Odorant binding protein from black-tail leafhopper and application of odorant binding protein in prevention and treatment of rice dwarf virus

Abstract

The invention provides an odorant binding protein sourced from cicada and application of the odorant binding protein in prevention and treatment of rice dwarf disease. 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 NcinOBP8 of the black tail leafhopper and a volatile matter released by rice induced by a rice dwarf virus have high affinity; the 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 NcinOBP8 is silenced, the rice volatile matter does not attract the black-tail leafhopper nymphs after 2 hours. Therefore, the silent cicada NcinOBP8 can influence the perception of the cicada on the rice volatile matters.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural disease prevention and control, and particularly relates to an odor-binding protein derived from black-tailed leafhopper and an application thereof in preventing and controlling rice dwarf disease. Background Art

[0002] Rice dwarf virus (RDV) is a serious rice disease caused by rice dwarf virus. Nephotettix cincticeps ) is spread through the plant's lining. Rice plants infected with RDV are significantly stunted, with increased number of shoots, dark green leaves, and short, thick, and stiff leaves. Several milky white, dotted lines appear on the leaf sheaths or leaves, parallel to the veins. From the seedling to the tillering stage, the disease generally prevents heading. Later, the disease manifests as necked-up ears with small, barren grains. The occurrence of rice dwarf disease seriously hinders high and stable rice yields.

[0003] Currently, the prevention and control of rice dwarf disease is extremely difficult due to the lack of highly effective virus-resistant germplasm resources and the small size and easy hiding of the vector insects. Currently, the main method of controlling rice dwarf disease outbreaks is to use chemical agents to control the vector insects. However, the excessive use of chemical agents has led to frequent "3R" problems.

[0004] RNA pesticides can be designed with double-stranded RNA targeting specific gene sequences of RDV or key genes of insect vectors, achieving "targeted control" by precisely silencing viral replication or blocking the ability of insects to transmit the virus. RNA pesticides are highly specific and only act on target viruses or vectors, avoiding the killing of non-target organisms. Compared with traditional chemical pesticides, they are environmentally friendly, degrade quickly, and leave no residue. They are not prone to developing drug resistance and can silence viral genes before vector transmission, making up for the difficulty of monitoring during the incubation period. Therefore, RNA pesticides are expected to become a key technology for green control, promoting the transformation of rice dwarf disease control from passive insecticide to active blocking, and providing a new path for sustainable agriculture. However, the ability to efficiently screen and identify key target genes that regulate vector transmission is a key factor affecting the practical application of this technology. Summary of the Invention

[0005] The present invention aims to provide an odor-binding protein derived from the black-tailed leafhopper and its application in preventing and controlling rice dwarf disease. The provided odor-binding protein plays an indispensable role in the process of the black-tailed leafhopper sensing rice volatiles. By regulating the expression level of the protein, the black-tailed leafhopper's ability to sense rice volatiles can be regulated.

[0006] The amino acid sequence of the odorant binding protein NcinOBP8 provided by the present invention is shown in SEQ ID NO: 1; the odorant binding protein NcinOBP8 MLLVSASVFVVLVVICVKGEDDCMPPKPGEKHIHPPECCKLEGVSGIYPDAMKVAAGKCREKFPHPPHLPKPSGSPPPGPPPRSPEMKKYITCMAECVFTEVGVVKDGKLDKDAAMKSFKTDNKELTSTISAAIDKCFTTYEKDVDPSLECKSGAAEMEKCVMREIFLSCPSSMWNDSAECTALKTKIDKCPQMPVMLMGPPGPRSPRPH (SEQ ID NO: 1); The odorant-binding protein described above has a nucleotide sequence of one of its encoding genes as follows: ATGTTGCTTGTTAGTGCCTCTGTTTTTGTGGTATTAGTTGTTATATGTGTGAAGGGAGAAGATGACTGCATGCCACCTAAACCCGGAGAGAAACACATTCACCCGCCGGAGTGTTGCAAGCTGGAGGGAGTTAGTGGGATCTATCCAGATGCGATGAAAGTCGCAGCGGGAAAGTGTCGAGAGAAATTTCCTCACCCTCCACATCTACCTAAACCCTCTGGTTCCCCACCCCCTGGACCACCCCCTCGGAGCCCAGAGATGAAGAAGTACATTACCTGCATGGCAGAGTGCGTGTTTACAGAAGTAGGAGTAGTCAAGGATGGTAAATTGGACAAGGATGCAGCGATGAAGAGCTTCAAAACCGACAACAAGGAACTGACCTCCACCATCTCCGCTGCAATAGACAAGTGTTTCACCACGTACGAGAAGGACGTAGACCCCAGCTTGGAGTGCAAGAGTGGGGCTGCAGAGATGGAGAAGTGCGTGATGCGGGAAATCTTCCTCAGTTGTCCATCATCAATGTGGAATGACTCTGCAGAATGCACCGCACTCAAAACCAAGATCGATAAATGTCCGCAAATGCCCGTCATGCTCATGGGTCCCCCAGGACCTCGCAGTCCCCGTCCTCATTAG (SEQ ID NO: 2); When 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: GAAGATGATTGTATGCCGCCGAAACCGGGTGAAAAACATATTCATCCGCCGGAATGTTGTAAACTGGAAGGCGTGAGTGGTATTTATCCGGATGCCATGAAAGTTGCAGCAGGTAAATGCCGCGAAAAATTTCCGCATCCGCC GCATCTGCCGAAACCGAGCGGTAGCCCGCCGCCTGGTCCGCCTCCTAGAAGTCCGGAAATGAAAAAATATATTACCTGTATGGCGGAATGTGTTTTTACCGAAGTTGGTGTGGTGAAAGATGGCAAACTGGATAAAGATGCAG CAATGAAAAGTTTTAAGACCGATAATAAGGAGCTGACCAGTACCATTAGCGCCGCAATTGATAAATGCTTTACCACCTATGAAAAGGATGTTGATCCGAGTCTGGAATGTAAAAGTGGTGCAGCCGAAATGGAAAAATGTGTT ATGCGTGAAATTTTCCTGAGTTGCCCGAGTAGCATGTGGAATGATAGCGCAGAATGCACCGCACTGAAAACCAAAATTGATAAATGTCCGCAGATGCCGGTTATGCTGATGGGTCCGCCGGGTCCGCGCAGCCCTAGACCTCAT (SEQ ID NO: 3); The present invention discovered through a fluorescence competition binding experiment that the black-tailed leafhopper odor-binding protein NcinOBP8 has a high affinity for a volatile substance released by rice induced by rice dwarf virus.

[0007] The volatile substance, as specifically described in the examples, is (E)-β-caryophyllene.

[0008] Furthermore, RNAi experiments revealed that rice volatiles induced by rice dwarf virus attracted black-tailed leafhopper nymphs injected with dsEGFP, but after silencing the dsRNA of NcinOBP8, rice volatiles no longer attracted black-tailed leafhopper nymphs; therefore, knocking out or silencing the black-tailed leafhopper NcinOBP8 can affect the black-tailed leafhopper's perception of rice volatiles.

[0009] The present invention also provides a dsRNA for silencing the expression of the black-tailed leafhopper odor-binding protein NcinOBP8, the sequence of which is shown in SEQ ID NO: 4.

[0010] GTTGCTTGTTAGTGCCTCTGTTTTTGTGGTATTAGTTGTTATATGTGTGAAGGGAGAAGATGACTGCATGCCACCTAAACCCGGAGAGAAACACATTCACCCGCCGGAGTGTTGCAAGCTGGAGGGAGTTAGTGGGATCTATCCAGATGCGATGAAAGTCGCAGCGGGAAAGTGTCGAGAGAAATTTCCTCA CCCTCCACATCTACCTAAACCCTCTGGTTCCCCACCCCCTGGACCACCCCCTCGGAGCCCAGAGATGAAGAAGTACATTACCTGCATGGCAGAGTGCGTGTTTACAGAAGTAGGAGTAGTCAAGGATGGTAAATTGGACAAGGATGCAGCGATGAAGAGCTTCAAAACCGACAACAAGGAACTGACCTCCAC (SEQ ID NO: 4).

[0011] The primer pairs used to synthesize dsRNA have the following sequence information: F: 5-GTTGCTTGTTAGTGCCTCTGTTT-3 (SEQ ID NO: 5), R: 5-GTGGAGGTCAGTTCCTTGTTGT-3 (SEQ ID NO: 6).

[0012] The present invention discovered through fluorescence competition binding experiments that the black-tailed leafhopper odorant-binding protein NcinOBP8 has a high affinity for a volatile released by rice induced by rice dwarf virus. RNAi experiments also revealed that rice volatiles induced by rice dwarf virus attracted black-tailed leafhopper nymphs injected with dsEGFP. However, after silencing NcinOBP8, the rice volatiles no longer attracted black-tailed leafhopper nymphs after two hours. Therefore, silencing NcinOBP8 can affect the black-tailed leafhopper's perception of rice volatiles. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1: NcinOBP8 protein expression results (Coomassie Brilliant Blue staining), in the figure, M is a protein marker; - is no induction (negative control); A / N is the expression of the protein in the supernatant after induction with BL21 competent cells; B / N is the expression of the protein in the supernatant after induction with T7E competent cells; A / D is the expression of the protein in the precipitate after induction with BL21 competent cells; B / D is the expression of the protein in the precipitate after induction with T7E competent cells.

[0014] Figure 2 : NcinOBP8 protein purification results (Coomassie Brilliant Blue staining), in the figure, M is the protein marker; NcinOBP8 is the size of the target protein fragment after purification.

[0015] Figure 3 : Binding curve of NcinOBP8 protein and 1-NPN.

[0016] Figure 4 : Scatchard plot of NcinOBP8 protein binding to 1-NPN.

[0017] Figure 5 : Binding curve of NcinOBP8 and rice volatiles.

[0018] Figure 6 :NcinOBP8 interference efficiency detection diagram.

[0019] Figure 7 : Diagram of the leafhopper olfactory behavior selection device.

[0020] Figure 8 : Behavioral responses of leafhoppers to paraffin oil or rice volatiles after injection of dsEGFP or dsNcinOBP8. DETAILED DESCRIPTION

[0021] The present invention screened and obtained a black-tailed leafhopper odor-binding protein NcinOBP8, determined that the NcinOBP8 protein of the black-tailed leafhopper was silenced by dsRNA, and determined that the black-tailed leafhopper with reduced NcinOBP8 protein expression had a reduced perception effect on virus-induced rice volatiles.

[0022] In the embodiment of the present invention, the changes in target gene expression after RNA interference were analyzed using RT-qPCR reaction, and the data of RT-qPCR were analyzed using 2 -△△CtCalculations were performed using the independent-samples t-test in SPSS 27.0. Analysis of the rice volatile selection behavior of black-tailed leafhopper nymphs after RNA interference was performed using SPSS 27.0, fitting a generalized linear mixed model (GLMM) with a Poisson distribution to analyze data variance.

[0023] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0024] Example 1: Recombinant expression of odorant binding protein NcinOBP8 The flavor binding protein NcinOBP8, whose amino acid sequence is SEQ ID NO: 1 and whose encoding gene has the nucleotide sequence SEQ ID NO: 2, was screened and obtained from the black-tailed leafhopper and recombinantly expressed. The specific steps of recombinant expression are as follows: 1) Using the codon-optimized sequence of NcinOBP8 of the black-tailed leafhopper SEQ ID NO: 3 as a template, PCR amplification was performed using designed primers to obtain a PCR product fragment.

[0025] Table 1: Information of primers for NcinOBP8 synthesis Name Sequence (5'→3') Length NcinOBP8-1 ACAAGGCCATGGCTGATATCGGATCCGAAGATGATTGTATGCCGCCGAAACCGGGTGAAAAACATATTCATCCGCCGGA 79 NcinOBP8-2 TGCTGCAACTTTCATGGCATCCGGATAAATACCACTCACGCCTTCCAGTTTACAACATTCCGGCGGATGAATATG 75 NcinOBP8-3 CCATGAAAGTTGCAGCAGGTAAATGCCGCGAAAAATTTCCGCATCCGCCGCATCTGCCGAAACCGAGCGGTAGCC 75 NcinOBP8-4 TCCGCCATACAGGTAATATATTTTTTCATTTCCGGACTTCTAGGAGGCGGACCAGGCGGCGGGCTACCGCTCGGTTTC 78 NcinOBP8-5 ATTACCTGTATGGCGGAATGTGTTTTTACCGAAGTTGGTGTGGTGAAAGATGGCAAACTGGATAAAGATGCAGCAATGA 79 NcinOBP8-6 GCATTTATCAATTGCGGCGCTAATGGTACTGGTCAGCTCCTTATTATCGGTCTTAAAACTTTTCATTGCTGCATCTTTA 79 NcinOBP8-7 CCGCAATTGATAAATGCTTTACCACCTATGAAAAGGATGTTGATCCGAGTCTGGAATGTAAAAGTGGTGCAGCCGAAAT 79 NcinOBP8-8 CGCTATCATTCCACATGCTACTCGGGCAACTCAGGAAAATTTCACGCATAACACATTTTTCCATTTCGGCTGCACCACT 79 NcinOBP8-9 CATGTGGAATGATAGCGCAGAATGCACCGCACTGAAAACCAAAATTGATAAATGTCCGCAGATGCCGGTTATGCTGATG 79 NcinOBP8-10 CAGTGGTGGTGGTGGTGGTGCTCGAGTTAATGAGGTCTAGGGCTGCGCGGACCCGGCGGACCCATCAGCATAACCGGCA 79 2) Expression Vector Construction: The PCR product fragment and the pET-28a plasmid were double-digested with restriction endonucleases NcoI and XhoI, respectively. The digested products were recovered by agarose gel electrophoresis and ligated with T4 DNA ligase to generate the recombinant plasmid. 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, gently pipetting to mix thoroughly, and incubated on ice for 30 min. Heat shock was performed in a water bath at 42°C for 45 s, followed by 1-2 min on ice. 800 μL of prewarmed LB medium was added and incubated at 37°C at 158 ​​rpm for 50-60 min. Centrifuge at 6000 rpm for 4 min, remove a portion of the supernatant (650 μL), resuspend the remaining culture (150 μL), mix thoroughly, and plate onto a resistant LB plate. Invert the plate and incubate at 37°C for 12-16 h until a single colony (positive clone) appears.

[0026] 3) Small-scale expression and identification of positive clones: Single colonies containing the recombinant plasmid were selected and inoculated into 5 mL of LB liquid medium (containing resistance) and cultured overnight at 37°C. 1 mL of the culture was stored at -20°C. A portion of the culture was used as a control, and the remaining culture 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 then at 18°C ​​overnight. The cells were harvested by centrifugation at 8000 rpm for 3 min, resuspended in 1 mL of PBS, and disrupted by sonication at 200 W power, 3 s on, 4 s pause, and 25-30 min. The supernatant and precipitate were separated and collected by centrifugation at 12000 rpm and 4°C for 10 min. 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 analyzed by SDS-PAGE.

[0027] Express test results as Figure 1 As shown, the protein was expressed in both competent cells, with a molecular weight of approximately 38.6 kDa (including 20.0 kDa of tag protein). The size of the protein band after removing the tag protein was consistent with the size of the target gene protein.

[0028] 4) Protein purification and amplification: 100 μL of positive bacterial strains stored at -20°C were inoculated into 50 mL of LB liquid medium (containing resistance) and cultured with shaking for 16 h; 50 mL of bacterial solution was inoculated into 1000 mL of LB liquid medium and 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 h; the cells were collected by centrifugation at 8000 rpm for 15 min, resuspended in 50 mL of pre-chilled PBS buffer, and the protease inhibitor PMSF was added; the cells were ultrasonically disrupted with the parameters set to 200 W power, 3 s working, 4 s pause, and time for 20 min; centrifuged at 12000 rpm at 4°C for 30 min 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 mL The supernatant protein solution was loaded at a flow rate of 100 μg / min. The column was washed with PBS (pH 7.4) until the flow-through was protein-free (G250 assay solution did not change color). Elution was performed with PBS (pH 7.4) containing 30 mM, 50 mM, and 300 mM imidazole, respectively. The eluates were collected in sections until the G250 assay solution did not change color. 10 μL of the flow-through from each section was analyzed by SDS-PAGE electrophoresis. The collected eluates were dialyzed and concentrated, and then analyzed by SDS-PAGE electrophoresis at the final concentration. The purification results of the target protein NcinOBP8 are shown in Figure 2 (Coomassie Brilliant Blue staining). The purified product is approximately 38.6 kDa (including a 20.0 kDa tag protein). After removing the tag protein, the protein band is consistent with the target gene protein size.

[0029] Example 2: Fluorescence competition binding assay between NcinOBP8 and rice volatiles 1. Determination of the binding ability of NcinOBP8 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. The dissociation constant between NcinOBP8 and 1-NPN was determined by adding 250 μL of 20 mmol / L Tris-HCl (pH 7.4) buffer 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, between NcinOBP8 and 1-NPN was calculated according to the Scatchard equation. 1-NPN Based on the experimental results, the binding curve of NcinOBP8 and 1-NPN was drawn as follows: Figure 3 , the Scacca plot of NcinOBP8 binding to 1-NPN is shown in Figure 4 .

[0030] 2. Determination of the binding ability of NcinOBP8 to odorant ligands Add 250 μL of 20 mmol / 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 NcinOBP8 and 1-NPN in sequence. Record the changes in fluorescence intensity at different concentrations. Repeat three times for each experiment.

[0031] Assuming that NcinOBP8 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 NcinOBP8 and odor 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 NcinOBP8 and 1-NPN.

[0032] The results of the competition binding assay were as follows Figure 5As shown, the binding constant K of NcinOBP8 and (E)-β-caryophyllene i The binding activity was strong.

[0033] Example 3: Preparation of dsRNA of Black-tailed Leafhopper NcinOBP8 Based on the open reading frame of the NcinOBP8 gene of the black-tailed leafhopper (SEQ ID NO: 2), primers were designed to synthesize dsRNA with the sequence of SEQ ID NO: 4.

[0034] F: 5'-taatacgactcactatagggGTTGCTTGTTAGTGCCTCTGTTT-3' (SEQ ID NO: 5), R: 5'-taatacgactcactatagggGTGGAGGTCAGTTCCTTGTTGT-3' (SEQ ID NO: 6).

[0035] 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 RNA were measured using an ultra-micro UV spectrophotometer (N60). Reverse transcription was performed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) according to the manufacturer's instructions to synthesize the first-strand cDNA.

[0036] The synthesized black-tailed leafhopper cDNA was used as a template and PCR amplification was performed using the primers used for synthesizing dsRNA.

[0037] 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.

[0038] 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.

[0039] After the PCR reaction was completed, the amplification results were detected by agarose gel electrophoresis, and the PCR products were recovered and purified using a DNA purification kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0). After sequencing verification, the cDNA product prepared by the first set of primers was selected as the template for in vitro transcription of dsRNA.

[0040] The purified PCR product was used as a template for in vitro transcription of dsRNA to prepare dsRNA.

[0041] 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 incubated at 37°C for 2 h.

[0042] 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.

[0043] The dsRNA was then purified and dissolved in 40 μL of RNase-free H2O. The dsRNA was stored in a -20°C refrigerator to obtain the dsRNA of the gene NcinOBP8. The nucleotide sequence is as follows: GTTGCTTGTTAGTGCCTCTGTTTTTGTGGTATTAGTTGTTATATGTGTGAAGGGAGAAGATGACTGCATGCCACCTAAACCCGGAGAGAAACACATTCACCCGCCGGAGTGTTGCAAGCTGGAGGGAGTTAGTGGGATCTATCCAGATGCGATGAAAGTCGCAGCGGGAAAGTGTCGAGAGAAATTTCCTCA CCCTCCACATCTACCTAAACCCTCTGGTTCCCCACCCCCTGGACCACCCCCTCGGAGCCCAGAGATGAAGAAGTACATTACCTGCATGGCAGAGTGCGTGTTTACAGAAGTAGGAGTAGTCAAGGATGGTAAATTGGACAAGGATGCAGCGATGAAGAGCTTCAAAACCGACAACAAGGAACTGACCTCCAC (SEQ ID NO: 4).

[0044] Example 4: Role of NcinOBP8 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.

[0045] The third instar nymphs of the black-tailed leafhopper injected with dsEGFP were used as the control group. Specifically, equal concentrations of dsEGFP were injected. dsEGFP refers to the dsRNA of the enhanced green fluorescent protein gene (EGFP), and its sequence is as follows: CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCA (SEQ ID NO: 7).

[0046] Forty-eight hours after injection, black-tailed leafhopper nymphs treated with 5000 ng / μL of dsNcinOBP8 and dsEGFP were collected. Each group consisted of 15 nymphs, 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:

[0047] The quantitative primers for NcinOBP8 are as follows: F: 5'-GGGCTGGACAAATGCG-3', R: 5'-CGGCGTCTACCTTGATGT-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.

[0048] RT-qPCR reaction instrument LightCycler® 96 Instrument (Roche) The reaction conditions were 95°C for 30 s, 95°C for 15 s, and 60°C for 30 s for 39 cycles, with three technical replicates for each sample.

[0049] The results showed that compared with the control group, the expression of dsNcinOBP8 gene in the dsRNA injection treatment group was significantly decreased ( Figure 6 ).

[0050] 72 hours after the injection, use Figure 7 The function of NcinOBP8 was verified by the olfactory apparatus as follows.

[0051] The effect of (E)-β-caryophyllene, a standard substance, diluted to 0.1 μg / μL (dissolved in paraffin oil) on the olfactory behavior of black-tailed leafhopper nymphs after interfering with NcinOBP8 was analyzed. 0.2 g of absorbent cotton was placed at each end of an olfactory device. 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 device for 4 hours, black-tailed leafhoppers were moisturized and starved before being inserted into the center of the device joint. Fifteen black-tailed leafhoppers 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 device. The experiment was conducted in a climatic chamber with five replicates and maintained moisture throughout the experiment: temperature: 27°C ± 1°C, relative humidity: 75 ± 5%.

[0052] 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.

[0053] The test results are as follows Figure 8As shown, (E)-β-caryophyllene strongly attracted dsEGFP-injected black leafhopper nymphs. However, after silencing NcinOBP8, the black leafhoppers were no longer attracted to (E)-β-caryophyllene (except for 2 hours). This suggests that NcinOBP8 plays a key role in the perception of (E)-β-caryophyllene by the black leafhopper. Inhibiting NcinOBP8 expression is expected to prevent the black leafhopper from feeding on susceptible rice plants and reduce the risk of virus transmission.

Claims

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

1.

2. A gene, characterized in that The gene encodes the odorant binding protein according to claim 1.

3. The gene according to claim 2, wherein The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A nucleic acid fragment encoding the odorant binding protein of claim 1 with the signal peptide removed, characterized in that: The sequence of the nucleic acid fragment is SEQ ID NO:

3.

5. Use of the odorant binding protein according to claim 1 in binding to the volatile (E)-β-caryophyllene induced by rice dwarf virus to release from rice.

6. A method for reducing the ability of black-tailed leafhoppers to sense volatile substances released by rice, characterized in that: The method is to reduce the content of the odor binding protein according to claim 1 in black-tailed leafhopper.

7. The method according to claim 6, wherein The volatile substance is (E)-β-caryophyllene.

8. The method according to claim 6, wherein The method is to reduce the content of the odorant binding protein with the amino acid sequence of SEQ ID NO: 1 in the black-tailed leafhopper by using dsRNA.

9. The method according to claim 8, wherein The dsRNA has a sequence of SEQ ID NO:

4.

10. The method according to claim 9, wherein The sequences of the synthetic primer pair for the dsRNA are SEQ ID NO: 5 and SEQ ID NO: 6.

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