Brown planthopper nlnpf gene and dsrna in the application of preventing and treating brown planthopper

Abstract

The application discloses a brown planthopper NlNPF gene and application of dsRNA of the brown planthopper NlNPF gene in prevention and treatment of the brown planthopper. The nucleotide sequence of the NlNPF gene is shown as SEQ ID NO. 1, the ORF sequence is shown as bases 55-354 of SEQ ID NO. 1, and the amino acid sequence of the NlNPF gene is shown as SEQ ID NO. 2. The dsRNA of the brown planthopper NlNPF gene can inhibit expression of the brown planthopper NlNPF gene, reduce feeding of the brown planthopper on rice, and reduce the survival rate of the brown planthopper. The application obtains the cDNA sequence of the brown planthopper NlNPF gene, adopts the RNAi technology of microinjection of the dsRNA, silences the brown planthopper NlNPF gene, causes the brown planthopper to produce the antifeeding effect, and after spraying of the nano-carrier dsRNA, the mortality of the brown planthopper is obviously increased, so the dsRNA of the gene has important application value in the field of prevention and treatment of the brown planthopper.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the NlNPF gene and its dsRNA in brown planthoppers and RNA interference against this gene in the control of brown planthoppers. Background Technology

[0002] Brown planthopper [Nilaparvata lugens] The brown planthopper (Hemiptera: Delphacidae) is a migratory rice pest capable of long-distance flight and exhibits strong adaptability to various environments. It is currently the leading rice pest in my country and many Southeast Asian countries (Sogawa, 1982; Velusamy et al., 1986). The brown planthopper is selective in its host plants, feeding and laying eggs only on rice or wild rice (Dyck et al., 1979; Hong et al., 2007; Wang et al., 2008). Brown planthoppers primarily damage rice plants by sucking sap from the phloem, severely impacting rice growth and development. The salivary sheaths formed during feeding block the vascular bundles, and the planthoppers secrete large amounts of honeydew after feeding. This honeydew contains abundant amino acids and sugars, which can infect the rice plant with bacteria (Li Chunfeng et al., 2019). Severely infested rice plants can suffer from paralysis and lodging, leading to "planthopper burn" and severe yield reductions or even total crop failure. In addition to their own health problems, brown planthoppers also act as vectors for rice viral diseases such as rice clump dwarf virus and toothed leaf dwarf virus, which can have catastrophic consequences for rice yields (Hibino, 1996; Cheng Xiannian et al., 2003). Since the 1980s, the area affected by brown planthoppers in my country has been approximately 13.3-20 million hectares. 2 Brown planthoppers cover approximately half of my country's rice-growing area, causing serious losses to my country's rice production (Li Ruduo et al., 1996). In the past thirty years, the main characteristics of brown planthopper damage to rice in my country have been threefold: increased outbreak frequency, expanded damage range, and increased damage severity (Wang Peng et al., 2013).

[0003] RNAi, or RNA interference, is a technique that uses double-stranded RNA (dsRNA) to induce silencing of target gene mRNA, thereby affecting the normal physiological activities of the target organism. RNAi research began in 1995 when Guo et al. at Cornell University, after interfering with the nematode *Caenorhabditis elegans* using both sense and antisense RNA, found that both types of RNA interference caused silencing in the nematode. Caenorhabditis elegansParl gene expression was downregulated (Guo et al., 1995). In 1998, to reveal this phenomenon, Fire et al. at the Carnegie Institution of Washington injected purified single-stranded RNA into nematodes. Caenorhabditis elegans It was found that there was only a weak inhibitory effect, while injecting purified dsRNA into nematodes... Caenorhabditis elegans In their study, purified dsRNA was found to have a strong inhibitory effect on the expression of related genes. This confirmed that the inhibition of Parl gene expression by positive RNA in Guo and Kemphues' experiment was due to trace amounts of dsRNA contamination, leading to experimental results that did not match the theory, thus becoming the earliest RNAi phenomenon (Fire et al., 1998). To date, due to its ease of operation, relatively low cost, and more importantly, its specificity and high efficiency, RNAi technology has been widely applied in various fields of insect research (Bellés, 2010), such as verifying insect gene function, pest control, and developing new targets for pesticides (Tian Honggang et al., 2012; Wang Huidong et al., 2012).

[0004] Currently, the technique of using RNAi to interfere with insects is relatively mature. In insect-related research, the application of RNAi technology mainly focuses on gene function, RNAi-induced transgenic insect-resistant plants, and disease control in beneficial insects (Baum et al., 2007; Tian et al., 2009; Chen et al., 2010; Hunter et al., 2010; Yao et al., 2010). There are three main methods for introducing RNAi into insects: feeding, injection, and tissue culture. The optimal method must be selected based on different experimental needs. For example, the experiment silencing the salivary protein C002 in the pea aphid (Acyrthosiphonpisum) was the first study on the function of salivary proteins in piercing-sucking insects. The experiment found a significant relationship between the pea aphid's feeding and the C002 salivary protein (Mutti et al., 2006). Ji Rui (2013) found that silencing the salivary protein Nl1860 using RNA interference technology significantly reduced honeydew production and significantly increased mortality. However, RNA interference also has limitations in studying salivary proteins. For example, certain genes in some insects only appear in specific instars, stages, or tissues. For instance, in the tobacco hawk moth (Manduca sextai) and silkworm (Bombyx mori), genes are difficult to silence in biological tissues other than hemolymph (Eleftherianos et al., 2007; Miller et al., 2008; Huang Xiaohui, 2016). On the other hand, some salivary protein genes are essential for the normal development of insects. If RNA interference is performed on these genes, the insects will die rapidly, making it difficult to conduct experiments on the role of the gene in insect feeding.

[0005] In practical applications, dsRNA cannot exert its full effect due to problems such as rapid degradation and inefficient absorption in insects (Garbutt et al., 2013; Luo et al., 2013; Ren et al., 2014; Wynant et al., 2014). Therefore, improving the stability and cellular uptake capacity of dsRNA is key to realizing RNAi technology. Currently, nanoparticle (NP)-mediated RNAi control technology is gradually becoming a hot topic in pest control. As early as 2009, researchers discovered that dsRNA, when encapsulated in lipids, could produce a highly efficient RNAi effect in fruit flies through feeding. Moreover, this phenomenon has been verified in other species. In the Asian corn borer (Ostriniafurnacalis), feeding RNAi mediated by a cationic fluorescent nanoparticle (FNP) can effectively silence the expression of key developmental genes in the subspecies, resulting in a high lethality rate (He et al., 2013). In the soybean aphid (Aphis glycines), researchers used polymer-encapsulated dsRNA to overcome the insect's body wall barrier. In the Gambian mosquito (Anopheles gambiae), chitosan significantly increased the sensitivity of the insects to dsRNA feeding (Zhang et al., 2010). These examples illustrate the promising applications of nanoparticle-borne dsRNA. Summary of the Invention

[0006] The purpose of this invention is to provide the cDNA sequence and protein sequence of the NlNPF gene cloned from the brown planthopper. By silencing the NlNPF gene in the brown planthopper through microinjection and adjuvant-mediated RNAi, the feeding of the brown planthopper on rice is reduced, thereby protecting the rice crop.

[0007] The first objective of this invention is to provide a brown planthopper NlNPF gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the ORF sequence is shown in bases 55-354 of SEQ ID NO.1.

[0008] A second objective of this invention is to provide a protein encoded by the NlNPF gene of the brown planthopper, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The third objective of this invention is to provide a dsRNA for the NlNPF gene of the brown planthopper, which is a double-stranded RNA composed of nucleotides with the sequence shown in SEQ ID NO.1 that can inhibit the expression of the NlNPF gene and nucleotides with their reverse complementary sequences.

[0010] Preferably, the PCR amplification primer set for the dsRNA is: dsNlNPF-F: GGATCCTAATACGACTCAC TATAGG GCATCAAATCCGAGACAATTC and dsNlNPF-R: GGATCCTAATACGACTCACTATA GG CTGAAAGAGTAAATCTGCTCCAAC, the underlined region is the T7 RNA polymerase promoter sequence.

[0011] Preferably, the nucleotide sequence of the dsRNA is as shown in SEQ ID NO.3.

[0012] The fourth objective of this invention is to provide the application of an agent that inhibits the expression of the NlNPF gene in brown planthoppers in the preparation of products for the control of brown planthoppers.

[0013] Preferably, the product for controlling brown planthoppers is a dsRNA that inhibits the expression of the NlNPF gene in brown planthoppers, and can be the dsRNA described above.

[0014] Preferably, the product for controlling brown planthoppers involves introducing dsRNA into brown planthoppers to inhibit the expression of the NlNPF gene, thereby reducing the feeding and survival rate of brown planthoppers and achieving the control of brown planthoppers.

[0015] The fifth objective of this invention is to provide a method for controlling brown planthoppers using dsRNA that inhibits the expression of the NlNPF gene in brown planthoppers, wherein the dsRNA is introduced into the brown planthopper.

[0016] Preferably, the above-mentioned dsRNA is prepared into a nanocarrier dsRNA and sprayed onto rice infested with brown planthoppers.

[0017] Preferably, the nanocarrier dsRNA is prepared by mixing layered double hydroxides with dsRNA.

[0018] The present invention has the following positive and beneficial technical effects:

[0019] Experiments have shown that this invention yielded the cDNA sequence of the NlNPF gene in brown planthoppers. By employing RNAi technology with microinjection of dsRNA, the NlNPF gene in brown planthoppers was silenced, resulting in a feeding refusal effect in brown planthoppers. Clearly, the dsRNA of this gene has significant application value in the field of brown planthopper control. Attached Figure Description

[0020] Figure 1The expression patterns of NlNPF in different instars and tissues of brown planthopper were as follows: A: NlNPF expression levels in nymphs (1-5 instars) and adults (days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15); B: NlNPF expression levels in the ovary (Ov), head (Hd), midgut (Mg), epidermis (In), fat body (Fb), and legs (Lg) of brown planthopper.

[0021] Figure 2 Analysis of the relative expression level of the NlNPF gene after microinjection.

[0022] Figure 3 Measurement of feeding holes of brown planthoppers.

[0023] Figure 4 The honeydew amount and acquired body weight of brown planthoppers feeding on rice under different treatments are: A: honeydew amount of brown planthoppers feeding on rice under different treatments; B: acquired body weight of brown planthoppers feeding on rice under different treatments.

[0024] Figure 5 The effect of nanocarrier dsRNA on the mortality rate of brown planthoppers. Detailed Implementation

[0025] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0026] Unless otherwise specified, the techniques used in the embodiments are conventional techniques well known to those skilled in the art. Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available.

[0027] The brown planthoppers used in the following examples were raised at the Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, at a rearing temperature of 26±2℃.

[0028] Example 1: Cloning of the NlNPF gene in brown planthopper

[0029] Total RNA was extracted from brown planthoppers and reverse-engineered into cDNA. Primers were designed based on this sequence, and the 5' and 3' end sequences of the candidate gene were obtained using the TaKaRa 5' and 3' Full RACE kit. The transcription start and termination sites of the candidate gene were determined, and the full-length cDNA sequence of the gene was assembled. Primers NlNPF-F and NlNPF-R were resynthesized based on the full-length cDNA sequence, and the full-length cDNA of NlNPF was amplified (as shown in SEQ ID NO.1). The ORF was predicted, and its ORF sequence is shown as bases 55-354 of SEQ ID NO.1.

[0030] NlNPF-F: CACCTCACTTCTCACTTGTT;

[0031] NlNPF-R:CTCCAAGTTTATTCAAGAAA.

[0032] Example 2: Expression patterns of the NlNPF gene in brown planthoppers at different ages and in different tissues

[0033] Total RNA was extracted from six different tissues (ovary, head, midgut, epidermis, fat body, and foot) of the brown planthopper using Trizol in an RNase-free environment. After reverse transcription into first-strand cDNA, the RNA was detected using quantitative real-time PCR. The results are as follows: Figure 1 As shown in B, NlNPF was expressed at the highest level in the head of the brown planthopper, followed by the midgut, expressed in small amounts in the fat body, and almost not expressed in the ovary, epidermis, and foot.

[0034] Total RNA was extracted from brown planthoppers at different instars (1st-5th instar nymphs and short-winged female brown planthoppers at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 days after emergence), then reverse transcribed into cDNA, and detected using quantitative real-time PCR. Results are as follows: Figure 1 As shown in A, NlNPF was expressed at higher levels in 4th-5th instar nymphs and adults 1-4 days after emergence, with the highest expression on the first day of adulthood, followed by a gradual decrease in expression.

[0035] Primers for real-time PCR of the internal reference gene Nlactin:

[0036] QNlactin-F:ATGAAACCGTCTACAACTCG;

[0037] QNlactin-R: GCATCCTGTCGGCAATAC.

[0038] Primers for NlNPF real-time PCR:

[0039] QNlNPF-F:AGACTGCTCTACTATTGGTATGCTG;

[0040] QNlNPF-R: TGGAATTGTCTCGGATTTGATG.

[0041] Example 3: Preparation of dsRNA (dsNlNPF) for silencing the NlNPF gene in brown planthoppers and dsGFP for control.

[0042] 1. Using the cDNA obtained in Example 1 as a template, PCR amplification was performed using dsNlNPF-F and dsNlNPF-R as primers to obtain the PCR amplification product.

[0043] dsNlNPF-F (forward primer):

[0044] GGATCCTAATACGACTCACTATAGG GCATCAAATCCGAGACAATTC;

[0045] dsNlNPF-R (reverse primer):

[0046] GGATCCTAATACGACTCACTATAGG CTGAAAGAGTAAATCTGCTCCAAC,

[0047] The underlined area is the T7 RNA polymerase promoter sequence.

[0048] 2. Using the GFP-containing plasmid (Takara, Japan, Code No. 3270) already available in the laboratory as a template, PCR amplification was performed using dsGFP-F and dsGFP-R primers to obtain the PCR amplification product.

[0049] dsGFP-F (forward primer):

[0050] GGATCCTAATACGACTCACTATAGG TGAATTAGATGGTGATGTTA;

[0051] dsGFP-R (reverse primer):

[0052] GGATCCTAATACGACTCACTATAGG TCACCTTCAAACTTGACTTC,

[0053] The underlined area is the T7 RNA polymerase promoter sequence.

[0054] 3. Recover the amplification products from steps 1 and 2 respectively, add A, ligate to the pMD18-T (TAKARA) vector, and send positive clones for sequencing. Obtain the correct clone, use this clone plasmid as a template, and amplify it again using the primers described above. Purify and concentrate the amplification product to a concentration of 1 μg / μL. This product serves as the template for dsRNA synthesis. Use T7 RiboMAX. TM The Express RNAiSystem (Promega, USA, P1700) kit was used to synthesize dsRNA.

[0055] 4. According to the instructions of the above kit, add 10 μL of transcription buffer, 1 μL each of ATP, CTP, GTP, and UTP (100 mM), 1.25 μL of RNase inhibitor, 1 μL of T7 RNA Polymerase, 1.5 μg of template, and DEPCH2O to a final volume of 49 μL. Gently tap to mix and centrifuge briefly. Place in a PCR apparatus and program: 37℃ for 4 h; 75℃ for 5 min; store at 16℃. Aspirate 1 μL and perform gel electrophoresis. Once the target band is detected, proceed to the next step.

[0056] 5. Following the instructions, add 6 μL of 10×Reaction buffer, 12 μL of DNase, 0.5 μL of RNase, and 1.5 μL of DEPC H2O. Mix thoroughly, centrifuge briefly, and place in a PCR instrument at 37°C for 30 min. Then, remove and add 1 μL of EDTA (Fementas), return to the PCR instrument, and stop the reaction at 65°C for 5 min. Aspirate 1 μL, dilute 10-fold, and use 2 μL for gel electrophoresis and 2 μL for concentration determination using a Nano Drop UV spectrophotometer. If the detection band is a single bright band, and the OD260 / 280 is between 1.8 and 2.0, the dsRNA quality is good and RNA phenol-chloroform extraction can be performed.

[0057] 6. Perform routine phenol-chloroform extraction to remove proteins. Adjust the concentration of dsRNA to 5 μg / μL, aliquot into 10 μL tubes, and store at -80℃. This yields dsNlNPF targeting NlNPF and dsGFP targeting GFP. The nucleotide sequence of dsNlNPF is shown in SEQ ID NO.3; the nucleotide sequence of dsGFP is shown in SEQ ID NO.4.

[0058] Example 4: Microinjection of dsNlNPF and Effect Detection

[0059] 1. Plate preparation: Weigh 1.5g of agar powder and add it to 100mL of water, boil, pour into a glass petri dish, and let it solidify for later use.

[0060] 2. Injection: Take newly emerged female brown planthoppers of similar growth on their first day and place them in a test tube. Anesthetize them with CO2 for 20 seconds. Do not blow CO2 directly onto the planthoppers to avoid collisions and damage. Then, place the planthoppers on a 1.5% agar plate with their abdomens facing upwards. Inject dsNlNPF and dsGFP separately using a Nanoliter 2010 microinjector according to the instructions. The injection site is between the anterior and mesothorax. The injection volume is 46 μL (5 μg / μL).

[0061] 3. After injecting brown planthoppers with dsRNA, samples were taken starting 12 hours after injection, with three samples taken every 12 hours for a total of 36 hours. Brown planthoppers that were not injected and those injected with dsGFP were taken as controls. The changes in gene expression levels were verified by quantitative real-time PCR.

[0062] The specific procedures are as follows: After sampling, RNA is extracted and reversed to cDNA. The reversed DNA is then performed using the TAKARA PrimeScript RTreagent Kit with gDNA Eraser (catalog number RR047A) according to the instructions to obtain the reversed product. 5 μL of the reversed cDNA is taken, diluted 10-fold, and then subjected to real-time quantitative PCR as follows. The PCR reaction is conducted on a CFX96 Touch... TM The real-time PCR detection system (Bio-Rad) was used, following this reaction system: DNase / RNase-Free ddH2O 2.9 μL, 2×Supermix 4 μL, Primers (5 mM) 0.6 μL, cDNA template 0.5 μL. Reaction conditions: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5-10 s, 65℃ annealing and extension for 30 s, repeated for 40 cycles. Finally, melting curves were performed at 65℃-95℃, increasing by 0.5℃ for 5 s at each step, to determine the specificity of the amplified products. 2 -△△Ct The method is to conduct data analysis.

[0063] Nlactin primer (internal reference gene):

[0064] QNlactin-F:ATGAAACCGTCTACAACTCG;

[0065] QNlactin-R: GCATCCTGTCGGCAATAC.

[0066] NlNPF quantitative primers:

[0067] QNlNPF-F:AGACTGCTCTACTATTGGTATGCTG;

[0068] QNlNPF-R: TGGAATTGTCTCGGATTTGATG.

[0069] 4. Results attached Figure 2As shown, compared with uninjected brown planthoppers and the control group injected with dsGFP, the relative expression level of the NlNPF gene in the microinjection group was significantly reduced from 24 h after injection, showing a significant difference compared with the control (P<0.05). The results indicate that microinjection of dsNlNPF can induce an RNAi effect on the NlNPF gene in brown planthoppers, leading to a significant decrease in gene expression.

[0070] Example 5: Detection of feeding behavior of brown planthoppers after microinjection

[0071] To further verify the effect of the NlNPF gene on feeding by brown planthoppers, we counted the number of feeding marks at fixed locations within 24 hours. First, brown planthoppers injected with NlNPF gene dsRNA 24 hours prior (see Example 4) were placed on 30-day-old rice seedlings to feed, with one brown planthopper per rice plant and 20 replicates. After 24 hours, the brown planthoppers were removed, and the rice stems from which the planthoppers had fed were soaked in 0.04% eosin Y for 12 hours. After 12 hours, the planthoppers were removed and the feeding marks were observed under a stereomicroscope. Normal brown planthoppers were used as the control group, and brown planthoppers injected with dsGFP 24 hours prior were used as the dsGFP group. The results are as follows: Figure 3 As shown, the number of feeding traces in the dsNlNPF group was significantly increased compared to the control group and the dsGFP group, indicating that dsNlNPF has an inhibitory effect on brown planthopper feeding.

[0072] Example 6: Detection of feed intake by brown planthoppers after microinjection

[0073] Brown planthoppers injected with dsRNA of the NlNPF gene for 24 hours (see Example 4 for details) were introduced onto 30-day-old rice seedlings for feeding. One brown planthopper was placed on each rice plant, with 20 replicates. Honeydew accumulation and acquired body weight were recorded over 24 hours. Normal brown planthoppers were used as the control group, and brown planthoppers injected with dsGFP for 24 hours were used as the dsGFP group. Results are as follows: Figure 4 As shown, the dsNlNPF group could not feed normally on rice, and its food intake was significantly lower than that of the control and dsGFP groups. Brown planthoppers injected with NlNPF gene dsRNA for 24 hours were transferred to rice, and their 24-hour acquired body weight was recorded. The body weight of the dsNlNPF group was significantly lower than that of the control and dsGFP groups. This indicates that silencing NlNPF in brown planthoppers significantly reduces their food intake; and there was no significant difference between the dsGFP and control groups. In summary, the expression level of NlNPF is positively correlated with the food intake of brown planthoppers.

[0074] Example 7: Mortality rate of brown planthoppers after spraying with nanocarrier dsRNA

[0075] 0.01% layered double hydroxide (LDH) was mixed with dsRNA (5 μg / μL) at a volume ratio of 1:10 and allowed to stand at room temperature for 20 minutes to prepare the dsRNA nanocarrier (LDH_dsNlNPF). Brown planthoppers were inoculated onto 30-day-old rice seedlings, with one planthopper per plant and 30 replicates. The prepared LDH_dsNlNPF was then sprayed evenly to cover the entire rice plant. The mortality rate of the brown planthoppers was recorded every other day for 7 days. Normal brown planthoppers were used as the control group, and brown planthoppers sprayed with 0.01% LDH solution were used as the LDH group. Results are as follows: Figure 5 As shown, compared with the control group and the LDH group, the mortality rate of the LDH_dsNlNPF group was significantly higher on day 2, reaching 41.11% by day 7. This indicates that the nanocarrier dsRNA can continuously kill pests.

[0076] SEQ ID NO.1 (cDNA nucleotide sequence of NlNPF)

[0077]

[0078] MLTLKTALLLVCCVLLWAWPSYCDSIGADSRMHASNPRQFQSPEDLRSYLDQLQLYYA VAGRPRFGKRVGYNGRPSSWVPSQDSPVDDQLEQIYSFREK

[0079] SEQ ID NO. 3 (nucleotide sequence of dsNlNPF)

[0080] GCATCAAATCCGAGACAATTCCAATCTCCGGAGGATCTCAGGTCTTATCTGGACCAGTTGCAGCTGTACTACGCAGTTGCTGGTAGACCCAGATTTGGAAAAAGAGTTGGGTATAATGGCAGACCTAGCAGTTGGGTTCCTTCTCAGGATTCTCCAGTTGACGATCAGTTGGAGCAGATTTACTCTTTCAGSEQ ID NO. 4 (nucleotide sequence of dsGFP)

[0081] TGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGCCTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGA.

Claims

1. Suppress brown planthoppers NlNPF The application of gene-expressing agents in the preparation of products for controlling brown planthoppers, characterized by: The suppression of brown planthoppers NlNPF Gene expression agents are used by brown planthoppers. NlNPF The dsRNA of the gene, the nucleotide sequence of which is shown in SEQ ID NO.

3.

2. A method for suppressing brown planthoppers NlNPF A method for controlling brown planthoppers using gene-expressed dsRNA, characterized in that, The dsRNA was introduced into the brown planthopper, and the nucleotide sequence of the dsRNA is shown in SEQ ID NO.

3.

3. The method according to claim 2, characterized in that, The dsRNA was prepared into a nanocarrier dsRNA and sprayed onto rice plants infested with brown planthoppers.

4. The method according to claim 3, characterized in that, The aforementioned nanocarrier dsRNA is prepared by mixing layered double hydroxides with dsRNA.

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