G, H). Furthermore, the reproductive capacity of female brown planthoppers was significantly reduced after dsMthl2 treatment. Compared with the control group treated with dsEGFP, the oviposition rate of female brown planthoppers was significantly reduced after dsMthl2 treatment. Figure 5 In the middle (A), the pre-spawning period was significantly prolonged, and the spawning duration was significantly reduced. Figure 5 (B) Offspring number, hatching rate, and population growth index all decreased significantly. Figure 5 (China CI).

[0125] The experimental results of the above embodiments show that Methuselah-like 2 plays an important role in the biological processes of brown planthopper, such as molting and reproduction. Inhibiting its expression can effectively control the population of brown planthopper. Therefore, it is expected to play a full role in ecological control while suppressing pests, and can be widely used in crop breeding, biopesticide research and development and biological control.

[0126] The embodiments described above are only some, not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A brown planthopper G-protein-coupled receptor, Methuselah-like 2, characterized in that, The amino acid sequence of the G-protein coupled receptor Methuselah-like 2 is shown in SEQ ID NO.

2.

2. A gene encoding the brown planthopper G-protein-coupled receptor Methuselah-like 2 as described in claim 1, characterized in that, The gene is the full-length ORF sequence, and its nucleotide sequence is shown in SEQ ID NO.

1.

3. A primer pair for obtaining the gene as described in claim 2, characterized in that, The primer pair includes upstream and downstream primers, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

4.

4. The application of the inhibitor that inhibits the expression of the brown planthopper G-protein coupled receptor Methuselah-like 2 as described in claim 1 in the control of brown planthoppers.

5. The use of an inhibitor that suppresses the expression of the brown planthopper G-protein coupled receptor Methuselah-like 2 as described in claim 1 in the preparation of an insecticide.

6. The application according to claim 4 or 5, characterized in that, The inhibitor is a dsRNA synthesized based on the brown planthopper G-protein coupled receptor Methuselah-like 2.

7. The application according to claim 6, characterized in that, The dsRNA was prepared using a method comprising the following steps: Step 1) Design upstream and downstream primers based on the brown planthopper G-protein coupled receptor Methuselah-like 2. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.6, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

7. Step 2) Obtain a DNA fragment by PCR amplification, the nucleotide sequence of which is shown in SEQ ID NO.5; Step 3) After purification, the PCR amplification product is transcribed in vitro to synthesize the dsRNA.

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