Brown planthopper saliva gene NlSP55 and application thereof in biological control of brown planthopper

Abstract

The invention discloses a specific saliva gene NlSP55 of brown planthopper and application of the specific saliva gene NlSP55 in biological control of brown planthopper. Wherein the nucleotide sequence of the nilaparvata lugens saliva gene NlSP55 is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2. The dsRNA of the synthesized saliva gene NlSP55 is introduced into the brown planthopper body by using an RNA interference technology, so that the NlSP55 gene can be silent in a targeted manner, the feeding capacity of the brown planthopper is inhibited, the survival rate of the brown planthopper on sensitive rice and resistant rice is quickly reduced, and finally, the biological control effect of the brown planthopper is achieved. The invention specifically aims at the brown planthopper, is harmless to mammals, fishes and shrimps, natural enemy insects and pollination insects, and provides a new way for preventing and treating the brown planthopper.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a saliva gene NlSP55 specific to brown planthopper and an application thereof in biological control of brown planthopper. Background Art

[0002] Brown planthopper (Nilaparvata lugens) is a typical r-strategy insect with biological characteristics such as short generation cycle, high fecundity and high environmental adaptability. It has the potential to break out and become a pest in farmland ecosystems. At the same time, intensive agriculture, which is mainly characterized by large-scale planting of high-yield varieties and high nitrogen fertilizer and pesticide use, not only seriously weakens rice resistance, but also significantly weakens the environmental resistance of rice field ecosystems by killing natural enemies, reducing biodiversity, and strengthening some biological characteristics of brown planthoppers that adapt to rice field ecosystems (such as toxicity, fecundity, pesticide resistance, and high temperature resistance), resulting in a high population growth multiple of brown planthoppers. Therefore, exploring the damage mechanism of brown planthoppers and developing new brown planthopper control methods are of great significance to reduce the impact of rice planthoppers on rice production and ensure national food security.

[0003] Saliva is a bridge for insects and plants to interact, and is a powerful tool for brown planthoppers to feed. Brown planthoppers will continue to secrete watery and gelatinous saliva during the feeding process. These salivary components have multiple functions such as participating in the formation of insect salivary sheaths, in vitro digestion, detoxification metabolism, and inhibiting plant defense, and play an important role in insect feeding, virus transmission, etc. In the early stage, we successfully identified the salivary proteome of brown planthoppers and found a saliva gene (Nilaparvata lugens-specific salivary protein 55, NlSP55). NlSP55 is an orphan gene specific to brown planthoppers and has not been found in other species. The development of new pesticides targeting species-specific genes can help improve the specificity of pest control and reduce the impact on non-target organisms. At this stage, the function of the NlSP55 gene and its use in the control of brown planthoppers need to be further explored.

[0004] RNA interference (RNAi) is a gene silencing technology that triggers a series of molecular events in cells through double-stranded RNA (dsRNA), leading to the degradation or inhibition of specific mRNA, thereby reducing the expression level of specific genes. RNAi technology has shown great potential in many fields due to its high efficiency and specificity. In life science research, RNAi is used as a powerful tool to study gene function and explore its biological role by specifically silencing genes. In recent years, RNAi technology has also shown revolutionary progress in the field of agriculture, especially in pest management. By introducing specific dsRNA into pests, key genes can be specifically silenced, thereby controlling pest populations or reducing their ability to spread viruses. This RNAi-based biopesticide is environmentally friendly, has strong target specificity, and has a low residual risk, providing a new pest management strategy for modern agriculture. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a saliva gene NlSP55 unique to brown planthoppers and its application in the biological control of brown planthoppers. By targeting and silencing the saliva gene NlSP55 unique to brown planthoppers, the purpose of reducing the survival rate of brown planthoppers on different rice varieties and reducing the damage of brown planthoppers to rice is achieved, providing a sequence and data basis for the control of brown planthoppers.

[0006] In order to achieve the above object, the present invention cloned the coding protein sequence of the saliva gene NlSP55 unique to brown planthoppers, obtained the dsRNA of the NlSP55 gene by an in vitro synthesis method, introduced it into the body of brown planthoppers, and detected the expression level of the NlSP55 gene and the survival rate of brown planthoppers on different rice varieties. The present invention specifically adopts the following technical solutions:

[0007] The first aspect of the present invention provides a brown planthopper saliva gene NlSP55, the amino acid sequence encoded by the brown planthopper saliva gene NlSP55 is shown in SEQ ID NO.2, the nucleotide sequence is shown in SEQ ID NO.1, or a sequence having 90% homology with SEQ ID NO.1 and encoding an amino acid as shown in SEQ ID NO.2 after substitution.

[0008] A second aspect of the present invention provides an application of the above-mentioned brown planthopper saliva gene NlSP55 as a target in the biological control of brown planthoppers.

[0009] The third aspect of the present invention provides a dsRNA that interferes with the expression of the above-mentioned brown planthopper saliva gene NlSP55. The dsRNA is synthesized by an in vitro transcription method after being amplified using SEQ ID NO.5 and SEQ ID NO.6 as primers and the nucleotide sequence of the above-mentioned brown planthopper saliva gene NlSP55 as a template.

[0010] Preferably, the dsRNA consists of the nucleotide sequence shown in SEQ ID NO.12 and a nucleotide sequence that is reverse complementary to the nucleotide sequence shown in SEQ ID NO.12.

[0011] A fourth aspect of the present invention provides a drug for biological control of brown planthoppers, wherein the active ingredient of the drug at least comprises dsRNA that interferes with the expression of the brown planthopper saliva gene NlSP55.

[0012] A fifth aspect of the present invention provides a method for biological control of brown planthoppers, which inhibits the feeding ability of brown planthoppers on rice and reduces the survival rate of brown planthoppers on rice by interfering with the expression of the brown planthopper saliva gene NlSP55.

[0013] Preferably, the method introduces a solution containing dsRNA that interferes with the expression of the above-mentioned brown planthopper saliva gene NlSP55 or the above-mentioned drug for biological control of brown planthoppers into the body of the brown planthopper.

[0014] Preferably, the introduction method includes injection and / or feeding.

[0015] Preferably, the brown planthopper is a nymph.

[0016] Preferably, the rice includes brown planthopper-susceptible rice and brown planthopper-resistant rice.

[0017] The present invention has the following beneficial effects:

[0018] (1) The present invention discloses a saliva gene NlSP55 unique to brown planthoppers. By obtaining dsRNA of the NlSP55 gene through the present invention, after introducing it into brown planthoppers to silence the NlSP55 gene, the feeding ability of brown planthoppers can be significantly reduced, and the survival rate thereof can be decreased. This indicates that the saliva gene NlSP55 unique to brown planthoppers plays a key role in the feeding process of brown planthoppers, and at the same time has important value in the biological control of brown planthoppers.

[0019] (2) NlSP55 is an orphan gene specific to brown planthoppers and has not been found in other species. The present invention develops drugs and methods for biological control of brown planthoppers targeting genes specific to brown planthoppers, which helps to improve the specificity of pest control and reduce the impact on non-target organisms such as mammals, fish and shrimp, natural enemy insects and pollinating insects. It has the advantages of rapid effect, high mortality rate, and environmental friendliness, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is the experimental result of PCR amplification of NlSP55 gene in the embodiment of the present invention;

[0021] Figure 2This is a diagram showing the experimental results of dsRNA synthesis of NlSP55 gene in an embodiment of the present invention;

[0022] Figure 3 This is a graph showing the results of the dsRNA silencing efficiency test of the NlSP55 gene in an embodiment of the present invention, wherein dsGFP is a GFP gene dsRNA introduction group, and dsNlSP55 is a NlSP55 gene dsRNA introduction group;

[0023] Figure 4 This is a graph showing the survival rate of brown planthoppers on susceptible rice Nipponbare after the NlSP55 gene dsRNA was introduced into the brown planthoppers in the embodiment of the present invention, wherein dsGFP is a GFP gene dsRNA-introduced group, and dsNlSP55 is a NlSP55 gene dsRNA-introduced group;

[0024] Figure 5 This is a graph showing the survival rate of brown planthoppers on resistant rice ASD7 after the NlSP55 gene dsRNA was introduced in an embodiment of the present invention, wherein dsGFP is the GFP gene dsRNA-introduced group, and dsNlSP55 is the NlSP55 gene dsRNA-introduced group. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0026] Example 1: Cloning of the NlSP55 gene fragment of the brown planthopper

[0027] 1. Amplification of the NlSP55 gene fragment of brown planthopper

[0028] (1) Take an adult brown planthopper and place it on ice to anesthetize it; add pre-cooled 1×PBS (37 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4) on a slide, and immediately dissect the salivary glands of the planthopper under a stereoscope;

[0029] (2) Add 1 ml of Trizol (Takara) to the dissected salivary glands and grind them thoroughly; add 400 μl of chloroform and mix vigorously, then centrifuge at 12,000 rpm and 4°C for 15 minutes, carefully aspirate the top aqueous phase and transfer it to a new centrifuge tube; add an equal volume of isopropanol, mix well and let stand at room temperature; after 10 minutes, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant and add 1 ml of 75% ethanol to the precipitate; centrifuge at 9600 rpm for 5 minutes, discard the supernatant and add an appropriate amount of RNase-free water to the precipitate; determine the RNA concentration using NanoDrop;

[0030] (3) The extracted total RNA from the salivary glands of brown planthoppers was reverse transcribed using the ReverTra Ace q PCR RT Master with gDNA remover kit: First, the total RNA was denatured at 65°C for 5 min; 1 μg of total RNA was added to 2 μl of 4×DN Master Mix and made up to 8 μl with RNase-free water; after mixing, the mixture was incubated at 37°C for 5 min to remove contaminated genomic DNA; then, 2 μl of 5×RT Master Mix was added to the system, and after mixing, the mixture was incubated at 37°C for 1 h; finally, the mixture was denatured at 98°C for 5 min to inactivate the reverse transcriptase and finally obtain the salivary gland cDNA of brown planthoppers;

[0031] (4) Primers were designed based on the full-length sequence of NlSP55 gene as shown in Table 1. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.;

[0032] Table 1 NlSP55 gene PCR primers

[0033]

[0034] (5) Using the salivary gland cDNA of brown planthopper as a template, PCR amplification of the target gene was performed using the primers shown in Table 1. The specific amplification system was: Max Buffer 25μl, dNTP Mix (10mM each) 1μl, MaxSuper-Fidelity DNA Polymerase 1μl, upstream and downstream primers 1μl each, brown planthopper salivary gland cDNA template 1μl, and finally fill to 50μl with ddH2O. PCR amplification conditions are: 95℃ 3 minutes; 95℃ 15 seconds, 60℃ 1 minute, 35 cycles; 72℃ 10 minutes.

[0035] 2. Obtaining a monoclonal strain of the NlSP55 gene of the brown planthopper

[0036] (1) Separate the PCR products using 1% agarose gel electrophoresis ( Figure 1 ), and cut the target fragment with a blade;

[0037] (2) Use DNA agarose gel recovery kit (Shanghai Sangon Biotechnology, SK8131) to recover the target DNA: cut the agarose containing the target fragment from the agarose gel and place it in a centrifuge tube; add 5 times the volume of gel dissolving buffer B2 and incubate at 70°C for 10 minutes; add the dissolved gel solution to the adsorption column, centrifuge at 8,000g for 30 seconds, and discard the waste liquid; add 500μl wash solution, centrifuge at 9,000g for 30 seconds, and discard the waste liquid; centrifuge the empty adsorption column at 9,000g for 1 minute; place the adsorption column in a new centrifuge tube and add 30μl ddH2O in the center of the adsorption column; centrifuge at 10,000g for 2 minutes to finally obtain the purified brown planthopper NlSP55 gene DNA fragment;

[0038] (3) The DNA fragment of the NlSP55 gene of brown planthopper was connected to the pClone007 vector using a blunt-end cloning kit (Beijing Qingke Biotechnology Co., Ltd.): 1 μl of the DNA fragment of the NlSP55 gene of brown planthopper, 1 μl of the pClone007 Blunt simple vector, and 1 μl of 10× buffer were added to a 0.2 ml centrifuge tube, and finally ddH2O was added to make up to 10 μl. The sample was mixed and reacted at 25°C for 5 minutes.

[0039] (4) Add the ligation product to the competent medium, flick the tube wall with your hand, and place on ice for 30 minutes; transfer the centrifuge tube to a 42°C water bath for 90 seconds, quickly take it out and let it stand on ice for 2 minutes; add 500 μl of antibiotic-free LB medium to the centrifuge tube, shake at 180 rpm for 1 hour; take 100-200 μl of the bacterial solution and spread it on an LB plate containing ampicillin resistance;

[0040] (5) Pick a single colony and add it to 1 ml of LB liquid medium containing ampicillin resistance, shake at 180 rpm for 6 hours, and perform colony PCR on the bacterial solution. The reaction system is: Max Buffer 12.5μl, dNTP Mix (10mM each) 0.5μl, Max Super-Fidelity DNA Polymerase 0.5μl, upstream and downstream primers (as shown in Table 1) 0.5μl each, bacterial solution 1μl, and finally ddH2O to make up to 25μl. PCR amplification conditions are: 95℃ for 3 minutes; 95℃ for 15 seconds, 60℃ for 1 minute, 35 cycles; 72℃ for 10 minutes;

[0041] (6) The PCR products were separated by 1% agarose gel electrophoresis, and the positive recombinant clones were selected and sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing to obtain a 789 bp brown planthopper NlSP55 gene sequence. The obtained nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0042] ATGAAGCTTGTCCAAGTGACAGGATTCATTTTGTGGATTCACATCTTCACCAAATTGGTGTCCGGAGCAACACAGTATCGGATTTAGACATACGGAGTGTGCTGAGAAGTCGTCAAACAACAACCAAAAGCTTTGAATGACCTCACAAACGAACTTCTCACAA ATAAACCCCTCGTAGAGGACAACCAACCATCCGACAAAGGACTCCCGATTCCCGGAACTGAAGAGGGACTTCCACCGCTGTTGGCTGCTGCCTTGAAGTTGAACGAAGGAGAGGCCAAAGAAAATGACAAAGGAAAAGAGATAATAAGGGGAAAACGTTGCGGA GGAGGATGTGGTGGAGGAGGGTATAGAGGAGGATGTGGAAGAGGAGGTGGAGGTGGTGGAGGAGGAGGAGGATATGGTAGGAGTTACAGGGGGGGCTGTAGTTGTTCTGGGGATCGGGGTTATAGGAGGGGAGGAAGACGTGATGATTACAGCAGAGACAGAAG TAGGGGAAGAACGAAGAAGGTGATAATCGAGTATAGGGAGAAGAAGAGACGATAGTAGATCTACGGAACCGAGGAGGAGGAGTAGGAAGAGGGATAGTAGTAGGGGTAGAGATAGGGATCGTAGCAGGGATAAAGATCGTGATAGAGATCGCAATCGTGATA AGGATAGGGATAGGGATAGGGATAGGGATAGGAATCGTGATAAGGAAAGAGATAGGAAACAGCAGAACAACGATGACAAGAATACTCGCAGGAAAAAGCATTCTTCGTCGTCTGAAGATAGCAAGCATGATTAG(SEQ ID NO.1)

[0043] MKLVQVTGFILWIHIFTKLVSGATTVSDLDIRSVLRSRQTTPKALNDLTNELLTNKPLVEDNQPSDKGLPIPGTEEGLPPLLAAALKLNEGEAKENDKGKEIIRGKRCGGGCGGGGYRGGCGRGGGGGGGGGG YGRSYRGGCSCSGDRGYRRGGRRDDYSDRRSRGRTKKVIIEYREKKRDDSRSSTEPRRRSRKRDSSRGRDRDRSRDKDRDRDRNRDKDRDRDRDRNRDKERDRKQQNNDDKNTRRKKHSSSSEDSKHD(SEQ ID NO.2)

[0044] Example 2: dsRNA synthesis of the brown planthopper NlSP55 gene

[0045] 1. T7 Primer PCR Amplification and Purification

[0046] (1) Using the recombinant plasmid or bacterial solution containing the brown planthopper NlSP55 gene as a template, the target gene was amplified using primers with T7 promoter sequence (as shown in Table 2). The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. and the amplification system was Max Buffer 100μl, dNTP Mix (10mM each) 4μl, Max Super-Fidelity DNA Polymerase 4μl, upstream and downstream primers (SEQ ID NO.5 and SEQ ID NO.6) 4μl, recombinant plasmid or bacterial solution 4μl, and finally fill to 200μl with ddH2O. PCR amplification conditions are: 95℃ 3 minutes; 95℃ 15 seconds, 60℃ 1 minute, 35 cycles; 72℃ 10 minutes.

[0047] Table 2 Primers for double-stranded RNA synthesis of NlSP55 gene

[0048]

[0049] (2) The amplified product was separated by agarose gel electrophoresis and recovered by a DNA agarose gel recovery kit, and finally a large amount of a single NlSP55 gene fragment containing a T7 promoter was obtained, the sequence of which is shown in SEQ ID NO.7.

[0050] TAATACGACTCACTATAGGGATGAAGCTTGTCCAAGTGACAGGATTCATTTTGTGGATTCACATCTTCACCAAATTGGTGTCCGGAGCAACAACAGTATCGGATTTAGACATACGGAGTGTGCTGAGAAGTCGTCAAACAACACCAAAAGCTTTGAATGACCTC ACAAACGAACTTCTCACAAATAAACCCCTCGTAGAGGACAACCAACCATCCGACAAAGGACTCCCGATTCCCGGAACTGAAGAGGGACTTCCACCGCTGTTGGCTGCTGCCTTGAAGTTGAACGAAGGAGAGGCCAAAGAAAATGACAAAGGAAAAGAGATAATA AGGGGAAAACGTTGCGGAGGAGGATGTGGTGGAGGAGGGTATAGAGGAGGATGTGGAAGAGGAGGTGGAGGTGGTGGAGGAGGAGGAGGATATGGTAGGAGTTACAGGGGGGGCTGTAGTTGTTCTGGGGATCGCCCTATAGTGAGTCGTATTA (SEQ ID NO.7)

[0051] 2. Synthesis and purification of dsRNA

[0052] The dsRNA of the NlSP55 gene was synthesized and purified using the T7 High Yield RNA Transcription Kit from Novagen Biotech Co., Ltd. The specific method is as follows:

[0053] (1) Synthesize dsRNA using the NlSP55 gene fragment containing the T7 promoter obtained by PCR amplification as a DNA template. The reaction system is: 2 μl 10× Reaction Buffer, 2 μl ATP solution, 2 μl UTP solution, 2 μl CTPsolution, 2 μl GTP solution, 2 μl enzyme mix, 1 μg DNA template, and fill up to 20 μl with RNase-free water. After the reaction system is prepared, mix well and react at 37°C overnight.

[0054] (2) Add 1 μl of TURBO DNase to the reaction system to eliminate the DNA in the reaction system and react at 37°C for 15 min;

[0055] (3) Denaturing the reacted sample at 65°C for 5 min;

[0056] (4) Determine the dsRNA concentration using Nanodrop and determine the dsRNA quality using 1% agarose gel electrophoresis ( Figure 2 );

[0057] (5) The dsRNA of the NlSP55 gene was stored at 80°C for future use; the nucleotide sequence of the dsRNA of the NlSP55 gene is shown in SEQ ID NO.12.

[0058] AUGAAGCUUGUCCAAGUGACAGGAUUCAUUUUUGUGGAUUCACAUCUUCACCAAAUUGGUGUCCGGAGCAACAACAGUAUCCGGAUUUAGACAUACGGAGUGCUGAGAAGUCGUCAAACAACAACCAAAAGCUUUGAAUGACCUCACAAACGAACUUCUCACAAAUAAACCCCUCGUAGAGGACAACCAACCAUCCGACAAAGGACUCCCGAUUCCCGGAACUGA AGAGGGACUUCCACCGCUGUUGGCUGCUGCCUUGAAGUUGAACGAAGGAGAGGCCAAAGAAAAUGACAAAGGAAAAGAGAUAAUAAGGGGAAAACGUUGCGGAGGAGGAUGUGGUGGAGGAGGGUAUAGAGGAGGAUGUGGAAGAGGAGGUGGAGGUGGUGGAGGAGGAGGAGGAUAUGGUAGGAGUUACAGGGGGGGCUGUAGUUGUUCUGGGGAUCG(SEQID NO.12)

[0059] Example 3: Introduction of dsRNA of NlSP55 gene into brown planthopper

[0060] The dsRNA of the NlSP55 gene was introduced into the brown planthopper by microinjection. The specific method is as follows:

[0061] (1) Take the third instar nymph of brown planthopper and anesthetize it with CO2 for 10 seconds;

[0062] (2) A glass capillary (Wuhan Micro-Tan Scientific Instrument Co., Ltd.) was pulled to a suitable size using a capillary puller (P 97, Sutter Instrument). The program parameters used were set as follows: heat = 800, pull = 150, vel = 150, time = 80;

[0063] (3) Injecting dsRNA of the NlSP55 gene into a glass capillary using a micropipette tip (Eppendorf);

[0064] (4) The sampled glass capillary was installed in a microinjector (Eppendorf), and dsRNA was introduced into the brown planthopper under a stereoscope. The parameters of the microinjector were set as follows: injection pressure 1300 pah, injection time 0.3 s, and compensation pressure 10 pah;

[0065] (5) Using the same method, dsRNA of the jellyfish green fluorescent protein (GFP) was introduced into the brown planthopper as a negative control;

[0066] (6) After the brown planthoppers into which the dsRNA has been introduced have awakened, they are moved onto rice plants.

[0067] Example 4: Detection of dsRNA silencing efficiency of NlSP55 gene

[0068] On the 6th day after the dsRNA of the NlSP55 gene was introduced into the brown planthopper, the brown planthopper was collected and the expression level of the NlSP55 gene was determined. The specific method is as follows:

[0069] (1) The collected brown planthoppers were ground, total RNA was extracted using the Trizol method, and the cDNA of the brown planthopper was obtained by reverse transcription using the ReverTra Ace q PCR RTMaster with gDNA remover kit;

[0070] (2) Using Primer Primer 6.0 software, quantitative primers for the NlSP55 gene (SEQ ID NO.8 and SEQ ID NO.9) and the actin NlActin gene (SEQ ID NO.10 and SEQ ID NO.11) of the brown planthopper were designed, as shown in Table 3. The primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.;

[0071] Table 3 Quantitative PCR primers for NlSP55 gene and NlActin gene

[0072]

[0073] (3) Using the cDNA of brown planthopper as template, iTaq TMUniversal SYBR Green Supermix (Shanghai Yisheng Biotechnology Co., Ltd.) fluorescent quantitative detection reagent was used to determine the expression levels of the NlSP55 gene and the actin NlActin gene of the brown planthopper. The reaction system was configured as follows: 6.8 μl H2O, 10 μl 2×SYBR Green Supermix, 0.6 μl forward primer, 0.6 μl reverse primer, and 2 μl cDNA template. The 480 Real-Time PCR System ran the following program: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, extension at 60°C for 30 s, and a total of 40 cycles of denaturation and extension; a negative control of RNase-free water was set for each primer pair;

[0074] (4)Use 2 -ΔΔCt The expression level of NlSP55 in brown planthopper was calculated by the method, and the significant difference among different treatment groups was tested by Student's t-test.

[0075] (5) The experimental results showed that the expression level of NlSP55 gene in brown planthoppers introduced with NlSP55 gene dsRNA was significantly lower than that in the control ( Figure 3 ), indicating that NlSP55 gene dsRNA has a high silencing efficiency on NlSP55 gene of brown planthopper.

[0076] Example 5: Mortality Statistics and Phenomenon Records

[0077] The brown planthoppers injected with NlSP55 gene dsRNA were reared on the resistant rice variety ASD7 and the susceptible rice variety Nipponbare (3-4 leaf stage), with about 30 brown planthoppers placed in each group of rice, and three groups were repeated. The mortality rate was calculated daily. The brown planthopper rearing conditions were: temperature 26°C ± 0.5°C, relative humidity 50% ± 5%, and photoperiod of 16h:8h (day:night);

[0078] The experimental results are as follows Figure 4 and Figure 5 As shown, the brown planthopper (dsNlSP55) into which the NlSP55 gene dsRNA was introduced had no significant difference in the sensitivity of the rice variety Nipponbare ( Figure 4 ), or in the resistant rice variety ASD7 ( Figure 5 ), the survival curve was significantly different from that of the control (dsGFP) (p<0.01), and the survival rate was significantly reduced, indicating that NlSP55 gene dsRNA has potential application value in the control of brown planthoppers.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A brown planthopper saliva gene N1SP55, characterized in that: The amino acid sequence encoded by the brown planthopper saliva gene NlSP55 is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1, or a sequence having 90% homology with SEQ ID NO.1 and encoding amino acids as shown in SEQ ID NO.2 is substituted.

2. Application of the brown planthopper saliva gene N1SP55 as a target in the biological control of brown planthopper according to claim 1.

3. A dsRNA that interferes with the expression of the brown planthopper saliva gene N1SP55 according to claim 1, characterized in that: The dsRNA is synthesized by in vitro transcription after being amplified using SEQ ID NO.5 and SEQ ID NO.6 as primers and the nucleotide sequence of the brown planthopper saliva gene NlSP55 as a template as claimed in claim 1.

4. The dsRNA according to claim 3, characterized in that The dsRNA consists of the nucleotide sequence shown in SEQ ID NO.12 and a nucleotide sequence that is reverse complementary to the nucleotide sequence shown in SEQ ID NO.

12.

5. A drug for biological control of brown planthopper, characterized in that: The active ingredient of the medicine contains at least the dsRNA according to claim 3 or 4.

6. A method for biological control of brown planthopper, characterized in that: Interfering with the expression of the brown planthopper saliva gene NlSP55 described in claim 1, thereby inhibiting the brown planthopper's feeding ability on rice and reducing the survival rate of the brown planthopper on rice.

7. The method according to claim 6, characterized in that The solution containing the dsRNA according to claim 3 or 4 or the drug according to claim 5 is introduced into the body of brown planthopper.

8. The method according to claim 7, characterized in that The introduction method includes injection and / or feeding.

9. The method according to claim 8, characterized in that The brown planthopper is a nymph.

10. The method according to any one of claims 6 to 9, characterized in that: The rice includes brown planthopper-susceptible rice and brown planthopper-resistant rice.

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