Application of BdCA1 gene in controlling Bactrocera dorsalis

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

Technical Field

[0001] The invention belongs to the field of biotechnology and green pest control, and specifically relates to the application of the BdCA1 gene in controlling the citrus fruit fly. Background Art

[0002] Bactrocera dorsalis (Hendel), a member of the Tephritidae family of the order Diptera, is commonly known as the "needle bee". It originated in Asia and is a global quarantine pest. The insect has a very strong reproductive capacity, and a single female insect can lay 400 to 1,800 eggs in its lifetime. The female insect lays eggs inside the fruit. After the eggs hatch, the larvae feed on the fruit, causing it to rot or turn yellow and fall off, causing serious economic losses to the fruit and vegetable industry. Considering the harmful characteristics of the Bactrocera dorsalis, the prevention and control time should be chosen before laying eggs. For insects with the ability to migrate, the breeding period is a good time for prevention and control. During mating, insects rely on sex pheromones to identify and locate the opposite sex, so sex pheromones are crucial for insect reproduction. Insect sex pheromones are volatile chemicals released by insect secretory glands that can stimulate individuals of the opposite sex of the same species to produce courtship and corresponding physiological reactions. Previous studies have found that the rectal glands of male Bactrocera dorsalis can synthesize and release sex pheromones that attract females. These sex pheromones are synthesized with the help of Bacillus in the rectal glands. However, whether the physiological activities of the symbiotic bacteria Bacillus are regulated by the host and how the host regulates them still need further exploration.

[0003] Carbonic anhydrase (CA) is an enzyme widely found in bacteria, archaea and eukaryotes, which catalyzes the important physiological reaction of carbon dioxide hydration to bicarbonate and protons. Whether it plays a role in regulating insect sex pheromones remains unknown. Summary of the invention

[0004] The present invention has confirmed for the first time that host insects can regulate the physiological activities of symbiotic bacteria in the body by controlling the expression of conservative carbonic anhydrase genes. Specifically, after feeding carbonic anhydrase inhibitors and using RNAi technology to interfere with the expression of the BdCA1 gene of male citrus fruit flies, the pH value in the rectum decreases, thereby reducing the activity of the symbiotic bacteria Bacillus in the rectum, resulting in a significant decrease in the level of rectal synthetic pheromones and mating competitiveness. This shows that the expression of carbonic anhydrase is related to changes in the pH value of the insect rectum, and acidic conditions will inhibit the synthesis of symbiotic bacteria Bacillus, thereby affecting the mating ability of male insects. Therefore, the BdCA1 gene can be used as a green control target gene for citrus fruit flies.

[0005] The first object of the present invention is to provide an application of the BdCA1 gene in controlling the citrus fruit fly, characterized in that the BdCA1 gene encodes a protein with an amino acid sequence as shown in SEQ ID NO.3.

[0006] Preferably, the nucleotide sequence of the BdCA1 gene is as shown in SEQ ID NO.2.

[0007] Preferably, by reducing the expression level of the BdCA1 gene, the pH and synthesis of sex pheromones in the rectum of sexually mature Bactrocera dorsalis are reduced, thereby reducing the mating ability of sexually mature Bactrocera dorsalis.

[0008] Preferably, the reducing the expression level of the BdCA1 gene is by knocking out the BdCA1 gene or reducing the expression level of the BdCA1 gene by gene silencing technology.

[0009] The second object of the present invention is to provide a use of a carbonic anhydrase inhibitor in controlling Bactrocera dorsalis.

[0010] Preferably, the use inhibits the carbonic anhydrase activity in the rectum of sexually mature fruit flies by feeding carbonic anhydrase inhibitors, thereby reducing the pH in the rectum and the synthesis of sex pheromones, thereby reducing the mating ability of sexually mature fruit flies.

[0011] Preferably, the carbonic anhydrase inhibitor is acetazolamide, and the concentration is above 0.01 g / mL.

[0012] Preferably, the carbonic anhydrase inhibitor is an inhibitor that inhibits the activity of the BdCA1 protein, and the amino acid sequence of the BdCA1 protein is shown in SEQ ID NO.3.

[0013] Preferably, the sexually mature Bactrocera dorsalis is a male Bactrocera dorsalis that is 12 days old after emergence.

[0014] The third object of the present invention is to provide a method for controlling Bactrocera dorsalis, comprising the steps of reducing the expression level of the BdCA1 gene or feeding a carbonic anhydrase inhibitor, wherein the carbonic anhydrase inhibitor is an inhibitor that inhibits the activity of the protein having an amino acid sequence as shown in SEQ ID NO.3.

[0015] Beneficial effects of the present invention:

[0016] The present invention first discovered that when feeding carbonic anhydrase inhibitors, the pH value in the rectum of the citrus fruit fly will change from alkaline to acidic, which will inhibit the synthesis of sex pheromones by its rectal symbiotic bacteria and reduce its fertility. The present invention uses RNAi technology in the citrus fruit fly to significantly reduce the mRNA expression of the carbonic anhydrase gene (BdCA1) of the citrus fruit fly, and the pH is significantly reduced, so that the level of sex pheromones and mating competitiveness are significantly reduced. In the preparation of pest control agents for insect control, insect carbonic anhydrase inhibitors (substances that reduce carbonic anhydrase activity, or substances that degrade carbonic anhydrase, or substances that reduce the expression level of carbonic anhydrase) can be used, or RNAi technology can be used to reduce the mRNA expression of the BdCA1 gene, and the intestinal tract will form an acidic environment to inhibit the synthesis of sex pheromones by the symbiotic bacteria Bacillus, so that the fertility of male citrus fruit flies is significantly reduced, and the subsequent female insects lay eggs to control the harm to fruits, and in the control process, no drug resistance will be generated, no harm to humans and animals, and no pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a correlation analysis diagram of the rectal carbonic anhydrase activity and pH value of the male citrus fruit fly; wherein, A is a correlation analysis diagram of the rectal carbonic anhydrase activity and pH value of the male citrus fruit fly at different developmental stages, and B is a correlation analysis diagram of the rectal carbonic anhydrase activity and pH value of the male citrus fruit fly at different time points at 12 days old after eclosion; Spearman was used for correlation analysis of the samples.

[0018] Figure 2 This is the result of rectal pH value of male Bactrocera dorsalis after feeding carbonic anhydrase inhibitor; **** represents the significant difference at the level of P<0.0001 in the independent sample T test.

[0019] Figure 3 The following are the results of the content of sex pheromone in the rectum of male Bactrocera dorsalis after feeding with carbonic anhydrase inhibitors; A is the result of TMP content, and B is the result of TTMP content; ** represents the significant difference at the level of P<0.01 in the independent sample T test; **** represents the significant difference at the level of P<0.0001 in the independent sample T test.

[0020] Figure 4 This is a diagram showing the mating competition results of male Bactrocera dorsalis fed with carbonic anhydrase inhibitors; paired sample T test was used, and ** represents a significant difference at the level of P<0.01.

[0021] Figure 5 This is a heat map of differentially expressed CA genes at different developmental stages of male Bactrocera dorsalis.

[0022] Figure 6It is a correlation analysis diagram of the differentially expressed CA gene of the citrus fruit fly and the pH value; wherein, A is a correlation analysis diagram of the 2247 genes differentially expressed in the rectum of the citrus fruit fly male at different developmental stages and the pH value; B is a correlation analysis diagram of the 4179 genes differentially expressed in the rectum of the citrus fruit fly male at different developmental stages and the pH value; Spearman was used for correlation analysis of the samples.

[0023] Figure 7 This is a heat map of CA genes differentially expressed at different times in sexually mature male Bactrocera dorsalis.

[0024] Figure 8 It is a correlation analysis diagram of CA genes differentially expressed in the rectum of sexually mature male fruit flies and pH values; wherein, A is a correlation analysis diagram of 2247 genes differentially expressed in the rectum of sexually mature male fruit flies at different times and pH values; B is a correlation analysis diagram of 4179 genes differentially expressed in the rectum of sexually mature male fruit flies at different times and pH values; Spearman was used for correlation analysis of samples.

[0025] Fig. 9 This is a heat map of the relative expression levels of 2247 genes and 4179 genes in different parts of sexually mature male Bactrocera dorsalis.

[0026] Fig.10 is the phylogenetic tree of CA proteins in six species.

[0027] Fig.11 The figures are the relative expression level of BdCA1 and CA enzyme activity after silencing the BdCA1 gene in sexually mature male Bactrocera dorsalis; A is the result graph of BdCA1 gene expression after injection of dsCA1 into sexually mature Bactrocera dorsalis; B is the result graph of carbonic anhydrase activity after injection of dsCA1; *** represents significant difference at the level of P<0.001 in the independent sample T test; * represents significant difference at the level of P<0.05 in the independent sample T test.

[0028] Fig.12 This is the result of rectal pH value of sexually mature male Bactrocera dorsalis after injection of dsCA1; *** represents independent sample T test P<0.001.

[0029] Fig.13 The following are the results of the content of sex pheromone in the rectum of sexually mature male Bactrocera dorsalis after injection of dsCA1; A is the result of TMP content, and B is the result of TTMP content; *** represents the significant difference at the P<0.001 level by the independent sample T test; * represents the significant difference at the P<0.05 level by the independent sample T test.

[0030] Fig.14This is a diagram showing the mating competition results of sexually mature male Bactrocera dorsalis after injection of dsCA1; paired sample T test was used, and *** represents a significant difference at the level of P<0.001. DETAILED DESCRIPTION

[0031] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0032] Example 1: Relationship between carbonic anhydrase in the rectum of Bactrocera dorsalis and rectal pH

[0033] In order to verify whether the expressed carbonic anhydrase gene affects the pH value in the rectum of Bactrocera dorsalis, we measured the carbonic anhydrase activity and pH value of the rectum of male insects at different developmental stages (0, 3, 6, 9 and 12 days after eclosion) and sexual maturity (12 days after eclosion) at different time points (4:00, 8:00, 12:00, 16:00, 20:00 and 24:00) and compared their correlations using Spearman correlation analysis. The specific operations are as follows:

[0034] (1) In order to determine the carbonic anhydrase activity in the rectum of male insects at different developmental stages and time periods, 0.05 g of male rectum was collected by dissection at the above time points, and carbonic anhydrase was extracted using a carbonic anhydrase activity microassay kit (Tongwei, Shanghai, China) according to the instruction manual. The carbonic anhydrase activity in the sample was then determined using a microplate reader (BioTek Synergy H1, America).

[0035] (2) In order to determine the rectal pH value of male insects at different developmental stages and time periods, the rectal tissues of 10 male insects were collected by dissection at the above time and placed in a 1.5 mL centrifuge tube containing 30 μL of sterile enzyme-free water. After the sample was ground, it was centrifuged at 12,000 rpm for 10 min. The supernatant after centrifugation was transferred to a clean centrifuge tube, and then the pH value of the supernatant was measured using a micro pH meter (LabSen241-3SP).

[0036] The correlation analysis between the rectal carbonic anhydrase activity and pH value of male insects at different developmental stages showed that the rectal carbonic anhydrase activity of male insects at different developmental stages was highly positively correlated with the rectal pH value ( Figure 1 A in the figure). Correlation analysis was performed on the rectal carbonic anhydrase activity and pH value of sexually mature (12 days after eclosion) male insects at different time periods. The results showed that the rectal carbonic anhydrase activity of sexually mature male insects was moderately positively correlated with the rectal pH value within one day ( Figure 1 This indicates that the expression of carbonic anhydrase gene in the rectum of male Bactrocera dorsalis is related to the pH value of the rectum.

[0037] Example 2: Relationship between carbonic anhydrase activity of Bactrocera dorsalis and symbiotic bacteria synthesis of sex pheromones

[0038] In order to verify whether carbonic anhydrase regulates the physiological activities of the commensal bacterium Bacillus by controlling the rectal pH, this example uses a carbonic anhydrase inhibitor (acetazolamide) to reduce the activity of carbonic anhydrase and then detects various indicators in the intestine.

[0039] The specific operation is as follows: On the basis of normal feeding, sexually mature (12 days after eclosion) males were fed water containing acetazolamide (0.01 g / mL), and males raised normally were used as controls. Subsequent experiments were carried out 24 hours after feeding.

[0040] (1) To detect the pH value in the rectum of male insects, the rectums of 10 control group and 10 treatment group male insects were collected by dissection and placed in a 1.5 mL centrifuge tube containing 30 μL of sterile enzyme-free water. After the sample was ground, it was centrifuged at 12,000 rpm for 10 min. The supernatant after centrifugation was transferred to a clean centrifuge tube, and then the pH value of the supernatant was measured using a micro pH meter (LabSen241-3SP).

[0041] (2) In order to detect the level of sex pheromone in the male rectum, the rectums of 60 male insects in the control group and 60 male insects in the treatment group were dissected and collected, and placed in a brown chromatographic bottle containing 500 μL of chromatographic grade n-hexane. The samples were extracted in a shaker at 16°C and 200 rpm for 24 hours. The samples were then detected using an Agilent gas chromatograph-mass spectrometer (GC-MS). The system was Agilent 7890B / 5977B-GC / MSD. The gas chromatography column was an HP-5MS capillary column. Chromatographic conditions: injection port temperature 240°C; carrier gas was helium; column flow rate 1 mL / min; split injection, split ratio 10:1; column temperature program: starting temperature 50°C, maintained for 1 min, increased to 140°C at a rate of 5°C / min, and then increased to 250°C at a rate of 10°C / min and maintained for 3 min. Mass spectrometry conditions: EI power source, electron energy 70 eV, ion source temperature 230 ° C, interface temperature 280 ° C, scanning mass range 45-450 amu. The detected results were compared with the data provided by the NIST17.0 spectral library, and trimethylpyrazine (TMP) and tetramethylpyrazine (TTMP) were determined by comparing the retention time and mass spectrum.

[0042] (3) In order to detect the mating ability of male insects, the pronotum of male insects in the control group and the treatment group were marked differently. Fifteen marked male insects were placed in the same insect cage. At the same time, 15 sexually mature (12 days after eclosion) and unmated female insects were placed in each cage. This was repeated five times (cage). The number of successfully mated male insects in each cage was observed and recorded (successful mating refers to the presence of mating between male and female and the duration of mating is greater than 30 minutes).

[0043] The results showed that the pH value of the rectum of male worms fed with carbonic anhydrase inhibitors was significantly reduced ( Figure 2 ) The sex pheromone levels of male insects fed with carbonic anhydrase inhibitors ( Figure 3 ) and mating ability were significantly reduced ( Figure 4 ). These results suggest that carbonic anhydrase in the rectum of mature males can regulate the synthesis of sex pheromones by controlling rectal pH.

[0044] Example 3: Screening of CA genes specifically expressed in the rectum of sexually mature male fruit flies

[0045] Considering that male insects only release sex pheromones at night during sexual maturity, in order to further screen genes that regulate rectal pH, we performed transcriptome sequencing analysis on the rectum of male insects at different developmental stages and at different time points of the day in sexually mature male insects. The specific operation was as follows: healthy male Bactrocera dorsalis insects were selected at 0, 3, 6, 9 and 12 days after eclosion and at 4:00, 8:00, 12:00, 16:00, 20:00 and 24:00 when sexually mature (12 days after eclosion), and the rectal glands were dissected out and placed in sterile enzyme-free centrifuge tubes containing 200 μL TRIzol reagent, with 10 rectal glands per centrifuge tube, and each treatment was repeated 5 times. Grind with a tissue grinder. Then, RNA was extracted from the samples using the TRIzol method. Then, qualified samples were sent to the platform of Guangzhou Kidio Biotechnology Co., Ltd. for transcriptome sequencing. A double-end RNA-seq library was constructed according to the Illumina library construction protocol. The libraries were sequenced on the Illumina HiSeq2000 platform (Illumina, USA). Before assembly, the raw reads from each cDNA library were filtered to remove adapters, low-quality sequences, and reads contaminated by microorganisms. By establishing an index for the reference genome of Bactrocera dorsalis, the clean reads were mapped to the reference genome using HISAT2.2.4 software, set to "rna-strandness RF", and other parameters were default. The expression level of each gene was expressed in FPKM using StringTie. Differential gene expression analysis was performed using DESeq2 software. We identified genes with a FDR (false discovery rate) value less than 0.01 and an absolute value of the difference fold ≥ 2 as differentially expressed genes. Based on the results of the genome annotation, CA genes were further screened out from the differentially expressed genes.

[0046] In this example, CA genes were first screened from differentially expressed genes in male Bactrocera dorsalis at different developmental stages. Among the five CA genes, two CA genes (numbered 2247 and 4179) were expressed in the rectum of male insects gradually increasing with the number of development days, and were significantly highly expressed at sexual maturity (12 days after eclosion). Figure 5). Subsequently, the expression levels of the two CA (2247 and 4179) genes at different developmental stages were correlated with the pH values ​​at different developmental stages, and it was found that the expression in the rectum was highly positively correlated with the rectal pH at different developmental stages ( Figure 6 ). To further screen CA genes, according to the heat map of differentially expressed genes at different time points in the rectum of sexually mature male insects, only 2247 had a significant high expression at 8 pm (the peak of mating) among 2247 and 4179 genes ( Figure 7 ), and the expression of 2247 genes was moderately positively correlated with rectal pH ( Figure 8 A in Figure ), while the expression of 4179 genes was less correlated with rectal pH ( Figure 8 B). Considering that the rectum is the site of sex pheromone synthesis, the present invention performs quantitative PCR analysis on the expression levels of 2247 and 4179 in different parts of sexually mature (12 days after eclosion) male insects to further screen genes. The specific operation is: according to the CDs region of the 2247 gene and the 4179 gene of the citrus fruit fly, the primers used for synthesis are designed and synthesized using the Primer in NCBI. The designed primers are then sent to Guangzhou Shenggong Biological Co., Ltd. for synthesis for subsequent use. The primers and their sequences required for quantitative PCR are shown in Table 1.

[0047] Table 1 Primers and their sequences required for quantitative PCR

[0048]

[0049] Healthy and sexually mature (12 days after eclosion) male Bactrocera dorsalis were selected, and the head, thorax, abdomen, midgut and rectum of the male were dissected and placed in sterile enzyme-free centrifuge tubes containing 200 μL TRIzol reagent, with 5 tissues in each centrifuge tube, and each tissue was repeated 5 times. Grind with a tissue grinder. Subsequently, RNA was extracted from the sample using the TRIzol method and reversely transcribed into cDNA. The relative expression of the 2247 gene (primers 2247-F / 2247-R) was detected by RT-qPCR, and the internal reference gene primer was α-TUB. Considering that the rectum is the site of synthesis of sex pheromones, the analysis of specific expression in different tissues showed that the 2247 gene was specifically expressed in the rectum of sexually mature male Bactrocera dorsalis ( Fig. 9 ). These results indicate that gene 2247 may be a key gene for regulating rectal pH to control the synthesis of sex pheromones; gene 2247 was named Bactrocera dorsalis CA1 (BdCA1) gene.

[0050] Example 4: Phylogenetic tree construction and protein sequence analysis of Bactrocera dorsalis carbonic anhydrase

[0051] Amino acid sequence phylogenetic analysis was performed on 17 carbonic anhydrase genes from six species, including Drosophila melanogaster, Bactrocera dorsalis, Aedes aegypti, Manduca sexta, Apis mellifera, and Tribolium castaneum. The phylogenetic tree was constructed using MEGA11 and the maximum likelihood method (ML). The CA amino acid sequences were analyzed using the Poisson model and Uniformrates, and tested by bootstrap (500 replicates). The 17 genes were divided into two major families. The CA gene of Bactrocera dorsalis belongs to the α class ( Fig.10 ).

[0052] Example 5: Gene silencing test of Bactrocera dorsalis BdCA1 gene

[0053] (1) Design of primers related to the BdCA1 gene of Bactrocera dorsalis

[0054] According to the above results, the BdCA1 gene was subsequently selected for further gene silencing experiments. The nucleotide sequence of the BdCA1 gene is shown in SEQ ID NO.1, which is 1242bp long; the nucleotide sequence of its open reading frame (i.e., CDs sequence) is shown in SEQ ID NO.2, which is 819bp long and encodes a protein with an amino acid sequence as shown in SEQ ID NO.3. According to the CDs region of the BdCA1 gene of Bactrocera dorsalis, the primers used were designed and synthesized using Primer in NCBI. The designed primers were then sent to Guangzhou Sangon Biotech Co., Ltd. for synthesis for subsequent use. The primers used and their sequences are shown in Table 2.

[0055] Table 2 Primers and their sequences used in gene silencing experiments

[0056]

[0057] Note: N represents any base of A, T, C, or G.

[0058] (2) Synthesis of dsRNA of Bactrocera dorsalis BdCA1 gene

[0059] Healthy male Bactrocera dorsalis was selected, and the rectal glands were dissected out and placed in sterile enzyme-free centrifuge tubes containing 200 μL TRIzol reagent, with 10 rectal glands in each centrifuge tube, and repeated 5 times. Grind with a tissue grinder. Then, RNA was extracted from the sample using the TRIzol method. PrimeScript RT reagent Kit with g DNA Eraser (TaKaRa) reagent was used to reverse transcribe the extracted RNA into the first-strand cDNA according to the instructions. With this cDNA as a template, the CDs sequence of BdCA1 was amplified by PCR using the designed primers dsCA1-F / dsCA1-R. The PCR product was detected by electrophoresis using 1% (W / V) agarose gel prepared with 1×TAE buffer. Then, the agarose gel recovery kit was used to recover the fragments (OMEGA GelExtraction Kit), and the recovered fragments were connected to the Blunt vector. The ligation system was transformed into competent cells (pEASY-Blunt Zero Cloning Kit), and 8 positive clones were selected for plasmid recovery and sent to the sequencing company for sequencing. The plasmid with the correct sequencing result was used as a template and PCR amplification was performed using the designed primers (dsCA1-F / dsCA1-R). The PCR product obtained by amplification was transferred to the T7 RiboMAX TM The method of using the Express RNAi System is to first synthesize ssRNA and then generate dsRNA by hybridization of ssRNA. The specific operation method is referred to the instruction manual. The obtained dsRNA is stored in a -80℃ refrigerator for subsequent experimental steps.

[0060] (3) Injection of dsRNA of Bactrocera dorsalis BdCA1 gene

[0061] Sexually mature (12 days after eclosion) males were selected for injection to observe the phenotype. The synthesized dsRNA was injected into the abdomen of the males using a microsyringe to inject 1 μL (2000 μg) of dsCA1 and dsGFP respectively. After injection, the breeding conditions of the citrus fruit fly were the same as those of the wild type (light: dark time = 14h:10h, temperature 25±2℃, humidity 65%), and the experiment was carried out 48 hours later.

[0062] (4) Detection of the efficiency of Bactrocera dorsalis BdCA1 gene interference

[0063] The rectal glands of male worms injected with dsGFP and dsCA1 were collected for total RNA extraction. The control group was injected with dsGFP and the treatment group was injected with dsCA1. There were 5 biological replicates in each group, and 10 test worms in each biological replicate. The total RNA was reverse transcribed into cDNA, and the relative expression of BdCA1 was detected by RT-qPCR. The target gene primers were 2247-F / 2247-R, and the reference gene primers were α-TUB-F / α-TUB-R. The results showed that compared with the control group, the expression of BdCA1 gene in the treatment group was significantly reduced ( Fig.11 A).

[0064] At the same time, in order to further detect the interference efficiency after injection of dsCA1, the rectal carbonic anhydrase enzyme activity of male insects after interference with the BdCA1 gene was detected. The specific operation was to dissect and collect 0.05g of the rectum of male insects in the control group and the treatment group, respectively, and use the carbonic anhydrase activity microassay kit (Tongwei, Shanghai, China) to extract carbonic anhydrase according to the instruction manual, and then use a microplate reader (BioTek Synergy H1, America) to determine the carbonic anhydrase activity in the sample. The results showed that the carbonic anhydrase enzyme activity decreased significantly after injection of dsCA1 ( Fig.11 In B), enzyme activity detection simultaneously verified the downregulation of carbonic anhydrase gene expression by dsCA1.

[0065] Example 6: Relationship between Bactrocera dorsalis BdCA1 gene expression and symbiotic bacteria synthesis of sex pheromones

[0066] In order to further determine whether the BdCA1 gene in the rectum of Bactrocera dorsalis affects the synthesis of symbiotic bacteria sex pheromones by controlling the intestinal pH value, this example used RNAi technology to interfere with the mRNA expression of the BdCA1 gene and conducted the following experiments:

[0067] (1) Detection of pH value in the rectum of male insects after injection of dsCA1. The rectums of 10 male insects in the control group and 10 male insects in the treatment group were collected by dissection and placed in a 1.5 mL centrifuge tube containing 30 μL of sterile enzyme-free water. After grinding, the sample was centrifuged at 12,000 rpm for 10 min. The supernatant after centrifugation was transferred to a clean centrifuge tube, and then the pH value of the supernatant was measured using a micro pH meter (LabSen241-3SP).

[0068] (2) To detect the level of neutral pheromone in the rectum of male insects after injection of dsCA1, the rectums of 60 male insects in the control group and 60 male insects in the treatment group were dissected and collected, and placed in a brown chromatographic bottle containing 500 μL of chromatographically pure n-hexane. The samples were extracted in a shaker at 16°C and 200 rpm for 24 hours. The samples were then detected using an Agilent gas chromatograph-mass spectrometer (GC-MS). The system was Agilent 7890B / 5977B-GC / MSD. The gas chromatography column was an HP-5MS capillary column. Chromatographic conditions: injection port temperature 240°C; carrier gas was helium; column flow rate 1 mL / min; split injection, split ratio 10:1; column temperature program: starting temperature 50°C, maintained for 1 min, increased to 140°C at a rate of 5°C / min, and then increased to 250°C at a rate of 10°C / min and maintained for 3 min. Mass spectrometry conditions: EI electric source, electron energy 70eV, ion source temperature 230℃, interface temperature 280℃, scanning mass range 45-450amu. The detected results were compared with the data provided by the NIST17.0 spectral library, and trimethylpyrazine and tetramethylpyrazine were determined by comparing the retention time and mass spectrum.

[0069] (3) To test the mating ability of male insects after injection of dsCA1, the pronotum of male insects in the control group and the treatment group was marked differently. Fifteen marked male insects were placed in the same insect cage. At the same time, 15 sexually mature female insects that had not mated were placed in each cage. This was repeated five times (cages). The number of male insects that successfully mated in each cage was observed and recorded (successful mating refers to the occurrence of mating between male and female and the duration of mating is greater than 30 minutes).

[0070] The experimental results showed that after successfully reducing the expression of the BdCA1 gene in the rectum of the orange fruit fly ( Fig.11 A), carbonic anhydrase activity in the rectum of Bactrocera dorsalis ( Fig.11 B) and pH value ( Fig.12 ) was significantly reduced. The content of sex pheromone in the rectum of Bactrocera dorsalis ( Fig.13 ) and male mating ability decreased significantly ( Fig.14 ). These results indicate that the BdCA1 gene is a key gene that affects the reproductive capacity of the Bactrocera dorsalis by regulating the synthesis of sex pheromones in the rectum.

Claims

1. Application of the BdCA1 gene in controlling Bactrocera dorsalis, characterized in that: 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 in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: By reducing the expression level of the BdCA1 gene, the pH and synthesis of sex pheromones in the rectum of sexually mature citrus fruit flies are reduced, thereby reducing the mating ability of sexually mature citrus 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. 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: By feeding carbonic anhydrase inhibitors, the activity of carbonic anhydrase in the rectum of sexually mature citrus fruit flies is inhibited, so that the pH in the rectum and the synthesis of sex pheromones are reduced, thereby reducing the mating ability of sexually mature citrus 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: The method 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.

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