Cyctria megacephala odorant binding protein and application thereof

Abstract

According to the odorant binding protein for the black-tailed leafhopper and the application of the odorant binding protein, by interfering the expression quantity of the binding protein in the black-tailed leafhopper, the perceptual ability of the black-tailed leafhopper to volatile matters induced by rice dwarf viruses can be effectively inhibited, so that the effect of preventing and treating the rice dwarf viruses is achieved. The amino acid sequence of the odorant binding protein is SEQ ID NO: 1. According to the application disclosed by the invention, a fluorescent competitive binding experiment finds that the odorant binding protein NcinOBP7 of the black tail leafhopper and a volatile matter released by rice induced by a rice dwarf virus have high affinity; an RNAi experiment shows that the rice volatile matter induced by the rice dwarf virus has an attraction effect on the black-tail leafhopper nymphs injected with dsEGFP, but after the NcinOBP7 is silenced, the rice volatile matter does not attract the black-tail leafhopper nymphs any more. Therefore, the silence of the black-tail leafhopper NcinOBP7 can influence the perception of the black-tail leafhopper on the rice volatile matters, and subsequently speculates to inhibit the feeding of the black-tail leafhopper on the virus-infected rice and influence the transmission of viruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural pest control, and specifically relates to a black-tailed leafhopper ( Nephotettix cincticeps ) Odor-binding proteins and their applications. Background Art

[0002] The black-tailed leafhopper, a member of the order Hemiptera and family Cicadidae, is a common pest of rice. Not only does it directly feed on rice sap, causing damage, but it can also indirectly harm rice by spreading the rice dwarf virus (RDV). This dual threat severely impacts rice health and quality, threatening high and stable yields.

[0003] Odorant binding proteins (OBPs) are found in the olfactory sensilla lymph of insects. They are hydrophilic proteins with hydrophobic binding pockets. Their small molecular weight allows them to recognize volatile odors and sex pheromones from host plants, playing a crucial role in the first step of insect odor perception: binding to odorants to form complexes and transport them to olfactory neurons.

[0004] RNA interference (RNAi) is a mechanism of action triggered by short RNA fragments (siRNAs) that promotes the degradation of homologous mRNAs or inhibits their translation. First discovered in nematodes, RNAi has been shown to function in most organisms. RNAi technology can specifically inhibit gene expression, effectively silencing target genes to achieve pest and disease control. RNAi technology has been widely applied in gene function research, the development of transgenic insect-resistant plants, and the synthesis of new nucleic acid pesticides.

[0005] Identifying genetic targets for controlling the black-tailed leafhopper holds great potential. Due to the high specificity of these gene targets, this strategy promises precise control and holds great promise for its application in the field. However, the ability to efficiently screen and identify key target genes regulating host selection behavior is crucial for the practical application of this technology. Summary of the Invention

[0006] The present invention aims to provide a black-tailed leafhopper odor-binding protein NcinOBP7 and its application, which can effectively inhibit the black-tailed leafhopper's perception of volatiles induced by rice dwarf virus by interfering with the expression level of the binding protein in the black-tailed leafhopper.

[0007] The present invention first provides a black-tailed leafhopper odor-binding protein NcinOBP7, the amino acid sequence of which is as follows: MAHVYNWRLLDSPQSPTPARPTPATMSCRPILVVFCVVLALAQAKLDPEQVRKNFEACKTETGAPETYEEVIQQKKIPTSDKGMCLLKKREIYDSEGKYNPEGTKKYFLSVFDDRKDEYEKSVAIAEECSKIDVEGLDKCEAAVKHLTCAKTKAVQQNIKVDAVH (SEQ ID NO: 1); The nucleotide sequence of a gene encoding the odorant binding protein is as follows: ATGGCCCACGTATATAACTGGCGTCTCCTGGACTCTCCTCAGTCGCCGACTCCCGCACGTCCTACACCCGCCACCATGAGCTGCCGTCCCATCCTTGTCGTCTTCTGCGTCGTCCTCGCTCTAGCCC AGGCGAAACTTGACCCCGAGCAGGTGAGGAAGAACTTCGAAGCTTGCAAAACAGAAACCGGAGCTCCTGAGACCTACGAAGAAGTAATTCAGCAGAAGAAAATCCCTACAAGTGACAAGGGCATGTGC CTGGTCGAGTGTCTGCTAAAGAAGAGGGAAATTTACGACAGTGAGGGCAAGTACAACCCGGAGGGGACCAAGAAGTACTTCCTCAGCGTGTTTGACGACAGGAAGGATGAGTACGAAGAGTGTGG CCATCGCAGAGGAGTGCAGCAAGATAGACGTGGAGGGGCTGGACAAATGCGAAGCAGCTGTGAAGCACCTCACGTGCGCCAAGACCAAAGCTGTTCAGCAAAACATCAAGGTAGACGCCGTCCACTAA (SEQ ID NO:2); When recombinantly expressing the odorant binding protein, codon optimization was performed based on the protein amino acid sequence (SEQ ID NO: 1) and the signal peptide was removed. The optimized nucleotide sequence is as follows: AAGCTGGACCCTGAACAGGTTCGCAAAAATTTTGAAGCCTGCAAAACCGAAACCGGTGCCCCGGAAACCTATGAAGAAGTGATTCAGCAGAAAAAAATCCCGACCAGTGATAAAGGTATGTGTCTGGTTGAATGCCTGCTGAAAAAACGCGAAATTTATGATAGTGAAGGCAAATATAACCCGGAAG GCACCAAAAAATATTTTCTGAGTGTGTTTGACGACCGCAAAGATGAATATGAAAAAAGCGTTGCAATCGCCGAAGAATGTAGCAAAATTGATGTGGAAGGCCTGGATAAATGCGAAGCAGCCGTGAAACATCTGACCTGCGCCAAAACCAAAGCAGTGCAGCAGAATATTAAAGTGGATGCAGTTCAT (SEQ ID NO: 3); The present invention also provides a dsRNA for reducing the expression level of the odorant binding protein in black-tailed leafhoppers. The sequence of the provided dsRNA is as follows: CGTCCCATCCTTGTCGTCTTCTGCGTCGTCCTCGCTCTAGCCCAGGCGAAACTTGACCCCGAGCAGGTGAGGAAGAACTTCGAAGCTTGCAAAACAGAAACCGGAGCTCCTGAGACCTACGAAGAAGTAATTCAGCAGAAGAAAATCCCTACAAGTGACAAGGGCATGTGCCTGGTCGAGTGTCTGCTAAAGAAGAGGGAAATTTACGACAGTGAGGGCAAGTACA (SEQ ID NO: 4); The primer pairs used to synthesize the above dsRNA have the following sequence information: F: 5'-CGTCCCATCCTTGTCGTCTT-3' (SEQ ID NO: 5), R: 5'-TGTACTTGCCCTCACTGTCGTAA-3' (SEQ ID NO: 6).

[0008] The present invention also provides a method for reducing the perception ability of black-tailed leafhoppers to rice volatiles, wherein the method comprises reducing the expression level of the odor binding protein NcinOBP7 in the black-tailed leafhopper; Furthermore, the rice volatiles are volatiles released by rice induced by rice dwarf virus; As a specific description of the embodiment, the volatile substance is (E)-β-caryophyllene.

[0009] The method for reducing the expression level of the odorant binding protein NcinOBP7 in the caudal leafhopper is to use the dsRNA having the sequence SEQ ID NO: 4 to reduce the expression level; Furthermore, the method described above uses EGFP dsRNA as a control, wherein the sequence of EGFP dsRNA is as follows: CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCA (SEQ ID NO: 7).

[0010] The present invention discovered through fluorescence competition binding experiments that the black-tailed leafhopper odorant-binding protein NcinOBP7 has a high affinity for a volatile released by rice induced by rice dwarf virus. RNAi experiments revealed that rice volatiles induced by rice dwarf virus attracted black-tailed leafhopper nymphs injected with dsEGFP, but silencing NcinOBP7 no longer attracted the nymphs. Therefore, silencing NcinOBP7 impairs the leafhopper's perception of rice volatiles. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : NcinOBP7 protein expression results (Coomassie Brilliant Blue staining), M. Protein marker; -. No induction (negative control); A. Expression induced by BL21 competent cells; B. Expression induced by T7E competent cells; N. Supernatant after sonication; D. Precipitate after sonication.

[0012] Figure 2 : NcinOBP7 protein purification results (Coomassie Brilliant Blue staining), in the figure, M is a protein marker; NcinOBP7 is the target protein band after purification.

[0013] Figure 3 : Binding curve of NcinOBP7 protein and 1-NPN.

[0014] Figure 4 : Binding curve of NcinOBP7 and rice volatiles.

[0015] Figure 5 :NcinOBP7 interference efficiency detection results.

[0016] Figure 6 : Schematic diagram of the leafhopper olfactory behavior selection device.

[0017] Figure 7 : Behavioral responses of leafhoppers to paraffin oil or rice volatiles after injection of dsEGFP or dsNcinOBP7. DETAILED DESCRIPTION

[0018] The present invention uses RNA interference technology to silence the selected black-tailed leafhopper odorant binding protein NcinOBP7 with dsRNA, which can affect the black-tailed leafhopper's perception of virus-induced rice volatiles. Unless otherwise specified, terms used in this invention generally have the meanings commonly understood by those of ordinary skill in the art.

[0019] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0020] RNA was extracted using the Trizol extraction method (RNAiso PLus). The reverse transcription reagent (PrimeScript™ RTreagent Kit with gDNA Eraser (Perfect Real Time)) was purchased from TAKARA Biotechnology Co., Ltd., the dsRNA synthesis kit (T7 RNAi Transcription Kit) was purchased from Nanjing Novezan Biotechnology Co., Ltd., the kit used in the PCR reaction system (EX Taq™) was purchased from TAKARA Biotechnology Co., Ltd., and the DNA purification and recovery kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0) was purchased from TAKARA Biotechnology Co., Ltd.

[0021] The data processing method for the following examples was as follows: Paraffin oil was used as a control to record the effects of (E)-β-caryophyllene on the olfactory behavior of black-tailed leafhoppers after OBP7 disruption, as well as the olfactory responses of black-tailed leafhoppers injected with dsEGFP to (E)-β-caryophyllene. Data were analyzed using SPSS 27.0 statistical software, using a generalized linear mixed model (GLMM) with a Poisson distribution. RT-qPCR was used to detect the disruption efficiency of the NcinOBP7 gene, and the data were analyzed using 2 -△△Ct The calculations were performed using the Student's test using SPSS 27.0. Statistical analysis was performed using SPSS 27.0. The present invention will be described in further detail below with reference to specific examples and data. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0022] Example 1: Prokaryotic expression of the black-tailed leafhopper odorant binding protein NcinOBP7

[0023] The amino acid sequence of the black-tailed leafhopper odorant binding protein NcinOBP7 used in the present invention is SEQ ID NO: 1, The nucleotide sequence of its encoding gene is SEQ ID NO: 2.

[0024] The prokaryotic expression steps of NcinOBP7 include S1-S4 S1. Using the nucleotide sequence after codon optimization (SEQ ID NO: 3) as a template, primers were designed (Table 1) and PCR amplification was performed to obtain PCR product fragments.

[0025] Table 1: Sequence information of template design primers

[0026] S2. Expression vector construction: The PCR product fragment obtained in S1 and the pET-28a plasmid were double-digested with restriction endonucleases NcoI and XhoI, respectively. The cells were subjected to agarose gel electrophoresis and recovered. The double-digested products obtained in S2 were ligated with T4 DNA ligase to obtain recombinant plasmids. BL21 (DE3) and T7E competent cells were removed from the ultra-low temperature freezer and thawed on ice. 5 μg of plasmid was added to each cell, and the cells were mixed thoroughly by gentle pipetting. The cells were placed on ice for 30 min. Heat shock was performed in a water bath at 42°C for 45 s, and the cells were placed on ice for 1-2 min. 800 μL of preheated LB liquid medium was added, and the cells were incubated at 37°C and 158 rpm for 50-60 min. Centrifugation was performed at 6000 rpm for 4 min, and a portion of the supernatant (650 μL) was removed. The remaining bacterial liquid (150 μL) was resuspended and mixed, and then spread on a resistant LB plate. The plates were inverted and incubated at 37°C for 12-16 h until single colonies (positive clones) appeared.

[0027] S3. Small-scale expression and identification of positive clones: Single colonies containing the recombinant plasmid were selected and transferred to 5 mL of LB liquid medium (containing resistance) and cultured at 37°C overnight. 1 mL of bacterial solution was stored at -20°C. A portion of the bacterial solution was used as a control group, and the remaining bacterial solution was inoculated into 5 mL of LB liquid medium (containing resistance) and cultured at 37°C with shaking until the OD600 was approximately 0.6. IPTG inducer (final concentration 1 mM) was added and cultured with shaking at 37°C for 4 h and 18°C ​​overnight. The cells were collected by centrifugation at 8000 rpm for 3 min, resuspended in 1 mL of PBS, and ultrasonically disrupted with parameters set to 200 W power, 3 s working, 4 s pause, and 25-30 min. The supernatant and precipitate were separated and collected. 50 μL of 1× loading buffer was added to the precipitate, and 5× loading buffer was added to the supernatant. The sample was boiled in boiling water and 10 μL of the sample was taken for SDS-PAGE analysis.

[0028] Express test results as Figure 1 As shown, the results showed that the protein was expressed in both competent cells, the protein molecular weight was about 31.9kD, and the size of the protein band was consistent with the target gene protein after removing the tag protein size (20.0kDa).

[0029] S4. Protein purification and amplification: 100 μL of positive bacteria stored at -20°C was inoculated into 50 mL of LB liquid medium (containing resistance) and cultured with shaking for 16 hours; 50 mL of bacterial liquid was inoculated into 1000 mL of LB liquid medium, cultured at 37°C until OD600 was about 0.6, and the culture temperature was lowered to 18°C; IPTG inducer was added to a final concentration of 1 mM, and cultured with shaking at 18°C ​​for 12 hours; the bacteria were collected by centrifugation at 8000 rpm for 15 minutes, resuspended in 50 ml of pre-cooled PBS buffer, and the protease inhibitor PMSF was added; the bacteria were ultrasonically disrupted with the parameters set to 200 W power, 3 s working, 4 s pause, and 20 minutes; centrifuged at 12000 rpm at 4°C for 30 minutes to separate the supernatant and precipitate, and the supernatant and precipitate were collected; the supernatant protein solution was filtered with a 0.22 um filter for later use; a Ni-NTA column was prepared with 1 The supernatant protein solution was loaded at a flow rate of 1 mL / min; the column was washed with PBS buffer (pH 7.4) until the effluent contained no protein (the G250 detection solution did not change color); elution was performed with 30 mM, 50 mM, and 300 mM imidazole in PBS buffer (pH 7.4), respectively, and the eluate was collected in sections until the G250 detection solution did not change color; 10 μL of the flow-through at each stage was taken for SDS-PAGE electrophoresis detection, and the collected eluate was dialyzed and concentrated for final concentration SDS-PAGE electrophoresis detection.

[0030] Purification test of target protein NcinOBP7 The purification results are shown in Figure 2 (Coomassie Brilliant Blue staining). The size of the purified product is approximately 31.9 kDa. After removing the size of the tag protein (20.0 kDa), the protein band is consistent with the size of the target gene protein.

[0031] Example 2: Fluorescence competition binding assay between NcinOBP7 and rice volatiles 1. Determination of the binding ability of NcinOBP7 with fluorescent probes The purified recombinant protein was dissolved in 20 mmol / L Tris-HCl (pH 7.4) buffer to a final concentration of 1 mg / mL. The fluorescent probe N-Phenyl-1-naphthylamine (1-NPN) and the odorant (E)-β-caryophyllene were dissolved in methanol (chromatographic grade) to a final concentration of 1 mmol / L. First, the dissociation constant between NcinOBP7 and 1-NPN was determined: 250 μL of 20 mmol / L Tris-HCl (pH 7.4) buffer was added to a 96-well fluorescence plate. The protein solution was then added to a final protein concentration of 2 μM. Finally, the prepared 1-NPN solution was added, increasing the 1-NPN concentration from 2 to 20 μM. The excitation wavelength was set at 337 nm, and the scanning wavelength was 390–460 nm. After each addition of 1-NPN, the maximum fluorescence value was recorded, and the dissociation constant, K, of NcinOBP7 and 1-NPN was calculated according to the Scatchard equation. 1-NPN According to the experimental results, the binding curve of NcinOBP7 and 1-NPN was drawn as follows: Figure 3 .

[0032] 2. Determination of the binding ability of NcinOBP7 to odorant ligands Add 250 μL of 20 mM / L Tris-HCl (pH 7.4) buffer to a 96-well fluorescence plate, then add the protein solution to a final concentration of 2 μM. After the fluorescence value stabilizes, record the strongest fluorescence value. Then, add 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 6 μM, 8 μM, 12 μM, 16 μM and 20 μM odor standards to the mixed solution of NcinOBP7 and 1-NPN in sequence. Record the changes in fluorescence intensity at different concentrations. Repeat three times for each experiment.

[0033] Assuming that NcinOBP7 has 100% activity with the ligand and the binding ratio with the ligand is 1:1, the concentration of the ligand when the fluorescence intensity value drops to half of the initial value is calculated as IC. 50 The binding constant Ki of NcinOBP7 and ligand was calculated by the following formula: Ki = [IC 50 ] / (1+[1-NPN] / K 1-NPN) ); [1-NPN] is the concentration of free 1-NPN; K 1-NPN is the dissociation constant of NcinOBP7 and 1-NPN.

[0034] The results of the competition binding assay were as follows Figure 4 The results showed that the binding constant Ki It is 4.91±0.73, and the binding activity is strong.

[0035] Example 3: Preparation of dsRNA of Blacktail Leafhopper NcinOBP7 The preparation method of dsRNA of the black-tailed leafhopper odorant binding protein NcinOBP7 is as follows: Primers were designed based on the open reading frame of the NcinOBP7 gene of the black-tailed leafhopper (SEQ ID NO: 2) for synthesizing dsRNA (Table 2).

[0036] Table 2: dsNcinOBP7 synthesis primers

[0037] A newly emerged adult of the black-tailed leafhopper was collected individually and placed in a 1.5 mL centrifuge tube. Total RNA was extracted using the Trizol method. The concentration and quality of the RNA were measured using an ultra-micro UV spectrophotometer (N60). Reverse transcription was performed using a reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time)) according to the manufacturer's instructions to synthesize the first-strand cDNA.

[0038] The synthesized black-tailed leafhopper cDNA was used as a template and PCR amplification was performed using the primers used for synthesizing dsRNA.

[0039] The reaction system for PCR amplification was as follows: 10×EX Taq Buffer 2.5 μL, TaKaRa EX Taq 0.25 μL, dNTP Mixture 2 μL, upstream primer (10 μmoL / L) 1 μL, downstream primer (10 μmoL / L) 1 μL, cDNA / EGFP plasmid 2 μL, and ddH2O was added to make up to 26 μL.

[0040] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 40 s, and finally extension at 72°C for 10 min. The amplified product was stored at 4°C.

[0041] After the PCR reaction was completed, the amplification results were detected by agarose gel electrophoresis. After verification, only the first set of primers showed a single bright band at the target fragment size. The PCR product was further purified using a DNA purification kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0), and the cDNA product was used as a template for in vitro transcription of dsRNA.

[0042] The purified PCR product was used as a template for in vitro transcription of dsRNA to prepare dsRNA.

[0043] The in vitro transcription system for dsRNA was as follows: NTP Mix 8 μL, 10× Transcription Buffer 2 μL, T7 Enzyme Mix 2 μL, template 8 μL, and ddH2O was added to 20 μL; the mixture was placed at 37°C for 2 h.

[0044] After the reaction, a double enzyme digestion system was added to remove residual template DNA and single-stranded RNA. The double enzyme digestion system consisted of 17 μL RNase-free H2O, 1 μL DNaseⅠ, and 2 μL RNase T1 (10 U / μL). The mixture was incubated at 37°C for 30 min.

[0045] Then, the dsRNA was purified and finally dissolved with 40 μL RNase-free H2O and stored in a -20°C refrigerator. Finally, the dsRNA of NcinOBP7 with SEQ ID NO: 3 having the best interference effect was selected.

[0046] Example 4: Role of NcinOBP7 in Host Selection of Black-tailed Leafhopper The third-instar nymphs of the black-tailed leafhopper were selected and RNA interference was performed by microinjection. The dsRNA concentration was 5000 ng / μL, the injection volume was 50 nL, and the injection speed was 10 nL / s.

[0047] The control group was a 3rd instar nymph of the black-tailed leafhopper injected with dsEGFP, specifically, an equal concentration of dsEGFP was injected. Here, dsEGFP refers to dsRNA of the enhanced green fluorescent protein gene (EGFP). In the preparation method, the primers used to synthesize the green fluorescent protein dsRNA are as follows: dsEGFP-F: 5'-taatacgactcactataggggCACAAGTTCAGCGTGTCCG-3'; dsEGFP-F: 5'-taatacgactcactatagggTGCCTTCTTCTGCTTGTCG-3'.

[0048] The template used for PCR amplification was a plasmid containing the EGFP gene stored in the laboratory. The product obtained by PCR amplification was 409 bp in size, and the specific sequence was shown in SEQ ID NO: 7. The remaining reagents and methods used were the same as those used in the method for preparing dsNcinOBP7 in Example 4.

[0049] Forty-eight hours after injection, black-tailed leafhopper nymphs treated with 5000 ng / μL of dsNcinOBP7 and dsEGFP were collected. Fifteen nymphs formed a group, with three biological replicates collected for each treatment. RNA was extracted and purified from each group of black-tailed leafhopper nymphs, then reverse-transcribed into cDNA and diluted 10-fold to serve as a template for quantitative PCR. RT-qPCR analysis was performed using the following primers:

[0050] The quantitative primers for detecting NcinOBP7 are as follows: F:5' -TCCTGGACTCTCCTCAGTCG- 3', R: 5'-CAGGAGCTCCGGTTTCTGTT- 3'; The primers for the internal reference gene β-actin are as follows: F: 5'-GTGTTGGATTCTGGGTGATG- 3', R: 5' -GGTAGTCTGTAAGGTCTCG- 3'; The RT-qPCR system was (20 μL): 6.4 μL of ddH2O, 10 μL of TB Green (Takara), 0.8 μL of primers, and 2.0 μL of cDNA.

[0051] RT-qPCR reaction instrument LightCycLer® 96 Instrument (Roche) The reaction conditions were 95°C for 30 s; 95°C for 15 s, 60°C for 30 s, 39 cycles, with 3 technical replicates for each sample.

[0052] The results showed that compared with the control group, the expression of dsNcinOBP7 gene in the treatment group was significantly decreased ( Figure 5 ).

[0053] 72 hours after injection, the function of NcinOBP7 was verified using the olfactory setup shown in Figure 6. The specific method is as follows: The effect of (E)-β-caryophyllene, a standard substance, diluted to 0.1 μg / μL in paraffin oil on the olfactory behavior of black-tailed leafhopper nymphs after interfering with NcinOBP7 was analyzed. 0.2 g of absorbent cotton was placed at each end of the olfactory apparatus. 500 μL of 0.1 μg / μL (E)-β-caryophyllene was dripped onto the cotton wool at one end as the experimental group, while 500 μL of paraffin oil was dripped onto the cotton wool at the other end as the control group. After interfering with the olfactory apparatus, black-tailed leafhopper nymphs were moisturized and starved for 4 hours before being inserted into the center of the apparatus. Fifteen nymphs were inserted into each group. The number of choices made by the black-tailed leafhoppers in the experimental and control groups was recorded 2, 4, 6, 8, and 24 hours after interfering with the olfactory apparatus. The experiment was conducted in a laboratory under moisturized conditions at 27°C ± 1°C and a relative humidity of 75 ± 5%. Five replicates were performed.

[0054] The experimental and investigation methods of the olfactory response of black-tailed leafhoppers microinjected with dsEGFP to (E)-β-caryophyllene were the same as above.

[0055] The test results are as follows Figure 7 As shown, (E)-β-caryophyllene has a significant attraction to black-tailed leafhopper nymphs injected with dsEGFP, but after silencing NcinOBP7, black-tailed leafhoppers are no longer attracted to (E)-β-caryophyllene, indicating that NcinOBP7 plays a key role in the perception of (E)-β-caryophyllene by black-tailed leafhoppers.

Claims

1. A black-tailed leafhopper odor-binding protein, characterized in that The amino acid sequence of the odorant binding protein is SEQ ID NO:

1.

2. A coding gene, characterized in that The coding gene is used to encode the odorant binding protein according to claim 1, and its nucleotide sequence is SEQ ID NO:

2.

3. A gene encoding the odorant binding protein according to claim 1 with the signal peptide removed, characterized in that: The sequence of the coding gene is SEQ ID NO:

3.

4. A dsRNA for reducing the expression of the odorant binding protein according to claim 1 in black-tailed leafhopper, characterized in that The sequence of the dsRNA is SEQ ID NO:

4.

5. The dsRNA according to claim 4, wherein The sequences of the synthetic primer pair for the dsRNA are SEQ ID NO: 5 and SEQ ID NO:

6.

6. A method for reducing the sensitivity of black-tailed leafhoppers to rice volatiles, characterized in that: The method is to reduce the expression level of the odor binding protein according to claim 1 in black-tailed leafhopper.

7. The method according to claim 6, wherein In the method, the rice volatiles are volatiles released by rice induced by rice dwarf virus.

8. The method according to claim 7, wherein The volatile substance is (E)-β-caryophyllene.

9. The method according to claim 6, wherein The method is to use dsRNA to reduce the expression level of the odorant binding protein with an amino acid sequence of SEQ ID NO: 1, and the sequence of the dsRNA is SEQ ID NO:

4.

10. The method according to claim 6, wherein The method uses EGFP dsRNA as a control, wherein the sequence of EGFP dsRNA is SEQ ID NO: 7.

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