CN118755709B

CN118755709B - Application of BdCA1 gene in controlling Bactrocera dorsalis

Info

Publication number: CN118755709B
Application number: CN202410752700.XA
Authority: CN
Inventors: 程代凤; 桂诗宇
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2024-06-12
Filing date: 2024-06-12
Publication date: 2025-02-07
Anticipated expiration: 2044-06-12
Also published as: CN118755709A

Abstract

The present invention discloses the application of BdCA1 gene in the prevention and control of Bactrocera dorsalis. The present invention can change the rectal environment of male adult Bactrocera dorsalis from alkaline to acidic by feeding carbonic anhydrase inhibitor and interfering with BdCA1 gene expression by RNAi technology, thereby inhibiting the physiological activities of symbiotic bacteria, resulting in a significant decrease in the level of sex pheromone synthesis and mating ability of male insects. The present invention discloses for the first time the application of carbonic anhydrase as a target in pest control. The conservative carbonic anhydrase has important practical significance for pest control and can provide a new approach for green pest control.

Description

Application of BdCA gene in control of bactrocera dorsalis

Technical Field

The invention belongs to the field of biotechnology and pest green prevention and control, and particularly relates to application of BdCA gene in prevention and control of bactrocera dorsalis.

Background

Bactrocera dorsalis (Bactrocera dorsalis (Hendel)), commonly known as "needle bees", originated in Asia and is a world quarantine pest, of the Diptera (Diptera) Bactrocera genus (TEPHRITIDAE) of the subfamily Bactrocera genus (Dacinae). The insect has extremely strong fertility, and single-head females can lay eggs for 400-1800 eggs in one lifetime. Female insects lay eggs in fruits, after eggs are hatched, larvae eat to rot or yellow and fall off the fruits, so that serious economic loss of fruit and vegetable industry is caused. Considering the hazard characteristics of the bactrocera dorsalis, the prevention and control time should be selected before spawning. For insects with migratory ability, the breeding period is a better control opportunity. Insects rely on sex pheromones to identify and locate the dissimilarity during mating, and thus sex pheromones are critical to the reproduction of insects. Insect sex pheromones are volatile chemical substances released by the secretory glands of insects and can stimulate homoplasmic individuals to produce pugging and corresponding physiological reactions. Previous studies have found that sex pheromones attracting females can be synthesized and released in the rectal glands of the male worms of the fruit fly, and the sex pheromones are synthesized with the assistance of bacillus in the rectal glands. However, whether the physiological activities of the symbiotic bacillus are regulated by the host and the mode of regulating the host should be further discussed.

Carbonic anhydrase (Carbonic anhydrase, CA) is a widely occurring enzyme in bacteria, archaea and eukaryotes that catalyzes an important physiological reaction of carbon dioxide hydration to bicarbonate and protons. Whether it plays a role in regulating insect sex pheromones is still unknown.

Disclosure of Invention

The invention proves for the first time that host insects can regulate and control the physiological activity of in vivo symbiotic bacteria by controlling the expression of conserved carbonic anhydrase genes. Specifically, after feeding carbonic anhydrase inhibitors and interfering expression of the Bactrocera dorsalis BdCA gene by RNAi technology, the pH value in the rectum is reduced, so that the activity of symbiotic bacillus in the rectum is reduced, and the level and mating competitiveness of the rectum synthetic pheromone are obviously reduced. This suggests that carbonic anhydrase expression is associated with changes in insect rectal pH and that the acid conditions will inhibit the symbiotic bacillus synthetic pheromone, thereby affecting the ability of the male to mate. Therefore, bdCA gene can be used as a green control target gene of bactrocera dorsalis.

The first object of the invention is to provide an application of BdCA gene in controlling bactrocera dorsalis, which is characterized in that the BdCA gene codes for a protein with an amino acid sequence shown as SEQ ID NO. 3.

Preferably, the nucleotide sequence of BdCA gene is shown as SEQ ID NO. 2.

Preferably, by reducing the expression level of BdCA gene, the pH and sex pheromone synthesis in the rectum of the sexually mature bactrocera dorsalis is reduced, thereby reducing the mating ability of the sexually mature bactrocera dorsalis.

Preferably, the reduction of BdCA1 gene expression is by knocking out BdCA gene or by gene silencing techniques to reduce BdCA gene expression.

A second object of the present invention is to provide the use of carbonic anhydrase inhibitors for controlling bactrocera dorsalis.

Preferably, the application reduces the pH in the rectum and the synthesis of sex pheromone by feeding carbonic anhydrase inhibitor to inhibit carbonic anhydrase activity in the rectum of the sex mature bactrocera dorsalis, thereby reducing mating ability of the sex mature bactrocera dorsalis.

Preferably, the carbonic anhydrase inhibitor is acetazolamide, and the concentration is more than 0.01 g/mL.

Preferably, the carbonic anhydrase inhibitor is an inhibitor for inhibiting the activity of BdCA protein, and the amino acid sequence of BdCA protein is shown as SEQ ID NO. 3.

Preferably, the sexually mature bactrocera dorsalis is a bactrocera dorsalis male worm 12 days old after emergence.

The third object of the invention is to provide a method for controlling bactrocera dorsalis, which comprises the steps of reducing the expression level of BdCA gene or feeding carbonic anhydrase inhibitor, wherein the carbonic anhydrase inhibitor is an inhibitor for inhibiting the activity of a protein with an amino acid sequence shown as SEQ ID NO. 3.

The invention has the beneficial effects that:

the invention discovers for the first time that when carbonic anhydrase inhibitors are fed, the pH value in the rectum of the bactrocera dorsalis is changed from alkaline to acidic, which can inhibit the synthesis of sex pheromone of the symbiotic bacteria of the rectum, and reduce the fertility of the proctophyte. The RNAi technology is utilized in the bactrocera dorsalis, so that the mRNA expression of the bactrocera dorsalis carbonic anhydrase gene (BdCA 1) is obviously reduced, the pH is obviously reduced, and the sex pheromone level and mating competitiveness are obviously reduced. Insect carbonic anhydrase inhibitor (substance for reducing carbonic anhydrase activity, substance for degrading carbonic anhydrase, or substance for reducing expression level of carbonic anhydrase) or mRNA expression of BdCA gene is reduced based on RNAi technology in preparation of pest control agent, and intestinal tract forms acidic environment to inhibit synbiotic bacillus synthetic pheromone, so that fertility of fruit fly and fruit fly is obviously reduced, damage of subsequent female and insect spawning to fruits is controlled, drug resistance is not generated, harm to human and livestock is avoided, and environment is not polluted in the control process.

Drawings

FIG. 1 is a graph showing correlation analysis of activity and pH of the rectal carbonic anhydrase of the fruit fly and B is a graph showing correlation analysis of activity and pH of the rectal carbonic anhydrase of the fruit fly at different development periods and at different time points when 12 days are taken after emergence of the fruit fly, and the samples are subjected to correlation analysis by Spearman.

Fig. 2 is a graph of results of rectal pH of bactrocera dorsalis male after administration of carbonic anhydrase inhibitors, representing significant differences in P <0.0001 levels for independent sample T-test.

Fig. 3 is a graph showing results of the content of proctitive pheromone in bactrocera dorsalis after feeding carbonic anhydrase inhibitor, wherein a is a graph showing results of TMP content, and B is a graph showing significant differences in levels of P <0.01 in independent sample T test, and showing significant differences in levels of P <0.0001 in independent sample T test.

Fig. 4 is a graph of competition results for male bactrocera dorsalis mating fed carbonic anhydrase inhibitors, using paired sample T test, representing significant differences in levels of P < 0.01.

FIG. 5 is a thermal diagram of CA genes differentially expressed by Drosophila citri male insects at different stages of development.

FIG. 6 is a graph showing the correlation between the CA gene and the pH value of the differential expression of the fruit fly, wherein A is a graph showing the correlation between 2247 genes and the pH value of the differential expression of the fruit fly male worm in the rectum at different development periods, B is a graph showing the correlation between 4179 genes and the pH value of the differential expression of the fruit fly male worm in the rectum at different development periods, and the samples are subjected to correlation analysis by Spearman.

FIG. 7 is a thermal map of CA genes differentially expressed at different times in male insects of sexually mature fruit fly.

FIG. 8 is a graph showing the correlation between the CA gene and the pH value, which are differentially expressed in the rectum of the male sex-mature bactrocera dorsalis, wherein A is a graph showing the correlation between 2247 genes and the pH value, which are differentially expressed in the rectum of the sex-mature bactrocera dorsalis, B is a graph showing the correlation between 4179 genes and the pH value, which are differentially expressed in the rectum of the sex-mature bactrocera dorsalis, and the correlation of the sample is carried out by Spearman.

FIG. 9 is a heat map of the relative expression levels of 2247 genes and 4179 genes at different sites of the male worm of the sexually mature fruit fly.

FIG. 10 is a phylogenetic tree of CA proteins in 6 species.

Fig. 11 shows the relative expression level of BdCA and the CA enzyme activity after silencing the male worm BdCA1 gene of the sexually mature fruit fly, wherein A is a graph showing the result of the expression level of BdCA1 gene after injecting dsCA into the sexually mature fruit fly, B is a graph showing the result of the carbonic anhydrase activity after injecting dsCA1, wherein the difference between the T test of independent sample and the P <0.001 is significant, and the difference between the T test of independent sample and the P <0.05 is significant.

Fig. 12 is a graph of results of rectal pH after injection dsCA of sexually mature bactrocera dorsalis male worm with P <0.001 representing T test of independent samples.

Fig. 13 is a graph showing results of rectal pheromone content after injection dsCA a of male sex-mature bactrocera dorsalis, wherein a is a graph showing results of TMP content, and B is a graph showing significant differences in independent sample T test at P <0.001 level, and showing significant differences in independent sample T test at P <0.05 level.

Fig. 14 is a graph of mating competition results after injection dsCA of sexually mature bactrocera dorsalis male worm with a matched sample T test, representing significant differences in P <0.001 levels.

Detailed Description

The following examples are further illustrative of the invention and are not intended to be limiting thereof.

Example 1 relationship between intrarectal carbonic anhydrase and rectal pH of Bactrocera dorsalis

To verify whether expressed carbonic anhydrase genes affect pH in the rectum of bactrocera dorsalis, we determined and compared the enzyme activity of androstachys procyanidins with pH at different time points (4:00, 8:00, 12:00, 16:00, 20:00 and 24:00) during different development periods (days 0,3, 6, 9 and 12 after eclosion) and day of sexual maturation (12 days after eclosion) and performed a correlation analysis using Spearman correlation. The specific operation is as follows:

(1) To determine the carbonic anhydrase activities of the male rectum at different development periods and different time periods, 0.05g of male rectum was dissected and collected according to the above time, and the carbonic anhydrase activity in the sample was determined by using a carbonic anhydrase activity micro-assay kit (TONGUO, shanghai China) according to the instructions of use followed by an enzyme-labeled instrument (BioTek Synergy H1, america).

(2) To determine the male rectal pH at different developmental stages and for different time periods, 10 male rectal tissues were collected by dissection at the above time intervals and placed in 1.5mL centrifuge tubes with 30. Mu.L of sterile, enzyme-free water. After grinding the sample, it was centrifuged at 12,000rpm for 10min. The centrifuged supernatant was transferred to a clean centrifuge tube, and the pH of the supernatant was measured using a micropipette (LabSen 241-3 SP).

Correlation analysis of the androstane rectal carbonic anhydrase activity and pH at different developmental stages showed that the androstane rectal carbonic anhydrase activity at different developmental stages was highly positively correlated with rectal pH (a in fig. 1). The results of correlation analysis of rectal carbonic anhydrase activity and pH of sexually mature (12 days of day after emergence) males at different time periods showed that the rectal carbonic anhydrase activity of sexually mature males correlated moderately positively with rectal pH in one day (B in FIG. 1). This suggests that expression of the bactrocera dorsalis male rectal carbonic anhydrase gene correlates with rectal pH.

Example 2 relationship of Bactrocera dorsalis carbonic anhydrase Activity and symbiotic pheromone

In order to verify whether carbonic anhydrase regulates the physiological activity of symbiotic bacillus by controlling the pH of the rectum, the embodiment utilizes a carbonic anhydrase inhibitor (acetazolamide) to reduce the activity of the carbonic anhydrase and then detects various indexes in the intestinal tract.

Specifically, on the basis of normal feeding, sexually mature (12 days after emergence) males were fed with water containing acetazolamide (0.01 g/mL), and normal fed males served as controls. Subsequent experiments were performed after 24 hours of feeding.

(1) To detect pH in the male rectum, the rectum of 10 control and 10 treated male animals were dissected and collected, respectively, and placed in a 1.5mL centrifuge tube containing 30. Mu.L of sterile, enzyme-free water. After grinding the sample, it was centrifuged at 12,000rpm for 10min. The centrifuged supernatant was transferred to a clean centrifuge tube, and the pH of the supernatant was measured using a micropipette (LabSen 241-3 SP).

(2) To detect androstane rectal pheromone levels, 60 control and 60 treatment groups of androstane rectum were dissected and collected, respectively, and placed in a brown chromatographic bottle containing 500 μl of chromatographically pure hexane. Leaching for 24h in a shaker at 16℃and 200 rpm. The samples were then tested using an Agilent gas chromatograph-mass spectrometer (GC-MS). The system is Agilent 7890B/5977B-GC/MSD. The gas chromatographic column is HP-5MS capillary column. The chromatographic conditions include sample inlet temperature 240 deg.c, helium as carrier gas, column flow rate 1mL/min, split flow ratio 10:1, column temperature programming of initial temperature 50 deg.c, maintaining for 1min, raising temperature to 140 deg.c at 5 deg.c/min and maintaining for 3min at 10 deg.c/min to 250 deg.c. The mass spectrum conditions are EI power source, electron energy 70eV, ion source temperature 230 ℃, interface temperature 280 ℃ and scanning mass range 45-450amu. The detected results were aligned using data provided by NIST17.0 spectra library and Trimethylpyrazine (TMP) and tetramethylpyrazine (TTMP) were determined by comparing the retention time and mass spectra.

(3) In order to detect the mating ability of the male worms, different marks are carried out on the chest backboard of the male worms in the control group and the processing group, 15 marked male worms are respectively placed in the same worm raising cage, 15 female worms which are in sexual maturity (12 days after emergence) and are not mated are placed in each cage, the process is repeated for 5 times (cages), and the number of male worms which are successfully mated in each cage is observed and recorded (successful mating means that the male and female worms have mating phenomenon and the duration is longer than 30 min).

Experimental results showed that the male insect rectum pH was significantly reduced with carbonic anhydrase inhibitors (fig. 2). Sex pheromone levels (fig. 3) and mating ability of males fed with carbonic anhydrase inhibitors were both significantly reduced (fig. 4). These results indicate that mature male intrarectal carbonic anhydrase can regulate sex pheromone synthesis by controlling rectal pH.

Example 3 screening of specifically expressed CA Gene in the rectum of mature Male Bactrocera dorsalis

Considering that male worms release sex pheromones only at night in the sexual maturity period, to further screen genes regulating the pH of the rectum, we performed transcriptome sequencing analysis on the rectum of male worms at different development periods and different time points in the day of sexual maturity male worms. Specific procedures were performed by selecting healthy Bactrocera dorsalis from 4:00, 8:00, 12:00, 16:00, 20:00 and 24:00 growth at day ages 0, 3, 6, 9 and 12 after eclosion and sexual maturation (day age 12 after eclosion), dissecting out the rectal glands into sterile, sterile centrifuge tubes containing 200. Mu.L TRIzol reagent, 10 rectal glands per tube, 5 replicates per treatment. Grinding with a tissue grinder. The samples were then subjected to RNA extraction using the TRIzol method. The quality-acceptable samples were then sent to the Guangzhou Keto Biotechnology Inc. platform for transcriptome sequencing. The double-ended RNA-seq library was constructed according to the library construction protocol of Illumina. The library was sequenced on an Illumina HiSeq2000 platform (Illumina, USA). Prior to assembly, the original reads from each cDNA library were filtered to remove adaptors, low quality sequences, and microbial contaminated reads. CLEAN READS was mapped to the reference genome by creating an index to the bactrocera dorsalis reference genome using HISAT2.2.4 software, set to "ra-STRANDNESS RF", and the other parameters were defaults. The expression level of each gene was expressed as FPKM using STRINGTIE. Differential gene expression analysis was performed using DESeq2 software. We determined the gene whose FDR (false discovery rate) value is less than 0.01 and whose absolute value of fold difference is greater than or equal to 2 as the differentially expressed gene. Based on the results of genome annotation, CA genes were further selected from the differentially expressed genes.

In this example, the CA gene was first selected from the differentially expressed genes of the Male Bactrocera dorsalis at different developmental stages. Of the 5 CA genes, 2 CA genes (numbers 2247 and 4179) were expressed in the rectum of male worms progressively increased with the developmental days, and were significantly highly expressed at sexual maturity (12 days of age after eclosion) (fig. 5). The expression levels of the 2 CA (2247 and 4179) genes at different developmental stages were then respectively correlated with the pH values at different developmental stages, and the expression in the rectum was found to be highly positively correlated with the rectal pH at different developmental stages (FIG. 6). For further screening of CA gene, according to the differential expression gene heat map of sexually mature male rectum CA gene at different time points, of 2247 and 4179 genes, only 2247 had significantly high expression at 8 late (mating peak period) (FIG. 7), and the expression of 2247 gene was moderately positively correlated with rectum pH (A in FIG. 8), while the expression of 4179 gene was less correlated with rectum pH (B in FIG. 8). Considering that rectum is used as a sex pheromone synthesis part, the invention carries out quantitative PCR analysis on the expression quantity of 2247 and 4179 of different parts of sex mature (12 days of age after eclosion) male worms so as to further screen genes. Specifically, primers used for synthesis were designed based on CDs regions of Bactrocera dorsalis 2247 gene and 4179 gene using primers in NCBI. The designed primers were then sent to Guangzhou Biotechnology Inc. for synthesis for subsequent use. The primers and sequences required for quantitative PCR are shown in Table 1.

TABLE 1 primers and sequences required for quantitative PCR

The male sex mature (12 days of day after emergence) Bactrocera dorsalis with healthy growth was selected, and the head, chest, abdomen, midgut and rectum of the male were dissected and placed in sterile, sterile centrifuge tubes containing 200 μl TRIzol reagent, 5 tissues per tube, and 5 replicates per tissue. Grinding with a tissue grinder. Then, the TRIzol method is used to extract RNA from the sample, reverse transcribe it into cDNA, and RT-qPCR method is used to detect 2247 (primer 2247-F/2247-R) gene relative expression quantity, and the internal reference gene primer is alpha-TUB. Considering the rectum as the synthesis site of sex pheromone, analysis of different tissue-specific expression showed that 2247 gene was specifically expressed in rectum of sex mature bactrocera dorsalis (fig. 9). These results indicate that 2247 gene may be a key gene for regulating synthesis of rectum pH control pheromone, and that 2247 gene is named as Bactrocera dorsalis CA1 (BdCA 1) gene.

Example 4 construction of the carbon anhydrase phylogenetic Tree of Bactrocera dorsalis and protein sequence analysis

Amino acid sequence phylogenetic analyses of 17 carbonic anhydrase genes were performed in 6 species including Drosophila melanogaster (Drosophila melanogaster), bactrocera dorsalis (Bactrocera dorsalis), aedes aegypti (AEDES AEGYPTI), tobacco hornworm (Manduca sexta), western bee (APIS MELLIFERA), and Philippine Giraldii (Tribolium castaneum). The developmental tree was constructed using MEGA11 and Maximum Likelihood (ML), CA amino acid sequences were analyzed using Poisson model and uniformity, and examined by Bootstrap (500 replicates). These 17 genes are divided into two major families, and the CA gene of Bactrocera dorsalis belongs to class alpha (FIG. 10).

Example 5 Gene silencing assay of Bactrocera dorsalis BdCA gene

(1) Design of primer related to gene BdCA of bactrocera dorsalis

Based on the results described above, bdCA genes were subsequently selected for further gene silencing experiments. The nucleotide sequence of BdCA gene is shown as SEQ ID NO.1, the length is 1242bp, the nucleotide sequence of open reading frame (CDs sequence) is shown as SEQ ID NO.2, the length is 819bp, and the coding amino acid sequence is shown as SEQ ID NO. 3. According to CDs region of the Bactrocera dorsalis BdCA gene, primers in NCBI are used for designing and synthesizing the Primer. The designed primers were then sent to Guangzhou Biotechnology Inc. for synthesis for subsequent use. The primers used and their sequences are shown in Table 2.

TABLE 2 primers for Gene silencing assay and sequences thereof

Note that N represents A, T, C, G bases.

(2) Synthesis of Bactrocera dorsalis BdCA gene dsRNA

The male Bactrocera dorsalis with healthy growth is selected, the rectum gland is dissected out and placed in a sterile and asepsis centrifugal tube containing 200 mu L TRIzol reagent, and 10 rectum glands are arranged in each centrifugal tube for 5 times of repetition. Grinding with a tissue grinder. The samples were then subjected to RNA extraction using the TRIzol method. The RNA extracted was reverse transcribed into first strand cDNA using PRIMESCRIPT RT REAGENT KIT WITH G DNA ERASER (TaKaRa) reagents according to the instructions, and the CDs sequence of BdCA was amplified by PCR using the cDNA as a template and the designed primer dsCA-F/dsCA 1-R. The PCR products were electrophoretically detected on a 1% (W/V) agarose gel prepared with 1 XTAE buffer. The recovered fragments were then ligated into Blunt vector using agarose gel recovery kit (OMEGA Gel Extraction Kit kit), the ligation system was transformed into competent cells (pEASY-Blunt Zero Cloning Kit), 8 positive clones were picked for plasmid recovery and sent to sequencing company for sequencing. PCR amplification was performed using the plasmid with the correct sequencing result as a template and the designed primer (dsCA-F/dsCA-1-R). The PCR product obtained by amplification is synthesized into ssRNA according to the using method of the kit T7 RiboMAX ^TM Express RNAI SYSTEM, dsRNA is produced by hybridization of the ssRNA, and the specific operation method is referred to the specification. The resulting dsRNA was stored in a-80℃refrigerator for use in subsequent assay steps.

(3) Injection of Bactrocera dorsalis BdCA gene dsRNA

Sexually mature (12 days after emergence) males were selected for injection for phenotype observation. The synthesized dsRNA was injected into the abdomen of male worms using a microinjector at 1. Mu.L (2000. Mu.g) dsCA.sup.1 and dsGFP, respectively. The breeding conditions of bactrocera dorsalis after injection are the same as the wild type (light: dark time=14h:10h, temperature 25+ -2 ℃ and humidity 65%), and experiments are carried out after 48 hours.

(4) Bactrocera dorsalis BdCA gene interference efficiency detection

Total RNA extraction was performed by collecting male rectal glands injected dsGFP and dsCA1 respectively, 5 biological replicates per group were injected dsGFP and dsCA for treatment, 10 test insects per biological replicate were each, and reverse transcribed into cDNA, and the relative expression levels of BdCA1 were detected by RT-qPCR. Wherein the target gene primer is 2247-F/2247-R, and the internal reference gene primer is alpha-TUB-F/alpha-TUB-R. The results showed that the treated group BdCA had significantly reduced gene expression compared to the control group (a in fig. 11).

Meanwhile, in order to further detect the interference efficiency after dsCA is injected, the male worms interfering with BdCA1 genes are subjected to rectal carbonic anhydrase activity detection, specifically, 0.05g of male worms rectum of a control group and a treatment group are respectively dissected and collected, carbonic anhydrase activity is extracted according to the instruction of use by adopting a carbonic anhydrase activity micro-method detection kit (general fruit, shanghai China), and then carbonic anhydrase activity in a sample is detected by using an enzyme-labeled instrument (BioTek Synergy H1, american). As a result, the carbonic anhydrase activity was significantly decreased after dsCA a injection (B in FIG. 11), and the enzyme activity detection synchronously verified the down-regulation of the carbonic anhydrase gene expression by dsCA a.

Example 6 relationship of Bactrocera dorsalis BdCA.citri gene expression and symbiotic pheromone

To further determine whether the bactrocera dorsalis rectal BdCA1 gene affects symbiotic pheromone synthesis by controlling intestinal pH, the following experiment was performed after interfering with mRNA expression of BdCA gene by RNAi technology:

(1) The pH value in the male rectum after dsCA injections was measured, and the rectum of 10 control group and 10 treated group male were dissected and collected, respectively, and placed in a 1.5mL centrifuge tube containing 30. Mu.L of sterile, enzyme-free water. After grinding the sample, it was centrifuged at 12,000rpm for 10min. The centrifuged supernatant was transferred to a clean centrifuge tube, and the pH of the supernatant was measured using a micropipette (LabSen 241-3 SP).

(2) The levels of sex pheromone in the rectum of the male worms after dsCA injections were detected, the rectum of 60 control groups and 60 treated groups of male worms were dissected and collected, respectively, and placed in a brown chromatographic bottle containing 500. Mu.L of chromatographically pure hexane. Leaching for 24h in a shaker at 16℃and 200 rpm. The samples were then tested using an Agilent gas chromatograph-mass spectrometer (GC-MS). The system is Agilent 7890B/5977B-GC/MSD. The gas chromatographic column is HP-5MS capillary column. The chromatographic conditions include sample inlet temperature 240 deg.c, helium as carrier gas, column flow rate 1mL/min, split flow ratio 10:1, column temperature programming of initial temperature 50 deg.c, maintaining for 1min, raising temperature to 140 deg.c at 5 deg.c/min and maintaining for 3min at 10 deg.c/min to 250 deg.c. The mass spectrum conditions are EI power source, electron energy 70eV, ion source temperature 230 ℃, interface temperature 280 ℃ and scanning mass range 45-450amu. The detected results were compared using data provided by NIST17.0 spectra library and trimethylpyrazine and tetramethylpyrazine were determined by comparing the retention time and mass spectra.

(3) Detecting the mating ability of male worms after dsCA injections (dsCA), marking the chest backboard of male worms of a control group and a treatment group differently, placing 15 marked male worms in the same worm raising cage, simultaneously placing 15 sexually mature female worms which are not mated in each cage, repeating for 5 times (cages), and observing and recording the number of male worms which are successfully mated in each cage (the successful mating means that the male and female worms have mating phenomenon and the duration is more than 30 minutes).

The experimental results show that after successful reduction of BdCA gene expression in the bactrocera dorsalis rectum (a in fig. 11), carbonic anhydrase activity (B in fig. 11) and pH value (fig. 12) in the bactrocera dorsalis rectum are significantly reduced. Sex pheromone content (fig. 13) and male mating ability in the bactrocera dorsalis rectum were significantly reduced (fig. 14). These results indicate that BdCA gene is a key gene affecting the fertility of bactrocera dorsalis by regulating sex pheromone synthesis in the rectum.

Claims

1. Use of the BdCA1 gene in controlling Bactrocera dorsalis, wherein the BdCA1 gene encodes a protein with an amino acid sequence as shown in SEQ ID NO.3.

2. The use according to claim 1, characterized in that the nucleotide sequence of the CDS sequence of the BdCA1 gene is shown as SEQ ID NO.2.

3. The use according to claim 1, characterized in that the pH and synthesis of sex pheromones in the rectum of sexually mature fruit flies are reduced by reducing the expression level of the BdCA1 gene, thereby reducing the mating ability of sexually mature fruit flies.

4. The use according to claim 3, characterized in that the reducing the expression level of the BdCA1 gene is achieved by knocking out the BdCA1 gene or reducing the expression level of the BdCA1 gene by gene silencing technology.

5. Use of a carbonic anhydrase inhibitor in controlling Bactrocera dorsalis, characterized in that the carbonic anhydrase inhibitor is acetazolamide which inhibits the activity of BdCA1 protein, and the amino acid sequence of the BdCA1 protein is shown in SEQ ID NO.3.

6. The use according to claim 5, characterized in that the carbonic anhydrase activity in the rectum of sexually mature fruit flies is inhibited by feeding carbonic anhydrase inhibitors, so that the pH in the rectum and the synthesis of sex pheromones are reduced, thereby reducing the mating ability of sexually mature fruit flies.

7. The use according to claim 5, characterized in that the concentration of acetazolamide is above 0.01 g/mL.

8. The use according to claim 6, characterized in that the sexually mature Bactrocera dorsalis is a male Bactrocera dorsalis that is 12 days old after emergence.

9. A method for controlling Bactrocera dorsalis, characterized in that it comprises the steps of reducing the expression level of the BdCA1 gene according to claim 1 or feeding a carbonic anhydrase inhibitor, wherein the carbonic anhydrase inhibitor is acetazolamide.