Olfactory receptor of bactrocera dorsalis for methyl eugenol and use thereof

Abstract

Provided is the use of olfactory receptor BdorOR94bl as a target in the control of Bactrocera dorsalis. By means of functional genomics, electrophysiology, behavioristics, and traditional chemical ecology, the role of methyl eugenol in the mating behavior of Bactrocera dorsalis and the key olfactory receptor for male flies to recognize methyl eugenol are comprehensively analyzed. For the first time, the key olfactory receptor BdorOR94bl of Bactrocera dorsalis for recognizing methyl eugenol is identified, and the genomic coding region sequence and transcribed amino acid sequence information of the key olfactory receptor BdorOR94bl is obtained, which not only lays an important foundation for the subsequent screening of more stable and efficient structural analogs by taking the gene as a target, but also provides a foundation for the development of nucleic acid pesticides.

Description

Bactrocera minima olfactory receptor for methyl eugenol and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Bactrocera minima olfactory receptor for methyl eugenol and application thereof. BACKGROUND

[0002] Methyl eugenol (ME, cas:93-15-12) is a phenylpropanoid compound, which was first discovered in citronella oil and has an attractive activity to fruit flies. In subsequent long-term trapping experiments, it was found that ME has an attractive effect on a variety of fruit flies of the genus Bactrocera, especially Bactrocera minima. In terms of the attractive effect of Bactrocera minima, ME has the characteristics of male specificity, long-distance attraction, trace attraction, strong feeding, and high concentration without repelling. After being fed, ME is converted into a derivative in the rectal gland of Bactrocera minima, and serves as a component of sex pheromone, thereby improving their mating success rate. The strong attractive characteristics enable ME to play an important role in the population control of Bactrocera minima. In the field, ME is combined with various types of attractants, food lures, and insecticides to achieve good results in the prevention and control of Bactrocera minima, and a male annihilation technique (MAT) based on ME is formed.

[0003] Although there have been many reports on the attractiveness of ME and the behavioral function of its derivatives, the molecular mechanisms behind these effects and their ecological significance are still unclear. First, the role of ME derivatives in improving mating success has not been fully understood, especially the advantages in the classic courtship aggregation behavior of male insects, i.e., lek behavior, and the benefits to female insects. Second, the key olfactory receptors for recognizing ME have not been identified, and the chemical communication mechanism has not been elucidated. In addition, although ME has achieved certain results in the prevention and control of Bactrocera minima, it still has defects, such as being proved to have a certain cell carcinogenicity. Therefore, more detailed research on the ecological significance of ME and its olfactory mechanism is still needed, and more safe and effective attractants need to be explored to improve the trapping and killing technique, such as finding better structural analogs. SUMMARY

[0004] The present application comprehensively analyzes the role of ME in the mating behavior of Bactrocera minima and the key olfactory receptors of male insects for recognizing ME by using functional genomics, electrophysiology, behavior, and traditional chemical ecology methods. The key receptors of Bactrocera minima for sensing methyl eugenol and their detailed gene sequence information are determined; a non-sensitive strain of methyl eugenol is constructed through the key receptors; the key ecological significance of lek is determined; and the value of feeding ME in the reproduction of Bactrocera minima is determined.

[0005] The first aspect of the present application aims to provide the use of odorant receptor BdorOR94b1 as a target.

[0006] The second aspect of the present application aims to provide the use of BdorOR94b1 inhibitor in preventing and treating B. dorsalis and / or preparing a product for preventing and treating B. dorsalis.

[0007] The third aspect of the present application aims to provide a method for regulating the sensitivity of B. dorsalis to methyl eugenol.

[0008] The fourth aspect of the present application aims to provide a method for constructing a methyl eugenol-insensitive strain.

[0009] To achieve the above-mentioned objects, the technical solutions adopted by the present application are as follows:

[0010] The first aspect of the present application provides the use of odorant receptor BdorOR94b1 as a target in at least one of a1) to a5):

[0011] a1) preventing and treating B. dorsalis;

[0012] a2) screening or preparing a product for preventing and treating B. dorsalis;

[0013] a3) regulating the lek behavior of B. dorsalis;

[0014] a4) preparing a lek behavior regulator of B. dorsalis;

[0015] a5) regulating the sensitivity of B. dorsalis to a stimulating odor molecule.

[0016] The term "lek behavior" refers to the male aggregation behavior "mating rally".

[0017] In some embodiments of the present application, odorant receptor BdorOR94b1 is used as a target to regulate the olfactory sensitivity of B. dorsalis to a stimulating odor molecule.

[0018] In some embodiments of the present application, the stimulating odor molecule comprises a plant odor and derivatives thereof or an insect sex pheromone and analogs thereof.

[0019] In some embodiments of the present application, the stimulating odor molecule comprises methyl eugenol or a derivative thereof.

[0020] In some embodiments of the present application, the derivative comprises trans-coniferyl alcohol (ECF) and 2-Allyl-4,5-dimethoxy phenol (DMP).

[0021] In some embodiments of the present application, the nucleotide sequence of the odorant receptor BdorOR94b1 is shown as SEQ ID NO: 31, and the amino acid sequence thereof is shown as SEQ ID NO: 32.

[0022] In a second aspect of the present application, there is provided a use of a BdorOR94b1 inhibitor in preventing and / or treating B. dorsalis and / or in preparing a product for preventing and / or treating B. dorsalis.

[0023] In some embodiments of the present application, the BdorOR94b1 inhibitor is at least one of a substance that inhibits the activity of BdorOR94b1, a substance that degrades BdorOR94b1, or a substance that reduces the expression level of BdorOR94b1.

[0024] In some embodiments of the present application, the substance that reduces the expression level of BdorOR94b1 comprises at least one of b1) to b13):

[0025] b1) at least one of siRNA, dsRNA, miRNA, ribozyme, shRNA, CRISPR / Cas system targeting BdorOR94b1;

[0026] b2) a nucleic acid molecule encoding b1);

[0027] b3) an expression cassette comprising the nucleic acid molecule of b2);

[0028] b4) a recombinant vector comprising the nucleic acid molecule of b2);

[0029] b5) a recombinant vector comprising the expression cassette of b3);

[0030] b6) a recombinant cell comprising the nucleic acid molecule of b2);

[0031] b7) a recombinant cell comprising the expression cassette of b3);

[0032] b8) a recombinant cell comprising the recombinant vector of b4);

[0033] b9) a recombinant cell comprising the recombinant vector of b5);

[0034] b10) a recombinant microorganism comprising the nucleic acid molecule of b2);

[0035] b11) a recombinant microorganism comprising the expression cassette of b3);

[0036] b12) a recombinant microorganism comprising the vector of b4);

[0037] b13) a recombinant microorganism comprising the vector of b5).

[0038] In some embodiments of the present application, the CRISPR / Cas system comprises an sgRNA targeting BdorOR94b1 or a biological material related to sgRNA;

[0039] The biological material related to sgRNA comprises at least one of c1)-c12):

[0040] c1) a nucleic acid molecule encoding sgRNA;

[0041] c2) an expression cassette containing the nucleic acid molecule of c1);

[0042] c3) a recombinant vector containing the nucleic acid molecule of c1);

[0043] c4) a recombinant vector containing the expression cassette of c2);

[0044] c5) a recombinant cell containing the nucleic acid molecule of c1);

[0045] c6) a recombinant cell containing the expression cassette of c2);

[0046] c7) a recombinant cell containing the recombinant vector of c3);

[0047] c8) a recombinant cell containing the recombinant vector of c4);

[0048] c9) a recombinant microorganism containing the nucleic acid molecule of c1);

[0049] c10) a recombinant microorganism containing the expression cassette of c2);

[0050] c11) a recombinant microorganism containing the recombinant vector of c3);

[0051] c12) a recombinant microorganism containing the recombinant vector of c4).

[0052] In some embodiments of the present application, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 23-SEQ ID NO: 26.

[0053] In some embodiments of the present application, the CRISPR / Cas system further comprises a Cas protein and / or a biological material related to Cas protein;

[0054] The biological material related to Cas protein comprises at least one of d1)-d12):

[0055] d1) a nucleic acid molecule encoding Cas protein;

[0056] d2) an expression cassette containing the nucleic acid molecule of d1);

[0057] d3) a recombinant vector comprising the nucleic acid molecule of d1);

[0058] d4) a recombinant vector comprising the expression cassette of d2);

[0059] d5) a recombinant cell comprising the nucleic acid molecule of d1);

[0060] d6) a recombinant cell comprising the expression cassette of d2);

[0061] d7) a recombinant cell comprising the recombinant vector of d3);

[0062] d8) a recombinant cell comprising the recombinant vector of d4);

[0063] d9) a recombinant microorganism comprising the nucleic acid molecule of d1);

[0064] d10) a recombinant microorganism comprising the expression cassette of d2);

[0065] d11) a recombinant microorganism comprising the recombinant vector of d3);

[0066] d12) a recombinant microorganism comprising the recombinant vector of d4).

[0067] In some embodiments of the present application, the expression vector comprises an expression cassette (expression framework).

[0068] In some embodiments of the present application, the recombinant vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, a fosmid, an artificial chromosome.

[0069] In some embodiments of the present application, the plasmid vector can be an optional plasmid, the viral vector can be an optional virus, and the cell vector does not include reproductive material.

[0070] In some embodiments of the present application, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d and Cas14, including any recombinant variants thereof, in particular selected from Cas9, including any recombinant variants thereof.

[0071] In a third aspect, the present application provides a method for modulating the susceptibility of B. dorsalis to methyl eugenol, comprising the step of knocking down / knocking out the odorant receptor BdorOR94b1 of B. dorsalis using a gene editing technique.

[0072] In some embodiments of the present application, the gene editing technology comprises ZFNs, TALENs or CRISPR / Cas9 technology.

[0073] In some embodiments of the present application, the CRISPR / Cas9 technology is used to knock down / knock out the odorant receptor BdorOR94b1, comprising the following steps: injecting sgRNA and Cas9 protein into B. dorsalis embryos.

[0074] In some embodiments of the present application, the nucleotide sequence of the sgRNA at the sgRNA target site of the exon of the BdorOR94b1 gene is shown in SEQ ID NO:23-SEQ ID NO:26.

[0075] In some embodiments of the present application, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:23-SEQ ID NO:26.

[0076] In a fourth aspect of the present application, a method for constructing a methyl eugenol-insensitive strain is provided, comprising the following steps: genetically editing B. dorsalis by the method of the third aspect of the present application, then crossing the G0 generation individuals surviving from the embryos after injection to adults with wild type individuals, and passing generations to screen mutants, i.e. obtaining a methyl eugenol-insensitive strain.

[0077] In some embodiments of the present application, the specific method for screening mutants is as follows: the G0 generation individuals surviving from the embryos after injection to adults are detected for gene editing, the G1 generation individuals produced by crossing the successfully edited G0 generation with wild type are reserved, the G1 generation is detected for gene editing, and the successfully edited G1 generation is crossed with wild type to produce G2 generation, the G2 generation individuals with consistent genotypes are self-crossed, and homozygous mutants can be screened in the G3 generation.

[0078] The present application has the following advantages:

[0079] The application comprehensively analyzes the role of ME in the mating behavior of B. dorsalis and the key olfactory receptors of male insects for recognizing ME by using functional genomics, electrophysiology, behavior and traditional chemical ecology methods. The key odor receptor BdorOR94b1 of B. dorsalis for recognizing methyl eugenol is identified for the first time, and the genomic coding region sequence and the amino acid sequence information of the transcription are obtained. This not only provides an important basis for subsequent screening of more stable and efficient structural analogs taking the gene as a target, but also provides a basis for the development of nucleic acid pesticides. It provides reliable molecular targets for developing new and efficient female or bisexual trapping agents of B. dorsalis, and can provide a new and feasible way for the prevention and control of B. dorsalis. This has great significance for understanding the molecular mechanism and ecological value of plant secondary metabolites in regulating insect sexual behavior, and also has important value for screening more stable and efficient structural analogs taking the gene as a target.

[0080] The method provided by the application can successfully obtain an ME-insensitive strain, which can be applied to the prevention and control system of the sterile insect technique (SIT) of B. dorsalis, so as to achieve the prevention and control of B. dorsalis.

[0081] The ecological value of ME to B. dorsalis is determined. The application compares the attraction difference of male artificial lek groups in the fed / unfed state to female insects. It is found that the attraction of male insects after feeding to female insects is obviously stronger than that of unfed male insects, and it is proved that the difference in attraction is influenced by the rectal gland of B. dorsalis. GC-MS analysis of the rectal gland extract shows that ME can derive 4,5-dimethoxy-2-(2-propenyl) phenol (DMP) and trans-coniferyl alcohol (ECF) in the body after feeding. Among them, ECF is a key compound for enhancing the attraction to female insects.

[0082] The significance of the classical courtship behavior of B. dorsalis, lek, on population reproduction is determined. The differences in courtship and mating behaviors of B. dorsalis in groups and individuals are compared to determine the advantages of group effect on the reproduction of B. dorsalis. The results show that courtship and mating behaviors in groups are more likely to occur than in individuals. To further study the role of lek behavior in offspring quality, the present application provides different growth conditions of male insects in groups, including normal, malnutrition, white eye and white pupa four different types. The selection of female insects is evaluated by competitive mating, and the quality of offspring of the four types of male insects is compared. It is found that in the lek group, female insects will prefer to select healthy male insects. And the number and quality of offspring produced by healthy male insects are better. These results prove the important role of lek behavior in initiating mating and providing healthy advantages for females, which helps to improve the quality and quantity of offspring of B. dorsalis. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 is that the olfactory receptors (ORs) expressed in the antennae mediate the attraction of ME to male B. dorsalis; wherein Figure 1a is a schematic diagram of three trap experiments; Figure 1b is a comparison of the trapping rates of ME to male and female B. dorsalis; Figure 1c is a comparison of the trapping rates of ME to WT and BdorOrco - / - male insects; Figure 1d is a comparison of the trapping rates of ME to WT and BdorIR8a - / - male insects; Figure 1e is a schematic diagram of a four-arm olfactometer; Figure 1f is a comparison of the tendency behaviors of WT male insects, WT male insects with removed maxillary palps, and WT male insects with removed antennae to ME, the time is 8:00-9:00, and the dose of ME is 10 μg; all the above behavior experiments N=5, 30 B. dorsalis in each repeat, the data results are expressed as mean ± standard error, the data difference analysis uses unpaired t test, * represents P<0.05, ** represents P<0.01, **** represents P<0.0001, and ns represents no significant difference.

[0084] Figure 2 is the construction of BdorOrco - / - , BdorIR8a - / - mutants and an example of a behavior testing device; wherein Figure 2a is a schematic diagram of the gene structure of BdorOrco guide RNA (sgRNA) target site position; Figure 2b is gDNA amplification and sgRNA synthesis of the target region of BdorOrco, wherein Figure 2b1 is gDNA amplification of the target region of BdorOrco, Figure 2b2 is PCR assembly of BdorOrco sgRNA in vitro transcription template, and Figure 2b3 is BdorOrco sgRNA in vitro transcription, purification product detection; Figure 2c is the sequence peak chart of the target region of wild type BdorOrco + / + ; Figure 2d is a BdorOrco+ / - Target region sequence peak plot; Fig. 2e is a homozygous mutant Bdor Orco - / - Target region sequence peak plot; Fig. 2f is a schematic diagram of Bdor IR8a gene structure and sgRNA target site position; Fig. 2g is Bdor IR8a target region gDNA amplification and sgRNA synthesis, in which, Fig. 2g1: Bdor IR8a target region gDNA amplification, Fig. 2g2: PCR assembly Bdor IR8a sgRNA in vitro transcription template, Fig. 2g3: Bdor IR8a sgRNA in vitro transcription, purified product detection; Fig. 2h is wild type Bdor IR8a + / + Target region sequence peak plot; Fig. 2i is wild type Bdor IR8a + / + and a heterozygous mutant Bdor IR8a + / - , a homozygous mutant Bdor IR8a - / - Target region amplification gel plot alignment; Fig. 2j is a homozygous mutant Bdor IR8a - / - Target region sequence peak plot; Fig. 2k is an example of a trap experiment, the left is a trap experiment device, and the right is a result statistics of a trap bottle; Fig. 1 is an example of a four-arm olfactometer behavior experiment, which respectively shows the distribution state of the real fly at 0, 1 and 5 minutes.

[0085] Figure 3 BdorOR94b1 is a specific receptor for methyl eugenol (ME); Figure 3a is a schematic diagram of the screening process of receptors based on the change of mRNA level expression after stimulation; Figure 3b is a comparison of the expression level of antennal olfactory receptors (ORs) before and after ME stimulation; Figure 3c is the time course of BdorOR94b1 expression level under ME stimulation, n = 3, data expressed as mean ± standard error, data analysis using one-way ANOVA, using Tukey's multiple comparison test (differences are indicated by different letters, a = 0.05); Figure 3d is the expression level of BdorOR94b1 in different peripheral sensory organs of male and female flies; Figure 3e is a schematic diagram of the heterologous expression system of Drosophila ab3A null neurons; Figure 3f is a representative single sensillum recording (SSR) response of Drosophila ab3A neurons expressing BdorOR94b1 to 2-heptanone, ethyl hexanoate and ME; Figure 3g is a quantitative analysis of the response of Drosophila ab3A receptors to ME with or without BdorOR94b1 expression, in the figure, "+" and "-" represent the presence and absence of OR22abGAL4 and UAS-BdorOR94b1, respectively, data results are expressed as mean ± standard error, N = 10-14 in each group. The differences were statistically analyzed using unpaired t test (**** represents P < 0.0001); Figure 3h is the SSR response of BdorOR94b1 to 64 compounds, 100 μg of each compound, N = 9; i is the dose response curve of BdorOR94b1 to ME, N = 9, data expressed as mean ± standard error.

[0086] Figure 4 shows the evaluation of ME-stimulated transcriptome data and supplementary data on the SSR response of transgenic Drosophila. Figure 4a is a box plot of gene expression distribution in the ME-stimulated transcriptome samples, showing the FPKM values ​​of gene expression levels in all RNA samples. Figure 4b is a correlation plot among ME-stimulated transcriptome samples, depicting the Pearson correlations among all RNA samples; the color scale represents the Pearson correlation coefficient, with red indicating high correlation and blue indicating low correlation. Figure 4c is a principal component analysis (PCA) plot among ME-stimulated transcriptome samples, showing the differences between the treatment and control groups. Figure 4d is a heatmap of all differentially expressed genes in the ME-stimulated transcriptome, showing differentially expressed genes between the ME treatment and control groups. Rows represent genes with significant expression differences between the two groups, while columns correspond to individual samples in each group. The color scale represents gene expression levels, with red representing high expression and blue representing low expression. Figure 4e shows the response of the ab3A sensor of the UAS-BdorOR94b1 Drosophila strain to 2-heptanone and ethyl hexanoate. Representative SSR responses of hexanoate and ME; f is OR22ab Gal4 Representative SSR responses of the ab3A sensor of the UAS-BdorOR94b1 Drosophila strain to different doses of ME.

[0087] Figure 5 shows the detailed knockout process of the BdorOR94b1 mutant and supplementary data on the SSR response of the knockout strain to ME; Figure 5a shows the gene structure of BdorOR94b1, with the target site location shown; Figure 5b shows the synthesis process of BdorOR94b1 sgRNA; Figure 5c shows the wild-type BdorOR94b1. + / + Target region sequence peak diagram; Figure 5d shows wild-type BdorOR94b1 + / + With mutant heterozygote BdorOR94b1 + / - , homozygous BdorOR94b1 - / - Target region amplification gel image alignment; Figure 5e shows the mutant homozygote BdorOR94b1. - / - Target region sequence peak diagram; Figure 5f is a schematic diagram of SSR recordings in *Bactrocera dorsalis*; Figure 5g shows the quantification of SSR responses to 64 different odorants using s, BdorOR94b1. - / -Figure 5 is BdorOR94b1 is a key receptor mediating the behavioral responses to methyl eugenol (ME); wherein Figure 5a is a schematic diagram of the EAG responses of B. dorsalis to ME; Figure 5b is the representative EAG responses of B. dorsalis males to different concentrations of ME; Figure 5c is the EAG dose-response histogram of B. dorsalis males to ME, N = 12 responses recorded for each male, data results are expressed as mean ± SEM; Figure 5d is a schematic diagram of the single sensillum recording (SSR); Figure 5e is the representative SSR responses of B. dorsalis trichoid sensilla (s.trichoid, N = 109) and basiconic sensilla (s.basiconica, N = 26) to ME; Figure 5f is the SSR responses of WT and BdorOR94b1

[0088] Figure 6 is BdorOR94b1 is a key receptor mediating the behavioral responses to methyl eugenol (ME); wherein Figure 6a is a schematic diagram of the EAG responses of B. dorsalis to ME; Figure 6b is the representative EAG responses of B. dorsalis males to different concentrations of ME; Figure 6c is the EAG dose-response histogram of B. dorsalis males to ME, N = 12 responses recorded for each male, data results are expressed as mean ± SEM; Figure 6d is a schematic diagram of the single sensillum recording (SSR); Figure 6e is the representative SSR responses of B. dorsalis trichoid sensilla (s.trichoid, N = 109) and basiconic sensilla (s.basiconica, N = 26) to ME; Figure 6f is the representative SSR responses of WT and BdorOR94b1 - / - basiconic sensilla (s.basiconica) of B. dorsalis to ME, 4,5-dimethoxy-2-(prop-2-en-1- yl)phenol (DMP) and Heptanal; Figure 6g is the SSR responses of WT and BdorOR94b1 - / - basiconic sensilla (s.basiconica) of B. dorsalis to ME, 4,5-dimethoxy-2-(prop-2-en-1- yl)phenol (DMP) and Heptanal; Figure 6g is the SSR responses of WT and BdorOR94b1 - / - basiconic sensilla (s.basiconica) of B. dorsalis to ME, 4,5-dimethoxy-2-(prop-2-en-1- yl)phenol (DMP) and Heptanal; Figure 6g is the SSR responses of WT and BdorOR94b1 - / - Figure 6 is BdorOR94b1 is a key receptor mediating the behavioral responses to methyl eugenol (ME); wherein Figure 6a is a schematic diagram of the EAG responses of B. dorsalis to ME; Figure 6b is the representative EAG responses of B. dorsalis males to different concentrations of ME; Figure 6c is the EAG dose-response histogram of B. dorsalis males to ME, N = 12 responses recorded for each male, data results are expressed as mean ± SEM; Figure 6d is a schematic diagram of the single sensillum recording (SSR); Figure 6e is the representative SSR responses of B. dorsalis trichoid sensilla (s.trichoid, N = 109) and basiconic sensilla (s.basiconica, N = 26) to ME; Figure 6f is the representative SSR responses of WT and BdorOR94b1 - / - Figure 6 is BdorOR94b1 is a key receptor mediating the behavioral responses to methyl eugenol (ME); wherein Figure 6a is a schematic diagram of the EAG responses of B. dorsalis to ME; Figure 6b is the representative EAG responses of B. dorsalis males to different concentrations of ME; Figure 6c is the EAG dose-response histogram of B. dorsalis males to ME, N = 12 responses recorded for each male, data results are expressed as mean ± SEM; Figure 6d is a schematic diagram of the single sensillum recording (SSR); Figure 6e is the representative SSR responses of B. dorsalis trichoid sensilla (s.trichoid, N = 109) and basiconic sensilla (s.basiconica, N = 26) to ME; Figure 6f is the representative SSR responses of WT and BdorOR94b1

[0089] Figure 7 is that the derivative ECF of methyl eugenol enhances the attractiveness of lek to female insects after feeding on methyl eugenol; wherein Figure 7a is the basic design idea of the experiment, the cuboid behavior room is divided into A and B sides on average, and a small cage that can load attractant is placed on the left and right sides, there are 30 freely moving WT females in the behavior room, the density of male insects in the small cage is 10 or no insects during the experiment, and the rectal gland extract is directly smeared on the outer wall of the breathable small hole; Figure 7b is the competitive attraction of fed / unfed ME males to WT females; Figure 7c is the competitive attraction of rectal gland extract of fed / unfed ME males (with males in the small cage) to WT females; Figure 7d is the competitive attraction of rectal gland extract of fed / unfed ME males to WT females; Figure 7e is GCMS analysis of rectal gland extract after feeding on ME; Figure 7f is the competitive attraction of rectal gland extract of males with / without added trans-coniferyl alcohol (ECF) to WT females; Figure 7g is the competitive attraction of rectal gland extract of males with / without added 4,5-dimethoxy-2-(prop-2-en-1-yl) phenol (DMP) to WT females; Figure 7h is the competitive attraction of fed / unfed ME males to BdorOrco females; Figures 7b, 7c, 7d, 7e, 7f, 7g, and 7h all have N=5, 30 orange Drosophila suzukii in each repeat, and the data results are represented by mean ± standard error. - / -

[0090] Figure 8 is GC-MS analysis of rectal gland compounds after ME feeding; wherein Figure 8a is a schematic diagram of an artificial lek attraction testing device; Figure 8b is a photo of males feeding on ME; Figure 8c is a real object of the artificial lek attraction testing device; Figure 8d is a photo of male orange Drosophila suzukii rectal glands; Figure 8e is a female cage attracted by the cage; Figure 8f is GCMS analysis results of rectal gland extract of fed / unfed ME males, trans-coniferyl alcohol (ECF), and 4,5-dimethoxy-2-(prop-2-en-1-yl) phenol (DMP); Figure 8g is a total ion gas chromatography-mass spectrometry chromatogram of DMP; and Figure 8h is a total ion gas chromatography-mass spectrometry chromatogram of ECF.

[0091] ​Figure 9 shows the value of lek behavior to the population reproduction of *Bacteroides citrinum*. Figure 9a compares the number of matings between leks and individual matings, from 16:30 to 18:30, counting the number of matings in each time period. Figure 9b compares the wingbeat frequency between leks and individual males, from 16:30 to 18:30, counting the number of male wingbeats in each time period. Figure 9c compares the wingbeat frequency of normally reared males (Normal) with that of less competitive males (malnutrition, Bdorwhite). - / - BdorWP - / - The competitive mating experiment design was as follows: the female:male ratio was 1 (WT):4 (2 normal males and 2 less competitive males). The number of male types selected by the females in each group was counted, and each group was repeated 10 times. Figures 9d and 9e show the offspring evaluation experimental design of normal males and males with poor growth conditions. Ten healthy wild-type females were provided to 10 normal males and 10 males with poor growth conditions, respectively. After sufficient mating, the number of eggs laid and the hatching rate of the eggs were counted. For all experiments in Figures 9a, 9b, 9c, 9d, and 9e, N=5. All data results are expressed as mean ± standard error. The unpaired t-test was used for data difference analysis. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001, and ns indicates no significant difference.

[0092] Figure 10 shows the effects of malnutrition on the body size, pupal weight, egg production, and hatching rate of the oriental fruit fly. Figure 10a shows the construction process of malnourished males; Figure 10b compares the body size of adults and pupae of wild-type and malnourished males; Figure 10c compares the pupal weight of wild-type and malnourished males; Figure 10d compares the egg production of wild-type and malnourished males; Figure 10e compares the hatching rate of offspring eggs of wild-type and malnourished males. In the figures, ** indicates P < 0.01, and *** indicates P < 0.005. Detailed Implementation

[0093] The present invention will be further described in detail below through specific embodiments.

[0094] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0095] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions not noted in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are all conventional products which can be obtained by market purchase.

[0096] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0097] Embodiments

[0098] Experimental methods:

[0099] 1. Construction of odorant receptor co-receptor BdorOrco, ionotropic receptor co-receptor BdorIR8a loss-of-function lines

[0100] (1) Construction of BdorOrco, BdorIR8a mutants using CRISPR / Cas9

[0101] The construction of olfactory receptor mutants includes sgRNA synthesis, embryo injection and mutant screening.

[0102] The full-length structure of BdorOrco and BdorIR8a was predicted by the orange fruit fly genome assembled by the laboratory early sequencing group (National Biological Information Center GSA database, PRJCA020830). The gDNA of 10 males and 10 females was extracted, and the target region was amplified by specific primers (primers and conditions are shown in Table 1). The PCR product was cloned into a blunt vector (pEASy@-Blunt Cloning kit, Beijing Quanshi Gold Biology, item number: CB101-01), and the bacterial colony was sent for testing to determine the conserved region. According to the conserved region, we designed the target point interval of sgRNA cas9-AI with a length of 20 bp and containing a three-base sequence of NGG or CCN on the side. The sgRNA was synthesized using a commercial kit (GeneArt gRNA Kit, Thermo Fisher). TM

[0103] ​

[0104]

[0105] Note: underlined part is intron, non-underlined part is exon.

[0106] Fresh embryos were collected for injection, sgRNA (sequence see Table 1 target position) working concentration was 300 ng / μL, cas9 protein concentration was 150 ng / μL. The above reagents were mixed and then injected into embryos using FemotoJet and Inject Man4 system (Eppendorf, Hamburg, Germany). The injected embryos were placed in an incubator at 26.5°C, 60% humidity, and waited for hatching. The hatched larvae were picked up with a soft brush and fed with food.

[0107] The gDNA of the mutant adult mid-leg tissue was extracted for genomic detection and amplification (primers and conditions see Table 1). The PCR products were subjected to sanger sequencing, and those with obvious nested peaks near the target site were edited individuals. After the G0 generation hatched into adults, their genomic amplification was sequenced, and individuals with obvious nested peaks were paired with wild type. The G1 generation of adults obtained by pairing were subjected to genotype detection, and the lines that met the above requirements were saved and crossed with wild type again. The G2 generation of adults obtained by crossing were selected for self-crossing to homozygous. The homozygous G3 generation of BdorOrco - / - , BdorIR8a - / - The lines were saved for use to carry out experiments.

[0108] Table 1 PCR primers, conditions and target position

[0109] 2BdorOrco - / - , BdorIR8a - / - , antennaless, maxillary palpless male behavior response to ME

[0110] We tested the behavior response of BdorOrco - / - , BdorIR8a - / - male to ME using the trap experiment commonly used in Drosophila (see behavioral experiment for details), and found that the main receptor type for orange Drosophila to sense ME is olfactory receptor (OR). Subsequently, we proved that the peripheral olfactory tissue for sensing ME is mainly the antenna by removing the antenna and maxillary palp respectively in the four-arm olfactometer. These results indicate that ORs on the antenna are very important for orange Drosophila to sense ME.

[0111] (1) Olfactory stimulation, transcriptome sequencing and qPCRs verification

[0112] The odor stimulation experiment was carried out in a small cage with a size of (18 cm x 12.5 cm x 14 cm), and the experimental insects were 12-day-old sexually mature male insects. The Me treatment group and the control group each had 100 male insects. When the odor was stimulated, 100 male insects were divided into two small cages, with 50 male insects in each small cage to ensure that the male insects could fully contact Me. The stimulating compound Me was dissolved in paraffin oil (concentration 100 μg / μL), and 20 μL (100 μg / μL) of Me was added to a clean filter paper in the cage. The control cage was placed with paraffin oil. The antennae were collected immediately after 5 hours (the time was fixed at 9:00-14:00), and then frozen in liquid nitrogen and stored in a -80°C refrigerator for RNA extraction for transcriptome sequencing. The treatment group and the control group each had 3 replicates.

[0113] RNA sequencing was performed on the illumina Novaseq 6000 sequencing platform. The original data was filtered using the trimmomatic v0.39 software to remove adapters and low-quality sequencing sequences (sequence quality value less than 20 or sequence length less than 40 bp), and high-quality sequencing data was obtained. The high-quality sequencing data was aligned with the existing reference genome (NCBI project number: PRJCA020830) in the laboratory using the hisat2 v2.2.1 software, and the odor receptor genes aligned were quantified using the featureCounts v2.0.1 software to obtain the raw expression matrix, FPKM value matrix, and TPM value matrix of the aligned genes.

[0114] The FPKM value matrix was plotted using the R language ggplot2 v3.4.2 package, and whether the expression patterns were similar between samples was determined according to whether the central tendency of the FPKM values of the genes in different samples was uniform. The different samples were hierarchically clustered according to the FPKM value matrix using the R language hclust function, and the correlation between samples was determined according to the distance between samples. The correlation coefficient between samples was calculated according to the FPKM value matrix using the R language cor function, and the strength of the correlation between samples was determined by the size of the correlation coefficient. Principal component analysis was performed on the FPKM value matrix using the R language FactoMineR v2.8 package and the factoextra v1.0.7 package to determine the size of the difference between the ME treatment group and the control group.

[0115] Quantitative real-time PCR (qRT-PCR) was used to validate the differential expression of key odorant receptors from the transcriptome analysis. The stimulation gradient was set as 0 h, 2.5 h, 5 h, and 24 h according to the stimulation method described above. Total RNA was extracted from different stimulation groups using TriZol (Invitrogen, Carlsbad, CA, USA). Approximately 1 μg of total RNA was used as a template to synthesize cDNA. Specific qPCR primers (Table 2) were designed for qRT-PCR experiments. The total PCR system was 10 μL, including 5 μL of 2x Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.5 μL of each primer (10 μM), 1 μL of cDNA, and 3 μL of RNase-free water. The qPCR instrument was a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Primer sequences and PCR conditions are shown in Table 1. Dissociation curve analysis was performed before the main experiment to confirm the specificity of amplification. Three biological replicates were performed. Relative expression levels were calculated using 2 -ΔΔCT Relative expression levels were calculated using 2

[0116] Table 2 qRT-PCR primers and conditions

[0117] (2) Construction of transgenic Drosophila lines

[0118] Total RNA from male antennae was extracted by TriZol (Invitrogen, Carlsbad, CA, United States). Subsequently, RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) was used to synthesize cDNA from total RNA according to the manufacturer’s instructions. The cDNA was diluted 1:10 with RNase-free water and stored at -20 °C until use. TMFirst Strand cDNA Synthesis Kit (Thermo Scientific) cDNA reverse transcription. cDNA was diluted to 300 ng / μL and stored at -20 °C as cloning template. BdorOR94b1 was amplified using specific primers (see Table 1) with the following program: 98 °C 3 min, 98 °C 10 s, 60 °C 15 s, 35x, 72 °C 1 min 30 s, 72 °C 10 min. After purification, the product was cloned into pUAST-attB vector and the recombinant plasmid was purified (Qiagen midiprep kit). The plasmid was injected into y[1]M{vas-int.Dm}ZH-2A w[*] ; P{CaryP}attP2 background and the G0 generation was crossed with the balancer strain to select for the white marker. The W; sp / Cyo; UAS-BdorOR94b1 / TM2 effector strain was finally constructed after several generations of selection. The W; sp / Cyo; UAS-BdorOR94b1 / TM2 was crossed with the empty neuron strain W; 22abGAL4 / 22abGAL4; TM2 / TM6B. The homozygous individuals W; 22abGAL4 / 22abGAL4; UAS-BdorOR94b1 / UAS-BdorOR94b1 were finally used for single sensillum recordings.

[0119] 3. Analysis of antennal transcriptome upon ME odor stimulation

[0120] A down-regulated odorant receptor gene BdorOR94b1 was screened in the antennae upon ME odor stimulation through antennal transcriptome. The complete genomic sequence and the encoded amino acid sequence of BdorOR94b1 were obtained through bioinformatics techniques. The specific sequences are as follows:

[0121] The full-length of BdorOR94b1 genomic sequence is 1611 bp, with 4 exons (the part without underline in SEQ ID NO: 31) and 3 introns (the part with underline in SEQ ID NO: 31). The nucleotide sequence is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32.

[0122]

[0123] MAVKKWSPRNTSSMSRTASANIIIAVLKALGYWQWTRDPRQPYIEKVERAYRIVLHTTFPFTFIALMLTGVLLSRDLDEIGSILHVLLLTEFSLIVKTLHIWRKGGVAWRFMHEVANDPIYDLRQQSEWTKWQQAQRSFAIVSNTYFVAATTVVVFACIGAMMTPADVYVLPMNIYVPFDWHHPRRYWYAWTYNTIASLMT ATANAMLDLVNCYFMFHLSLLYKLIGWRLSALRRSANEPPVIEQMSEIFQMHMKVRRLTTECETLVSIPVFSQIILSSFILCFCGYRLQQMEIMENLSMLFSTVEFATVMAVQIFLPCYFGNKVTESSDALTDEIFNSDWTTFDVPTRRFMILYMELLKKPANLMSVNYFIIGVDIFAKTMKNAYSIFALVLNMNN(SEQ ID NO:32).

[0124] 4. Constructing a transgenic fruit fly strain expressing BdorOR94b1 in vitro.

[0125] Total RNA was extracted, and cDNA template was obtained after reverse transcription. The complete sequence of BdorOR94b1 was cloned, and a transgenic plasmid containing this sequence was constructed using the pUAST-attB plasmid as a template. Transgenic Drosophila expressing BdorOR94b1 protein was constructed in vitro using an ab3A empty neuron strain lacking endogenous receptor 22a. Single-sensory recordings (SSRs) were used to verify BdorOR94b1's sensitivity to mesenchymal receptors (ME) in vitro.

[0126] 5. Construct a loss-of-function mutant of BdorOR94b1.

[0127] Based on the genomic structure in 3, a knockout target site was set on the BdorOR94b1 exon using CRISPR / Cas9-mediated knockout technology to obtain a transcription frameshift termination deletion mutant, which was then cultured into a homozygous line. The specific process was the same as in 1 (3).

[0128] 6. Verify the behavioral response of mutants to ME through electrophysiology and behavioral studies.

[0129] Through the comparison of the differences in the response of the above mutant lines and wild type to ME by the tendency behavior experiment, electroantennogram recordings (EAG) and SSR technology, it is verified whether BdorOR94b1 participates in the function of recognizing ME in vivo.

[0130] (1) Behavior experiment

[0131] The flies used in the trap and four-arm behavior tests are all unmated adults at 12 days after eclosion, and are bred in small cages (18 cm x 12.5 cm x 14 cm) before the experiment, with the number of flies in each cage controlled at 40-45, and sufficient food and water provided. After reaching the age, healthy and active adults are selected for the tendency behavior experiment.

[0132] The trap experiment is carried out using artificial attractant bottles, and once the adults enter the trap, they cannot escape. The trap is placed in a large cage with a size of 18 cm x 12.5 cm x 14 cm. Two trap bottles are placed in each cage, trap 1 is the treatment group, and Me (100 μg / μL, 10 μL added) is placed as the test odor, and trap 2 is the control, and paraffin oil is placed as the odor solvent. The experimental time is from 9:00 to 10:00, the light intensity is controlled at 280-300 lux, the experimental environment conditions are temperature (26±1)℃, relative humidity (60±5)%. After the experiment, the number of adults in the trap is recorded, with 5 replicates in each group and 30 adults in each replicate.

[0133] The four-arm behavior experiment of fruit flies is carried out in a customized four-arm olfactometer, and a laboratory automatic high-definition camera system is used for recording. Before the experiment, the four-arm olfactometer is wiped clean, and the gas flow stability is tested in advance to ensure that the four-arm gas flow is stable at 0.4 L / min. During the behavior observation, the test flies are placed in the four-arm olfactometer, and after the ventilation, the flies are allowed to adapt to the airflow environment and are evenly distributed in the four quadrants. Then the airflow is closed again and the test odor methyl eugenol (1 μg / μL, 10 μL added) and the control paraffin oil are added as soon as possible, and the two arms on one side of the four-arm olfactometer are placed with the ME odor source, and the other side is placed with the paraffin oil. After ventilation, the tendency response of the flies within 10 minutes is recorded by camera. The experimental time is from 8:00 to 9:00, the light intensity is set to 280-300 Lux, the behavior room temperature is (26±1)℃, and the relative humidity is (60±5)%. After the experiment, the video is copied and the data is counted, and a total of 5 replicates are observed in each group, with 30 adults in each replicate.

[0134] (2) Electrophysiological recording

[0135] EAG recordings: Orange fruit fly male heads were removed and one end of the antennae was cut off in segments. Two glass electrodes were prepared and filled with 0.1 M KCI solution. The reference electrode was attached to the head cut and the recording electrode was attached to the antennal end cut. Twelve 12-day-old virgin males of each genotype were recorded. Antennal potential recordings were performed under a BX51 microscope (Olympus). Airflow stimulation was performed using a controller (CS-55, Syntech, Kirchzarten, Germany) with a stimulation airflow of 1.4 mL / min and a stimulation duration of 300 ms. Signals were collected using a universal probe preamplifier and then converted by a digital-to-analog converter (IDAC-4-USB, Syntech, Netherlands). The signals obtained were analyzed using EAG pro 2.0. The EAG response signal was the signal of the test compound minus the signal of paraffin oil.

[0136] SSR recordings: For Drosophila, the head and antennae were extended out of a 10-μL cut-off pipette tip, which was fixed with dental wax. A tungsten recording electrode was inserted into the base of the sensillum to test the ab3A null neurons with 2-heptanone and hexyl acetate as indicator odors. The reference electrode was inserted into the compound eye of the fly. Five to six flies and 10-14 sensilla were recorded for each GAL422ab / UAS-BdorOR94b1 and its GAL and UAS controls. The response spectrum of the BdorOR94b1 receptor and the dose response were recorded for six flies and 9-11 sensilla. For B. dorsalis, the head was extended out of a 200-μL cut-off pipette tip, which was fixed with dental wax, and the antennae were fixed to a glass slide with double-sided tape. The recording electrode was inserted into the base of the sensillum using a tungsten electrode, and the reference electrode was also inserted into the compound eye using a tungsten electrode. The response spectrum and the dose response of the ME sensillum were recorded for three to four wild-type males and five to six sensilla. For comparison of BdorOR94b1 and wild type, three wild-type males and 14 ME sensilla were recorded, and five mutant males and 48 ME sensilla were recorded. Sensillum recordings were performed under a BX51 microscope (Olympus). Odor stimulation and signal collection were as described for EAG. The signals obtained were processed using Autospike v3.9. Low-frequency filtering was set to 300 Hz, and high-frequency filtering was set to 2 kHz. The response was calculated as the number of action potentials increased after 1 s of stimulation.

[0137] Results

[0138] (1) The antennally expressed odor receptors mediate the attraction of ME to B. dorsalis males.

[0139] The oriental fruit fly exhibits sexual dimorphism in its response to ME (metaphorical olfactory receptor), with males showing a strong attraction to ME while females show no obvious response (Fig. 1a and Fig. 1b). This was achieved by constructing the olfactory receptor co-receptor BdorOrco and the BdorIR8a deletion mutant BdorOrco. - / - and BdorIR8a - / - (See Table 3, Figures 2a-2j for screening details). It was found that ME lost its attraction to male BdorOrco mutants (Figure 2), while knocking out BdorIR8a (Figure 2) had no significant effect on attraction (Figures 1c and 1d). Since the odor trap experiment could not observe the searching behavior of male oriental fruit flies for ME in real time, we used a modified four-walled olfactory instrument (Figure 1e). Removing the maxillary palps had almost no effect on the males' searching for ME, only a very slight decrease. Conversely, removing the antennae completely eliminated the directional behavior (Figure 1f).

[0140] Table 3. Survival and mutation data in the construction of *Bacteroides citrus* mutants.

[0141] (2) BdorOR94b1 was identified as a specific receptor for sensing ME.

[0142] This invention, through stimulated transcriptomics and qPCR, identified BdorOR94b1, the only receptor downregulated after ME stimulation (Figs. 3a-3c, 4a-4d). It is specifically expressed in the antennae of adult fruit flies (Fig. 3d). We further expressed the cloned BdorOR94b1 gene in the Drosophila ab3A empty neuron system lacking endogenous 22a (Fig. 3e), and single-sensillum recordings (SSRs) showed that ME strongly activated BdorOR94b1 in the ab3 sensor (Figs. 3f and 3g; Fig. 4e). The response of BdorOR94b1 is highly specific; among 64 odors associated with the oriental fruit fly, only ME and its analogue DMP activated this receptor, with ME being the most potent ligand and exhibiting a dose-response characteristic (Figs. 3h and 3i, 4f).

[0143] (3) The important role of the receptor in mediating ME attraction behavior was verified by constructing a BdorOR94b1 deletion mutant.

[0144] This invention utilizes CRISPR-Cas9 to construct the BdorOR94b1 deletion mutant (screening details are shown in Table 3, Figures 5a-5e), which has a 124-base deletion in exon 1 (Figures 5a-5e). Electroantennographic (EAG) results show that BdorOR94b1... - / -The mutant line lost the electrophysiological response to ME essentially (Fig. 6a-6c). SSR recording results proved that the neurons responding to ME were located in the basiconic sensilla (s. basiconica) (Fig. 6d and Fig. 6e). These olfactory receptor neurons (ORNs) only responded to ME and DMP, with the strongest response to ME in a dose-dependent manner, which is consistent with the findings of the Drosophila antennal lobe system (Fig. 5f-5i). When BdorOR94b1 was knocked out, these ORNs no longer responded to ME and DMP, and only showed a response to another neuron in the same sensillum that senses heptanal (Fig. 6f and Fig. 6g). The four-arm olfactometer behavioral experiments also showed that BdorOR94b1 - / - The tendency behavior to ME was essentially lost (Fig. 6h and Fig. 6i). These results suggest that BdorOR94b1 is an indispensable OR for B. dorsalis to sense ME.

[0145] (4) To investigate whether ME affects the mating behavior of B. dorsalis by promoting lek positioning, a series of behavioral experiments were conducted using artificial leks.

[0146] First, we compared the difference in female preference between leks with and without feeding male insects, and the results showed that females preferred to feed on males that had fed on ME (Fig. 7a and Fig. 7b). Further experiments proved that ME would derive two compounds, (E)-coniferyl alcohol (ECF) and 4,5-dimethoxy-2-(prop-2-en-1-yl) phenol (DMP), in male insects and store them in the rectal glands of male insects, and the key compound that enhances the attractiveness of male insects is ECF (Fig. 7c-7g, Fig. 8). And BdorOrco - / - Females lost the tendency behavior to this compound (Fig. 7h). These findings collectively suggest that ME-derived ECF plays a crucial role in mediating female lek positioning, and this process is also regulated by olfaction.

[0147] (5) To determine whether the lek positioning behavior of B. dorsalis provides females with sufficient opportunities to choose healthy mates, thereby improving the quality and reproductive success of offspring, a series of competitive mating experiments were conducted.

[0148] Since the mating behavior of B. dorsalis (Fig. 9a) and the occurrence rate of courtship behavior (Fig. 9b) in groups were significantly higher than in individuals alone, we wanted to determine whether females could choose male sexual partners based on quality, thereby improving the quality and survival rate of offspring. To verify this hypothesis, the present invention introduced two normal fruit flies and two poorly growing fruit flies (including three types, malnourished (Fig. 10), white-eyed mutant line Bdorwhite- / - and white pupa mutant line Bdorwp - / - The reproductive behavior of these lines was impaired to some extent), and then a female was provided to record its mating choice (Figure 9c). The results showed that the females preferred to choose normal and robust males compared to the three reproductive-deficient lines (Figure 9c). The impact of this mating choice on the fecundity was then evaluated, and it was found that mating with weak males not only resulted in a significant reduction in the number of eggs, but also led to a significant decrease in the hatching rate of larvae (Figures 9d and 9e). These results suggest that lek behavior plays a crucial role in initiating mating and providing females with fitness advantages, ultimately contributing to the reproductive success and quality of offspring of B. dorsalis.

[0149] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the scope of knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

Use of the odorant receptor BdorOR94b1 as a target in at least one of a1) - a5): a1) controlling B. dorsalis; a2) screening or preparing a product for controlling B. dorsalis; a3) modulating lek behavior of B. dorsalis; a4) preparing a modulator of lek behavior of B. dorsalis; a5) modulating the sensitivity of B. dorsalis to a stimulating odor molecule. Use according to claim 1, characterized in that Modulating the olfactory sensitivity of B. dorsalis to a stimulating odor molecule using the odorant receptor BdorOR94b1 as a target. The use according to claim 2, characterized in that The stimulating odor molecule comprises a plant odor and derivatives thereof or an insect pheromone and analogs thereof. Use according to claim 3, characterized in that The stimulating odor molecule comprises methyl eugenol or derivatives thereof; preferably, the derivatives comprise trans-thujanol and 4,5-dimethoxy-2-(2-propenyl)phenol. Use of a BdorOR94b1 inhibitor in controlling B. dorsalis and / or preparing a product for controlling B. dorsalis. Use according to claim 5, characterized in that The BdorOR94b1 inhibitor is at least one of a substance that degrades BdorOR94b1 or a substance that reduces the expression level of BdorOR94b1; Preferably, the substance that reduces the expression level of BdorOR94b1 comprises at least one of b1) - b5): b1) at least one of siRNA, dsRNA, miRNA, ribozyme, shRNA, CRISPR / Cas system targeting BdorOR94b1; b2) a nucleic acid molecule encoding b1); b3) a recombinant vector containing the nucleic acid molecule of b2); b4) a recombinant cell containing the nucleic acid molecule of b2); b5) a recombinant cell containing the recombinant vector of b3); b6) a recombinant microorganism containing the nucleic acid molecule of b2); b7) a recombinant microorganism containing the recombinant vector of b3). A method for modulating the sensitivity of B. dorsalis to methyl eugenol, comprising the step of knocking down / knocking out the odorant receptor BdorOR94b1 of B. dorsalis using a gene editing technology; Preferably, the gene editing technology comprises ZFNs, TALENs or CRISPR / Cas9 technology. The method of claim 7, wherein Knocking down / knocking out the odorant receptor BdorOR94b1 using CRISPR / Cas9 technology, comprising the step of injecting sgRNA and Cas9 protein into B. dorsalis embryos. The method of claim 8, wherein The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 23 to SEQ ID NO:

26. A method of constructing a methyleugenol-insensitive line, comprising the steps of: A G0 generation individual of B. dorsalis, which is obtained by injecting the embryo with the gene editing method of claim 8 or 9, is then mated with a wild type individual, and the mutant is screened by further generations, thereby obtaining a methyl eugenol-insensitive strain. A methyl eugenol-insensitive strain obtained by the method for modulating the sensitivity of B. dorsalis to methyl eugenol according to any one of claims 7-9. A methyl eugenol-insensitive strain obtained by the construction method of claim 10.

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