Odorant Receptor Protein of Repellent Compounds against Spodoptera frugiperda and Its Application

Abstract

The present invention provides a repellent compound odorant receptor protein of Spodoptera frugiperda and its application. The amino acid sequence of the odorant receptor protein is shown as SEQ ID No.4. Screening for repellent compounds using the repellent compound odorant receptor protein of the present invention can achieve the purpose of saving time, space, manpower and material resources, and improving the screening efficiency. This will provide more options for reducing the damage of Spodoptera frugiperda to farm crops, thereby achieving the purpose of green prevention and control of major agricultural pests.

Description

Technical Field

[0001] The present invention relates to the fields of proteins and nucleic acids, and particularly to the application of odorant receptor proteins in screening for repellent compounds. Background Art

[0002] The fall armyworm (Spodoptera frugiperda) is a highly destructive agricultural pest worldwide, belonging to the genus Spodoptera of the family Noctuidae in the order Lepidoptera. This species is native to the tropical and subtropical regions of the Americas. Due to its wide range of host plants, strong migratory ability, and high fecundity per female, it has rapidly spread in the past decade and has become a major transboundary migratory agricultural pest under global alert by the Food and Agriculture Organization of the United Nations.

[0003] Insects can use their sensitive olfactory systems to sense specific compounds in the environment and make corresponding behavioral responses. Screening and identifying highly efficient compounds that can repel or attract the fall armyworm will provide more options for the green control of the fall armyworm. Screening odor compounds using odorant receptor proteins has the advantages of significantly reducing the workload and shortening the screening cycle.

[0004] Therefore, exploring the odorant receptor proteins of insects has important application value. Summary of the Invention

[0005] One aspect of the present invention provides the SfruORV odorant receptor protein, and the amino acid sequence of the SfruORV odorant receptor protein is as shown in SEQ ID No. 4.

[0006] Another aspect of the present invention provides the nucleic acid encoding the SfruORV odorant receptor protein as described in one aspect of the present invention.

[0007] In a specific embodiment, the base sequence of the nucleic acid is as shown in SEQ ID No. 3.

[0008] A third aspect of the present invention provides the application of the SfruORV odorant receptor protein as described in one aspect of the present invention as a receptor protein for fall armyworm repellent compounds or the nucleic acid as described in claim 2 as a receptor gene for fall armyworm repellent compounds.

[0009] In a specific embodiment, the SfruORV odorant receptor protein or the nucleic acid is used for screening fall armyworm repellent compounds.

[0010] A fourth aspect of the present invention provides a method for screening fall armyworm repellent compounds using the SfruORV odorant receptor protein, which includes the following steps:

[0011] 1) Inject the cRNA solution into Xenopus oocytes to obtain Xenopus oocytes injected with cRNA, where the cRNA is the cRNA of the Spodoptera frugiperda SfruORV gene and the cRNA of the Spodoptera frugiperda SfruOrco gene;

[0012] 2) Use a two-electrode voltage clamp to identify the response of the Xenopus oocytes injected with cRNA to the odorant compound to be tested.

[0013] If the Xenopus oocytes injected with cRNA are activated by the odorant compound to be tested, then test whether the wild-type Spodoptera frugiperda shows an avoidance behavior towards the odorant compound to be tested. If the wild-type Spodoptera frugiperda shows an avoidance behavior, then determine the odorant compound to be tested as an avoidance compound;

[0014] If the Xenopus oocytes injected with cRNA are not activated by the odorant compound to be tested, then abandon the test on whether the wild-type Spodoptera frugiperda shows an avoidance behavior towards the odorant compound to be tested.

[0015] In a specific embodiment, in step 1), the mass ratio of the cRNA of the Spodoptera frugiperda SfruORV gene to the cRNA of the Spodoptera frugiperda SfruOrco gene is 1:1.5 to 1.5:1.

[0016] In a specific embodiment, the mass ratio of the cRNA of the Spodoptera frugiperda SfruORV gene to the cRNA of the Spodoptera frugiperda SfruOrco gene is 1:1.

[0017] A compound that can bind to an odorant receptor protein is called a ligand of this receptor protein. After the odorant receptor protein binds to the ligand, the ion channel of the receptor will open, and external cations will enter the cell, thereby converting the chemical signal into an electrical signal. However, only when the entering cations exceed a threshold can the neuron be activated. The greater the response of the odorant receptor protein to the ligand, the more cations enter the cell, and the stronger the converted electrical signal. After the neuron is activated, the electrical signal is conducted downstream along the axon and finally reaches the central nervous system, causing a corresponding behavioral response. In the present invention, octanal has the largest electrophysiological response value to this odorant receptor protein, and it can cause the Spodoptera frugiperda to show an avoidance behavioral response. Therefore, the response value for screening avoidance compounds is set to at least 0.6 times the response value of octanal or more; or further, it can be set to 0.8 times the response value of octanal or more; or still further, it can be set to 1 times the response value of octanal or more.

[0018] In a specific embodiment, in step 2), taking the electrophysiological response of the Xenopus oocytes to octanal as a positive control, when the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested ≥ 0.6 times the electrophysiological response value to octanal, then test whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested; when the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested < 0.6 times the electrophysiological response value to octanal, then abandon the test on whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested.

[0019] In a specific embodiment, in step 2), when the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested ≥ 0.8 times the electrophysiological response value to octanal, especially when the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested ≥ 1 times the electrophysiological response value to octanal, then test whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested; when the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested < 0.8 times the electrophysiological response value to octanal, then abandon the test on whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested.

[0020] In a specific embodiment, in step 2), the steps of testing whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested are as follows:

[0021] a) Dissolve the odor compound to be tested with a solvent to obtain an odor compound solution to be tested; use the solvent as a negative control;

[0022] b) Use a Y-tube to test the wild-type Spodoptera frugiperda against the odor compound to be tested;

[0023] c) When the number of wild-type Spodoptera frugiperda that choose the negative control is significantly greater than the number that choose the odor compound to be tested, then the odor compound to be tested is considered an avoidance compound.

[0024] In a specific embodiment, cRNA is synthesized through the following steps:

[0025] I) Obtain the total RNA of the antennae of adult Spodoptera frugiperda;

[0026] II) Synthesize the first-strand cDNA using the total RNA as a template;

[0027] III) Using the first-strand cDNA as a template, perform PCR amplification on the SfruORV gene, ligate the PCR product of the SfruORV gene to a cloning vector to obtain a cloning vector containing the SfruORV gene, and then cut the SfruORV gene from the cloning vector and ligate it to an expression vector to obtain an expression vector containing the SfruORV gene; or directly ligate the PCR product of the SfruORV gene to an expression vector to obtain an expression vector containing the SfruORV gene;

[0028] IV) Linearize the expression vector containing the SfruORV gene (it can be confirmed by taking 5 μl for electrophoresis that the linearization is complete) to obtain a linearized expression vector, and purify the linearized expression vector;

[0029] V) Synthesize cRNA using the purified linearized expression vector as a template to obtain the cRNA of the SfruORV gene;

[0030] VI) Prepare the cRNA of the SfruOrco gene using the same operating steps as in I) to V) above, except that the upstream primer and downstream primer used in step III) are the primers of the SfruOrco gene.

[0031] In a specific embodiment, the Xenopus oocytes injected with cRNA are cultured in a Xenopus oocyte medium in an 18 °C incubator for 3 to 4 days and then subjected to two-electrode voltage clamp detection.

[0032] The fifth aspect of the present invention provides the application of octanal in repelling Spodoptera frugiperda.

[0033] In a specific embodiment, the dosage range of octanal is 10 μg to 100 μg.

[0034] Advantages of the present invention:

[0035] When directly testing whether wild-type Spodoptera frugiperda has a repellent behavior towards the odor compound to be tested, especially when the odor compound to be tested contains hundreds or thousands of kinds, either parallel determination will occupy a large amount of space, or sequential testing will consume a large amount of time. The SfruORV gene obtained from Spodoptera frugiperda in the present invention is the receptor gene of the repellent compound of Spodoptera frugiperda. Then, based on the function of the SfruORV gene, compounds that have an electrophysiological response to it can be screened in advance using this receptor, narrowing the range of compounds to be screened, and then performing behavioral tests, which can achieve the purpose of saving time, space, manpower and material resources, and improving the screening efficiency. This will provide more options for reducing the damage of Spodoptera frugiperda to farm crops, thereby achieving the purpose of green prevention and control of major agricultural pests. Brief Description of the Drawings

[0036] Figure 1 Response spectrum of Xenopus oocytes co-expressing the SfruORV gene and the SfruOrco gene of Spodoptera frugiperda to different odor compounds.

[0037] Figure 2 Two-electrode voltage clamp recording of the response trajectory of Xenopus oocytes co-expressing the SfruORV gene and the SfruOrco gene of Spodoptera frugiperda to octanal at different doses.

[0038] Figure 3 Concentration gradient curve of the response of Xenopus oocytes co-expressing the SfruORV gene and the SfruOrco gene to octanal activation. Among them, the average value of the maximum response was normalized to 1, and the error bars were represented by SEM (n = 6).

[0039] Figure 4 Behavioral responses of female Spodoptera frugiperda to octanal at different doses. Among them, the abscissa values are used to represent the percentage of female Spodoptera frugiperda's choice between the negative control and octanal. N.S. indicates no significant difference (P > 0.05), and * indicates a significant difference (P < 0.05).

[0040] Figure 5 Behavioral responses of male Spodoptera frugiperda to octanal at different doses. Among them, the abscissa values are used to represent the percentage of male Spodoptera frugiperda's choice between the negative control and octanal. N.S. indicates no significant difference (P > 0.05), and * indicates a significant difference (P < 0.05). Detailed implementation mode

[0041] The above content of the present invention will be further described in detail below in the form of preferred implementation cases, but it does not constitute a limitation to the present invention.

[0042] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0043] Example 1

[0044] 1. Rearing and tissue collection of wild-type Spodoptera frugiperda

[0045] Wild-type Spodoptera frugiperda larvae were reared with conventional artificial feed, the indoor rearing temperature was 26 ± 1°C, the light was 16:8 (L:D), and the relative humidity was 55 ± 5%.

[0046] Newly emerged male and female insects were reared separately in different insect cages, and the adults were fed with 10% honey water. The indoor rearing temperature was 26 ± 1°C, the light was 16:8 (L:D), and the relative humidity was 55 ± 5%.

[0047] Adult antennae 1 to 3 days after eclosion were immediately frozen in liquid nitrogen and then stored at -70°C.

[0048] 2. RNA extraction from antennae of wild-type Spodoptera frugiperda

[0049] Before the experiment, the homogenizer needs to be washed in an ultrasonic cleaner in advance, wrapped with tin foil, and sterilized at 180°C for 3 h in an oven. The entire RNA extraction process is carried out under RNase-free conditions. Wear masks and gloves throughout the process. All pipette tips and centrifuge tubes are RNase-free. Extract RNA using the Trizol method. Finally, dissolve the extracted and dried RNA in 20 to 50 μL of RNase-free water (determined according to the precipitation amount) to obtain an RNA solution.

[0050] 3. Synthesis of the first-strand cDNA

[0051] The entire reverse transcription process is ensured to be carried out under RNase-free conditions. Using the extracted RNA solution as a template, synthesize the first-strand cDNA according to the instructions of the RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA) to obtain the first-strand cDNA product.

[0052] 4. Cloning of the SfruORV gene and SfruOrco gene of Spodoptera frugiperda

[0053] The nucleotide sequences of the SfruORV gene and SfruOrco gene are both from the antenna transcriptome of Spodoptera frugiperda. Using the cDNA obtained in Section 3 as a template, use primers SfruORV-F1 (SEQ ID No.1) and SfruORV-R1 (SEQ ID No.2) to perform PCR amplification on the SfruORV gene (SEQ ID No.3) of Spodoptera frugiperda. The amino acid sequence encoded by the SfruORV gene is shown as (SEQ ID No.4); use primers SfruOrco-F1 (SEQ ID No.5) and SfruOrco-R1 (SEQ ID No.6) to amplify the SfruOrco gene (SEQ ID No.7) of Spodoptera frugiperda. The amino acid sequence encoded by the SfruOrco gene is shown as SEQ ID No.8. The co-expression of the SfruORV gene and SfruOrco gene of Spodoptera frugiperda enables the protein encoded by the SfruORV gene to have a function.

[0054] The purified SfruORV and SfruOrco PCR products were ligated into the PCS2+ expression vector via EcoR I and Xhol I restriction sites, and then transformed into Trans1-T1 competent cells (TransGen Biotech), finally obtaining the positive recombinant plasmid PCS2+-ORV cloned with the SfruORV gene and the positive recombinant plasmid PCS2+-Orco cloned with the SfruOrco gene.

[0055] 5. Expression of odorant receptors in the Xenopus oocyte heterologous expression system

[0056] The recombinant plasmid PCS2+-ORV was digested with Not I endonuclease for single-enzyme digestion linearization. After detecting complete digestion by agarose gel electrophoresis, phenol-chloroform extraction was performed, and finally the precipitate was dissolved in RNase-free water to obtain the PCS2+-ORV linearized plasmid solution. The band homogeneity was detected by agarose gel electrophoresis, and then the concentration was measured. Using the mMESSAGE mMACHINE sp6 kit (Thermo Fisher Scientific, USA), SfruORV cRNA was synthesized with the PCS2+-ORV linearized plasmid as the template. The synthesized SfruORV cRNA was dissolved in RNase-free water, the concentration was measured, and then the SfruORV cRNA solution was diluted to 2000 ng / μL.

[0057] The recombinant plasmid PCS2+-Orco was linearized with Not I endonuclease, and then SfruOrco cRNA was synthesized with the linearized PCS2+-Orco plasmid as the template. The synthesized SfruOrco cRNA was dissolved in RNase-free water, the concentration was measured, and then the SfruOrco cRNA solution was diluted to 2000 ng / μL. The operation was referred to the synthesis of SfruORV cRNA.

[0058] The SfruORV cRNA dilution and the SfruOrco cRNA dilution were mixed in equal amounts to obtain a mixed solution. Healthy and mature Xenopus oocytes were selected and 27.6 nL of the mixed solution was injected using a microinjector (Nanoliter2000, USA), and then cultured in an incubator at 18 °C for 3 days.

[0059] 6. Screening of odorant compounds by two-electrode voltage clamp

[0060] 6.1 Prepare a 1 mol / L stock solution of each of the selected 62 volatile odor compounds in dimethyl sulfoxide (DMSO), and then prepare a perfusion solution with a final concentration of 10 -4 mol / L using 1×Ringer's solution (9.6 mM NaCl, 0.2 mM KCl, 0.5 mM MgCl₂·6H₂O, 0.5 mM HEPES, 0.6 mM CaCl₂, pH 7.6); among them, the 62 volatile odor compounds are, in sequence: butanol, pentanol, hexanol, heptanol, octanol, isobutanol, 2-methyl-butanol, isopentanol, 1-octen-3-ol, cis-2-hexenol, trans-2-hexenol, cis-3-hexenol, trans-3-hexenol, benzyl alcohol, 2-phenylethanol, geraniol, linalool, myrtenol, nerolidol, β-citronellol, S-cis-verbenol, (-)-borneol, (+)-borneol, butyraldehyde, hexanal, heptanal, octanal, nonanal, (E)-2-hexenal, benzaldehyde, phenylacetaldehyde, salicylaldehyde, citral, citronellal, cinnamaldehyde, hexyl acetate, trans-2-hexenyl acetate, cis-3-hexenyl acetate, geranyl acetate, methyl benzoate, methyl salicylate, methyl phenylacetate, methyl jasmonate, ocimene, myrcene, (-)-trans-caryophyllene, α-pinene, (-)-β-pinene, γ-terpinene, camphene, sabinene, E-β-farnesene, farnesene, (S)-(-)-limonene, β-ionone, verbenone, acetophenone, jasmonone, camphor, indole, eugenol, methyl eugenol.

[0061] 6.2 Use a two-electrode voltage clamp system, the OC-725C oocyte clamp (Warner Instruments, USA), to measure the compensatory current values generated by the Xenopus oocytes co-expressing the SfruORV gene and the SfruOrco gene in the above 62 volatile odor compounds in Section 5. Use the OC-725C two-electrode voltage clamp to record the responses of the oocytes to the volatile odor compounds, and use the amplifier Digidata 1440A combined with the software pCLAMP 10.0 (Axon Instruments Inc., California, USA) to record the response values and analyze the data. The data are expressed as mean ± standard error of the mean (SEM) (n = 6), and the results are shown in Figure 1 .

[0062] The results show that at 10 -4At the concentration of M, the Spodoptera frugiperda SfruORV protein showed the strongest response to octanal stimulation, with an average response value of 1565 nA; the response to nonanal ranked second, with an average response value of 508 nA, which was less than 1 / 3 of the octanal response value; weak responses were also observed to heptanol, octanol, 1-octen-3-ol, geraniol, linalool, heptanal, citral, hexyl acetate, and leaf acetate, with average response values of 120 nA, 87 nA, 133 nA, 110 nA, 168 nA, 242 nA, 167 nA, 120 nA, and 140 nA respectively; no response was observed to the other tested volatile odor compounds.

[0063] 6.3 Dose-response analysis of oocytes expressing SfruORV gene and SfruOrco gene to octanal

[0064] 1 mol / L octanal was serially diluted to final concentrations of 1.0×10 -8 、1.0×10 -7 、1.0×10 -6 、1.0×10 -5 、1.0×10 -4 、1.0×10 -3 mol / L. Two-electrode voltage clamp was used to record the dose-response traces of Xenopus oocytes co-expressing SfruORV gene and SfruOrco gene to the above gradient concentrations of octanal (n = 6). As Figure 2 shown, the response value increased with the increase of concentration. The software GraphPad Prism 5.0 was used to fit the response curve of response value to dose as Figure 3 shown, and the EC 50 value of octanal activating SfruORV protein was calculated to be 6.159×10 -6 mol / L.

[0065] Example 2

[0066] Preparation of repellent:

[0067] Octanal was used as the solvent with n-hexane to prepare a stock solution with a concentration of 100 μg / μL.

[0068] 100 μL of the octanal stock solution was taken and added to 900 μL of n-hexane, and mixed evenly to obtain octanal solution 1# with a concentration of 10 μg / μL.

[0069] 100 μL of the octanal solution with a concentration of 10 μg / μL was taken and added to 900 μL of n-hexane, and mixed evenly to obtain octanal solution 2# with a concentration of 1 μg / μL.

[0070] Absorb 100 μL of octanal solution with a concentration of 1 μg / μL, add 900 μL of n-hexane, mix well, and obtain octanal solution 3# with a concentration of 100 ng / μL.

[0071] Comparative Example 1

[0072] Use the solvent n-hexane as the negative control 4#.

[0073] Example 3

[0074] Y-tube experiment

[0075] Use the Y-tube to measure the behavioral selection response of male and female adult Spodoptera frugiperda to octanal.

[0076] The Y-tube is a colorless and transparent glass tube with a diameter of about 2.2 cm. The main arm and the two side arms are both 14 cm long, and the angle between the two side arms is 90°.

[0077] 1) Before the test, starve the test insects for 16 h (only feed water).

[0078] 2) The test is carried out 2 h after entering the dark period. The indoor temperature is 26 ± 1 °C, the humidity is 55 ± 5%, and a red light lamp (light intensity about 26 Lux) that Spodoptera frugiperda cannot recognize is placed above the Y-tube to provide illumination.

[0079] 3) Use a QC-1B air sampling instrument as the air flow power system (Beijing Institute of Labor Protection). Before the experiment starts, adjust the air flow rates of the two side arms to be the same, both 0.8 L / min. Among them, the gas flowing out of the air sampling instrument first passes through activated carbon filtration, then passes through a humidifying bottle for humidification, and finally reaches the two side arms of the Y-tube through a flow meter.

[0080] 4) Drop 10 μL of 10 μg / μl octanal (1#) and 10 μL of n-hexane (4#) into 1 mL of 10% honey water respectively, mix well, and then add the honey water containing octanal and n-hexane to two small cotton balls of about 1 cm 3 respectively. Place the cotton balls in the air for 10 min to allow the solvent to volatilize, and obtain octanal small cotton ball 1# and negative control small cotton ball 4# respectively. Then put the two cotton balls into the two side arms of the Y-tube respectively.

[0081] 5) Put a single adult Spodoptera frugiperda into the main arm of the Y-tube, turn on the air sampling instrument, and make the gas flow rates of the two side arms of the Y-tube both 0.8 L / min. Record the selection results of the test insects within 5 min. If the test insects reach the small cotton ball and feed within 5 min, it is regarded as making a choice. If the test insects stay in the main arm or walk back and forth, it is regarded as not making a choice.

[0082] 6) The number of male and female Spodoptera frugiperda adults tested separately was ≥ 32, with 1 tested each time. After every 3 tests, the side arms of the small cotton ball 1# of octanal and the small cotton ball 4# of negative control were swapped to eliminate the influence of directional effects on Spodoptera frugiperda. After every 10 tests, a new Y-tube was used. After the experiment, the Y-tube was cleaned and rinsed with absolute ethanol, and then dried in an oven at 150 °C for 2 h.

[0083] The 1# octanal solution at 10 μg / μl was replaced with the 2# octanal solution at 1 μg / μL or the 3# octanal solution at 100 ng / μL for separate tests.

[0084] The results were statistically analyzed and plotted. Among them, the behavioral response results of female Spodoptera frugiperda were as Figure 4 shown; the behavioral response results of male Spodoptera frugiperda were as Figure 5 shown.

[0085] The results showed that compared with the negative control, octanal at doses of 10 μg and 100 μg could produce a significant repellent effect on female Spodoptera frugiperda; while at 1 μg, octanal had no significant repellent effect on female Spodoptera frugiperda. Similarly, octanal at doses of 10 μg and 100 μg could produce a significant repellent effect on male Spodoptera frugiperda; while at 1 μg, octanal had no significant repellent effect on male Spodoptera frugiperda.

Claims

1. SfruORV odorant receptor protein, the amino acid sequence of the SfruORV odorant receptor protein is shown in SEQ ID No.

4.

2. Nucleic acid encoding the SfruORV odorant receptor protein as claimed in claim 1.

3. The nucleic acid according to claim 2, wherein The base sequence of the nucleic acid is shown in SEQ ID No.

3.

4. Use of the SfruORV odorant receptor protein as claimed in claim 1 or the nucleic acid as claimed in claim 2 or 3 in screening for Spodoptera frugiperda repellent compounds.

5. Method for screening Spodoptera frugiperda repellent compounds using the SfruORV odorant receptor protein, which comprises the following steps: 1) Inject the cRNA solution into Xenopus oocytes to obtain Xenopus oocytes injected with cRNA, where The cRNA is cRNA of the Spodoptera frugiperda SfruORV gene and cRNA of the Spodoptera frugiperda SfruOrco gene; wherein, the sequence of the SfruORV gene is shown in SEQ ID No. 3, and the sequence of the SfruOrco gene is shown in SEQ ID No. 7; 2) Identifying the response of the Xenopus oocytes injected with the cRNA to the odorant compound to be tested using a two-electrode voltage clamp. If the Xenopus oocytes injected with the cRNA are activated by the odorant compound to be tested, then testing whether the wild-type Spodoptera frugiperda exhibits a repellent behavior towards the odorant compound to be tested. If the wild-type Spodoptera frugiperda exhibits a repellent behavior, then determining the odorant compound to be tested as a repellent compound. If the Xenopus oocytes injected with the cRNA are not activated by the odorant compound to be tested, then abandoning the test on whether the wild-type Spodoptera frugiperda exhibits a repellent behavior towards the odorant compound to be tested.

6. The method according to claim 5, wherein In step 1), the mass ratio of the cRNA of the Spodoptera frugiperda SfruORV gene to the cRNA of the Spodoptera frugiperda SfruOrco gene is 1:1.5 to 1.5:

1.

7. The method according to claim 6, characterized in that, The mass ratio of the cRNA of the Spodoptera frugiperda SfruORV gene to the cRNA of the Spodoptera frugiperda SfruOrco gene is 1:

1.

8. The method according to claim 5, characterized in that, In step 2), using the electrophysiological response of the Xenopus oocytes to octanal as a positive control. When the electrophysiological response value of the Xenopus oocytes to the odorant compound to be tested is ≥ 0.6 times the electrophysiological response value to octanal, then testing whether the wild-type Spodoptera frugiperda exhibits a repellent behavior towards the odorant compound to be tested; when the electrophysiological response value of the Xenopus oocytes to the odorant compound to be tested < 0.6 times the electrophysiological response value to octanal, then abandoning the test on whether the wild-type Spodoptera frugiperda exhibits a repellent behavior towards the odorant compound to be tested.

9. The method according to claim 5, wherein In step 2), the electrophysiological response of the Xenopus oocytes to octanal is used as a positive control. When the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested ≥ 0.8 times the electrophysiological response value to octanal, then test whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested; when the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested < 0.8 times the electrophysiological response value to octanal, then abandon the test on whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested.

10. The method according to claim 5, wherein In step 2), the electrophysiological response of the Xenopus oocytes to octanal is used as a positive control. When the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested ≥ 1 times the electrophysiological response value to octanal, then test whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested; When the electrophysiological response value of the Xenopus oocytes to the odor compound to be tested < 1 times the electrophysiological response value to octanal, then abandon the test on whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested.

11. The method according to claim 5, characterized in that In step 2), the steps for testing whether the wild-type Spodoptera frugiperda has an avoidance behavior towards the odor compound to be tested are as follows: a) Dissolve the odor compound to be tested with a solvent to obtain an odor compound solution to be tested; use the solvent as a negative control; b) Use a Y-tube to test the wild-type Spodoptera frugiperda against the odor compound to be tested; c) When the number of wild-type Spodoptera frugiperda that choose the negative control is significantly greater than the number that choose the odor compound to be tested, then the odor compound to be tested is considered an avoidance compound.

12. The method according to claim 5, characterized in that, cRNA is synthesized through the following steps: I) Obtain the total RNA of the antennae of adult Spodoptera frugiperda; II) Synthesize the first-strand cDNA using the total RNA as a template; III) Using the first-strand cDNA as a template, perform PCR amplification on the SfruORV gene, ligate the PCR product of the SfruORV gene to a cloning vector to obtain a cloning vector containing the SfruORV gene, then cut the SfruORV gene from the cloning vector and ligate it to an expression vector to obtain an expression vector containing the SfruORV gene; or directly ligate the PCR product of the SfruORV gene to an expression vector to obtain an expression vector containing the SfruORV gene; IV) Linearize the expression vector containing the SfruORV gene to obtain a linearized expression vector, and purify the linearized expression vector; V) Synthesize cRNA using the purified linearized expression vector as a template, thereby obtaining the cRNA of the SfruORV gene; VI) Prepare the cRNA of the SfruOrco gene using the same operating steps as in I) to V) above, except that the upstream and downstream primers used in step III) are the primers of the SfruOrco gene.

Images