A biological control method for inhibiting Varroa mite parasitism

Abstract

The present application discloses a biological control method for inhibiting the parasitism of Varroa mites, which belongs to the field of biological control methods and is used for the control of Varroa mites. The method comprises: + ‑ATPase 1, vacuolar H + The expression gene of ATPase 2 was connected to the plasmid vector pBTK800 by restriction digestion to obtain the constructed pDS- Varroa Plasmids and electroporation of bee gut bacteria S.alvi The expression strain is obtained and expanded and then colonized in the bees, and finally one or more of the colonization stability test, the Varroa mite inhibition effect test, and the expression effect test are performed. The present application provides a new therapeutic target for the control of Varroa mites, uses honey bee intestinal engineered bacteria to establish a new plasmid-transmitted dsRNA pathway, significantly reduces the survival rate of honey bee Varroa mites, achieves long-term inhibition of Varroa mite parasitism, and reduces the pollution of the surrounding environment by existing methods.

Description

Technical Field

[0001] The present application belongs to the field of biological control methods, and more specifically, particularly relates to a biological control method for inhibiting Varroa mite parasitism. Background Art

[0002] Varroa destructor Varroa destructor Varroa mites (hereinafter referred to as Varroa mites) are a type of bee external parasitic mite. The globalization of bee trade and translocation of beekeeping have caused Varroa mites to spread rapidly around the world. Varroa mites are the most harmful to Western honey bees. They feed on bee hemolymph and fat bodies, reducing the fat and protein synthesis of bees and thus reducing their weight. They weaken the bees and shorten their lifespan. They are also carriers of bee viruses. Therefore, the prevention and control of Varroa mites is an essential part of bee breeding.

[0003] At present, the main control methods of Varroa mites include acaricide spraying, formic acid fumigation, essential oil disinfecting, etc. The acaricide spraying method is for example the patent document with Chinese patent publication number CN106163279A, which discloses a long-acting acaricide composition and a production method thereof, acaricide strips and a method for controlling Varroa destructor mites, and specifically discloses the following technical contents: the composition comprises oxalic acid, glycerol, tetracarbon dicarboxylic acid and formic acid. Patent document CN106163279A also relates to a cellulose strip embedded in the acaricide composition introduced into a beehive and a production method thereof. An example of the essential oil disinfecting method is the patent document with Chinese patent publication number CN116211915A, which discloses the use of camphor essential oil as a drug for preventing and controlling Varroa destructor mites, and specifically discloses that camphor essential oil has a good killing effect on Varroa destructor mites, the fumigation toxicity can penetrate the wax cover, and the mites can be treated without the need to imprison the queen and kill the offspring of the bee colony; it is highly volatile and has a low residue in bee products in the bee colony; this essential oil makes it difficult for Varroa destructor mites to develop drug resistance; and the cost of camphor essential oil is low.

[0004] However, the treatment schemes in the prior art cannot be effectively applied in the long term in actual use, cannot inhibit the parasitism of Varroa mites for a long time, and are likely to cause pollution to the surrounding environment of the bees. Summary of the invention

[0005] The purpose of the present application is to provide a biological control method for inhibiting the parasitism of Varroa mites, which provides a new therapeutic target for the control of Varroa mites, uses honey bee intestinal engineered bacteria to establish a new plasmid delivery dsRNA pathway, significantly reduces the survival rate of honey bee Varroa mites, achieves long-term inhibition of the parasitism of Varroa mites, and reduces the pollution of the surrounding environment by existing methods.

[0006] To achieve the above objectives, this application is implemented through the following technical solutions:

[0007] A biological control method for inhibiting Varroa mite parasitism described in the present application comprises the following steps:

[0008] S1, the mRNA fragment shown in the gene sequence SEQ ID NO.1 is used as the expression gene of chitin synthase 1, the mRNA fragment shown in the gene sequence SEQ ID NO.2 is used as the expression gene of chitin synthase 2, and the mRNA fragment shown in the gene sequence SEQ ID NO.3 is used as the expression gene of vacuolar H + -ATPase 1 expression gene, the mRNA fragment shown in the gene sequence SEQ ID NO.4 was used as the vacuolar H + -ATPase 2 expression gene was connected to the plasmid vector pBTK800 by enzyme digestion to obtain the constructed pDS- Varroa Plasmid, inserted into pDS- Varroa The expression gene sequence of the plasmid is shown in SEQ ID NO.5;

[0009] S2, the pDS- Varroa Plasmid electroporation into bee gut bacteria S. alv. To obtain the expression strain S. alv. : pDS- Varroa ;

[0010] S3. Expression strain S. alv. : pDS- Varroa Carry out expansion cultivation and colonize the bees after they leave the hive;

[0011] S4. The expression strain obtained in step 2 S. alv. : pDS- Varroa Conduct one or more of the following: colonization stability test, Varroa mite inhibition effect test, and expression effect test.

[0012] As one of the preferred technical solutions, the expression gene of chitin synthase 1, the expression gene of chitin synthase 2, and the vacuolar H + -ATPase 1 expression gene, vacuolar H + -ATPase 2 combination to add restriction sites at both ends of the gene insertion sequence. The company was commissioned to synthesize double-stranded DNA fragments, and the vector ligation kit was used to achieve restriction enzyme cleavage and ligation with the plasmid vector pBTK800 to complete the pDS- Varroa Construction of plasmids.

[0013] As one of the preferred technical solutions, in step S2 described in this application, pDS- Varroa Plasmid electroporation into bee gut bacteria S. alv. Before, the bee intestinal bacteria S. alv. Transformed into competent cells, competent cells were prepared by the following method:

[0014] S21. Honey bee intestinal bacteria S. alv. Columbia blood agar plates were used at 35°C and 5% CO 2 The intestinal symbiotic bacteria of honey bees were isolated from the incubator, and the strain was wkB2;

[0015] S22, the obtained bee intestinal bacteria S. alv. A single colony was added to 50 mL Columbia liquid medium and incubated at 35°C with 5% CO 2 Incubate in an incubator for 48 hours;

[0016] S23, after the incubation in step S32 is completed, the incubation solution is centrifuged at 7000 rpm for 10 min to collect the bacterial precipitate;

[0017] S24. Wash the separated bacterial precipitate three times with autoclaved double distilled water, and finally resuspend the bacterial pellet with 1 mL of autoclaved 10% glycerol, dispense 100 μL into a centrifuge tube, and store in a -80°C refrigerator.

[0018] As one of the preferred technical solutions, in step S2 described in this application, pDS- Varroa Plasmid electroporation into bee gut bacteria S. alv. The electrotransformation method is as follows: the electrotransformation process includes taking out 100 μL of bacterial solution from a -80°C refrigerator, adding 500 ng of plasmid and mixing thoroughly, incubating on ice for 20 min, adding 75 μL of the mixture into a 1 mm electrotransformation cup after the incubation, using a Bio-Rad instrument, after electrical stimulation according to the parameters of voltage 1800 V, capacitance 25 μF, and resistance 1 Ω, the bacterial cells are transferred to 50 mL of Columbia liquid culture medium, and incubated at 35°C, 5% CO 2 After overnight incubation, the bacterial cell samples were cultured on Columbia blood plates containing spectinomycin for 3 days to obtain the expression strains. S. alv. :pDS- Varroa , and stored it in glycerol and stored in a -80°C freezer.

[0019] As one of the preferred technical solutions, the colonization stability test in step S4 described in the present application includes obtaining a plasmid pDS-GFP that has been verified in the early stage and can stably express dsRNA but does not target host and parasite genes, and electroporating the pDS-GFP plasmid into the bee intestinal bacteria. S. alv. To obtain engineered bacteria S. alv. : pDS-GFP; Two experimental groups were set up using bees that had been treated with antibiotics. S. alv. : pDS- Varroa Vaccination group, S. alv.: In the pDS-GFP inoculation group, dead bees were cleaned up every day, and on the 1st, 3rd, and 5th day after bacterial colonization, the same number of hindgut tissues of bees were dissected from each feeding cup of each experimental group, ground with sterile PBS solution, and then gradiently diluted and smeared on Columbia blood plates containing spectinomycin at a concentration of 60 μg / mL. The number of CFU was counted after 3 days, and the experimental data were statistically analyzed to determine the colonization effect.

[0020] As one of the preferred technical solutions, the Varroa mite inhibition effect experiment in step S4 described in the present application includes obtaining a plasmid pDS-GFP that has been verified in the early stage and can stably express dsRNA but does not target host and parasite genes, and electroporating the pDS-GFP plasmid into the bee intestinal bacteria. S. alv. To obtain engineered bacteria S. alv. : pDS-GFP; The experiment used bees that had been treated with antibiotics and were divided into three treatment groups: S. alv. : pDS- Varroa Vaccination group, S. tax : pDS-GFP inoculated group and sucrose solution treated group. The inoculated bees were transferred to the feeding cup. After 2 days of feeding, the bees in the three treatment groups were respectively fed with CO 2 After anesthetization, feeding cups were redistributed within the group, and mites were placed on the body surface of each bee using a fine-tipped brush. Dead bees were cleaned every day during the experiment, and the number of dead bees and varroa mites was recorded until the eighth day of the experiment. The experimental data were counted and analyzed to determine the varroa mite inhibition effect.

[0021] As one of the preferred technical solutions, the expression effect test in step S4 described in the present application includes colonizing the expression strain on the first day of the bees leaving the hive. S. alv. : pDS- Varroa , inoculated with Varroa mites on the 3rd day after the bees left the hive; on the 4th day after inoculation, the surviving Varroa mites on the living bees were obtained, the RNA of a single Varroa mite was extracted, and the RNA was reverse transcribed, and the relative expression levels of the 4 target genes of Varroa mites were tested by a fluorescence quantitative kit, and the experimental data were counted and analyzed to determine the expression effect.

[0022] Compared with the prior art, the beneficial effects of this application are:

[0023] The expression strains described in this application S. alvi: pDS-Varroa It can effectively and stably colonize in the bee intestine, and can deliver dsRNA to the varroa mite, reduce the expression of the corresponding gene, effectively inhibit the parasitism of the varroa mite, and avoid the impact of the acaricide spraying method, formic acid fumigation method, essential oil disinfection method and other methods used in the prior art on the bee breeding environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The pDS- Varroa Plasmid maps.

[0025] Figure 2 The colonization of honey bee symbiotic bacteria ( S. alv. : pDS-Varroa, S. alv. : pDS-GFP) after plasminogen activator (P-PS) was added, and the two honey bee symbiotic bacteria effectively colonized the honey bee intestine at a similar rate (ANOVA, P>0.05).

[0026] Figure 3 It is the effect of engineered bee intestinal bacteria on the survival rate of Varroa mites and bees.

[0027] Figure 4 is the relative expression level of the four Varroa mite genes under different treatments.

[0028] Figure 5 is the corrected P value for pairwise comparison of relative gene expression levels of the four genes in Varroa mites. DETAILED DESCRIPTION

[0029] The technical solution described in this application is further described below in conjunction with the accompanying drawings and embodiments.

[0030] Example 1

[0031] Columbia blood agar plates were used at 35°C and 5% CO 2 Isolation of intestinal symbiotic bacteria of honey bees in an incubator S. alv. ( Snodgrassella VAT ), the strain name is wkB2. The conventional sugar shaking method is used to obtain the Varroa mites on the surface of the bees, and the Varroa mites are transferred to the bees anesthetized by carbon dioxide using a fine-pointed brush for temporary cultivation. The out-of-house bees recorded in this application refer to the sealed capped honeycombs that are about to be out of the house being taken out from the bee colony, placed in a non-sealed frame formed by the queen bee egg-laying controller, and cultured overnight in a constant temperature and humidity incubator at a temperature of 34±1℃ and a relative humidity of 75% to obtain the newly out-of-house bees.

[0032] A biological control method for inhibiting Varroa mite parasitism comprises the following steps:

[0033] Step 1: The mRNA fragment shown in the gene sequence SEQ ID NO.1 is used as chitin synthase 1 ( CHS1 ,LOC111254093) expression gene, and the mRNA fragment shown in the gene sequence SEQ ID NO.2 as chitin synthase 2 ( CHS2, LOC111246312), and the mRNA fragment shown in the gene sequence SEQ ID NO.3 was used as the vacuolar H + -ATPase 1 ( VATPS1 , LOC111245360) and the mRNA fragment shown in the gene sequence SEQ ID NO.4 was used as the vacuolar H + -ATPase 2 ( VATPS2 , LOC111254335) and were connected to the plasmid vector pBTK800 to obtain the constructed pDS- Varroa Plasmid, pDS- Varroa The expression gene sequence of the plasmid is shown in SEQ ID NO.5; Figure 1 As shown, dsRNA is inserted through the BASI restriction site.

[0034] Step 2: Add the pDS- Varroa Plasmid electroporation into bee gut bacteria S. alv. To obtain the expression strain S. alv. : pDS- Varroa ; In pDS- Varroa During the process of plasmid electroporation, it is necessary to first prepare the bee gut bacteria S. alv. of competent cells.

[0035] Bee gut bacteria S. alv. The competent cells were prepared by the following method: S21, honey bee gut bacteria S. tax Columbia blood agar plates were used at 35°C and 5% CO 2 The intestinal symbiotic bacteria of honey bees were isolated from the incubator, and the strain was wkB2;

[0036] S22, the obtained bee intestinal bacteria S. alv. A single colony was added to 50 mL Columbia liquid medium and incubated at 35°C with 5% CO 2 Incubate in an incubator for 48 hours;

[0037] S23, after the incubation in step S32 is completed, the incubation solution is centrifuged at 7000 rpm for 10 min to collect the bacterial precipitate;

[0038] S24. Wash the separated bacterial precipitate three times with autoclaved double distilled water, and finally resuspend the bacterial pellet with 1 mL of autoclaved 10% glycerol, dispense 100 μL into a centrifuge tube, and store in a -80°C refrigerator.

[0039] Intestinal bacteria in honey bees S. alv.After the competent cells were prepared, the following conditions were used to achieve pDS- Varroa Plasmid electroporation into bee gut bacteria S. alv. middle:

[0040] The electroporation process includes taking out 100 μL of bacterial solution from a -80°C refrigerator, adding 500 ng of plasmid and mixing thoroughly, incubating on ice for 20 min, adding 75 μL of the mixture into a 1 mm electroporation cup after the incubation, using a Bio-Rad instrument, and performing electrical stimulation according to the parameters of voltage 1800 V, capacitance 25 μF, and resistance 1 Ω, then transferring the bacterial cells to 50 mL of Columbia liquid medium and incubating at 35°C, 5% CO 2 After overnight incubation, the bacterial cell samples were cultured on Columbia blood plates containing spectinomycin for 3 days to obtain the expression strains. S. alv. : pDS- Varroa , and stored it in glycerol and stored in a -80°C freezer.

[0041] S4. The expression strain obtained in step 2 S. alv. : pDS- Varroa Conduct one or more of the following: colonization stability test, Varroa mite inhibition effect test, and expression effect test.

[0042] When performing the experiment described in step S4, it is first necessary to obtain a sufficient amount of honey bees that have left the hive, and place 50 honey bees that have left the hive in a 50 mL sterile centrifuge tube for each, and starve them for 2 hours.

[0043] The expression strain obtained S. alv. : pDS- Varroa The expression strain S. alv. : pDS- Varroa Dilute in sterile PBS, adjust the bacterial solution OD600 = 1, and use 1000 μL of bacterial solution and 1000 μL of 50% filtered tossing solution to mix evenly.

[0044] Take 500 μL of the mixture and add it to the centrifuge tube containing the starved bees. Gently shake the centrifuge tube for 20 seconds to allow the bees to fully contact the mixture. The inoculated bees need to be transferred from the centrifuge tube to a regular feeding cup and fed with 50% filtered sucrose solution containing spectinomycin (60 μg / mL) and artificially raised in a regular manner.

[0045] Under the above conditions, in step S4, the positive control used was a plasmid pDS-GFP that has been verified to stably express dsRNA but does not target host and parasite genes. The constructed plasmid vector was transfected into the bee intestinal bacteria. S. alv. , marked as engineered bacteria S. alv.: pDS-GFP.

[0046] The colonization stability test recorded in step S4 includes the following steps:

[0047] Step S41: The experiment uses bees that have been treated with antibiotics (spectinomycin) and sets up two experimental groups, namely S. alv. : pDS- Varroa Vaccination group, S. alv. : pDS-GFP inoculation group, each experimental group was set up with 90 bees for inoculation, and each feeding cup was fed with 30 inoculated bees, with a total of 3 cups of experimental bees.

[0048] Step S42: clean up dead bees in a timely manner every day.

[0049] Step S43, in the expression strain S. alv. : pDS- Varroa Bacteria and engineered bacteria S. alv. : On the first, third, and fifth days after the bees were colonized with pDS-GFP bacteria, the hindgut tissues of five bees were dissected from each feeding cup, ground with sterile PBS solution (phosphate buffer solution), and then graded and spread on Columbia blood agar plates containing spectinomycin (60 µg / mL). The number of CFU (colony forming units) was counted after 3 days. The experimental data were processed in R language, and the Kruskal-Wallis H test was used to test the expression strain S. alv. : pDS- Varroa Customized effect.

[0050] The Varroa mite inhibition effect experiment described in step S4 includes the following steps:

[0051] Step S44: The bees that have been treated with antibiotics (spectinomycin) are divided into three treatment groups: S. alv. : pDS- Varroa Vaccination group, S. alv. : The pDS-GFP inoculation group and the sucrose solution treatment group were tested for expression of the strain in the hive bees using the same method as the colonization stability test. S. alv. : pDS- Varroa Inoculation, engineering bacteria S. tax : pDS-GFP inoculation, the inoculated bees were transferred to feeding cups, each feeding cup was fed with 50 bees, and each treatment group had 2 feeding cups.

[0052] Step S45: After 2 days of feeding, i.e., on the third day of the experiment, the bees in the three treatment groups were respectively fed with CO 2After anesthetization and re-randomization, every 10 bees were housed in one petri dish, and each treatment group had 10 petri dishes; a mite was placed on the body surface of each bee using a fine-tipped brush.

[0053] Step S46: During the experiment, dead bees were cleaned up every day, and the number of dead bees and Varroa mites was recorded until the eighth day of the experiment. The survival curves of the experimental bees and Varroa mites were analyzed using the Kaplan-Meier estimation method in the R language survival package, and the FDR was corrected.

[0054] The expression effect test recorded in step S4 includes the following steps:

[0055] Step S47: Planting engineered bacteria on the first day after bees leave the hive S. alv. : pDS-GFP, inoculated with Varroa mites on the 3rd day, and obtained the surviving Varroa mites from the living bees on the 4th day of the experiment, extracted the RNA of a single Varroa mite using Quick-RNA MiniPrep kit (ZYMOResearch, Cat. No. R1055), reverse transcribed the RNA using a reverse transcription kit (Takara, Cat. No.RR037A), and finally tested it using a fluorescence quantitative kit (Bio-Rad, 172-5120). β-actin and GAPDH As internal reference genes, qPCR was used to detect the relative expression of four target genes of Varroa mites. The primers used for the genes were shown in SEQ ID NO.6 to SEQ ID NO.11. The gene amplification efficiency was calculated using the qPCR package in R language, and the relative expression was calculated using 2 ΔΔCT Methods Data were calculated and log2 transformed, and statistical analysis was performed using the Wilcoxon rank-sum test with FDR correction.

[0056] exist Figure 2 In the figure, part A shows the inoculated expression strain S. alv. : pDS- Varroa Survival curve of Varroa mites after inoculation with engineered bacteria S. alv. : pDS-GFP experimental group (N=90, Wilcoxon rank-sum test, adjusted P < 0.05) and sucrose solution experimental group (N=75, Wilcoxon rank-sum test, adjusted P <0.01) in Varroa mites compared with the expression strain inoculated S. alv. : pDS- Varroa The survival rate of Varroa mites was the lowest.

[0057] exist Figure 2 In the figure, part B shows the inoculated expression strain S. alv. : pDS- Varroa , Engineering bacteria S. tax : Survival rate of bees after inoculation of pDS-GFP and Varroa mites. S. alv. : pDS- Varroa Experimental group (N=97), engineered bacteria S. alv. : The pDS-GFP experimental group (N=100) and the sucrose solution experimental group (N=99) showed high survival rates, and there was no significant difference between the treatment groups (Wilcoxon rank sum test, adjusted P > 0.05). The X-axis indicates the number of days after inoculation with Varroa mites, the shaded area indicates the 95% confidence interval, ns indicates no significant difference, * indicates a significance level of 0.05, and ** indicates a significance level of 0.01.

[0058] exist Figure 3 In the figure, it shows that bees (N=90) colonized with honey bee symbionts ( S. alv. : pDS-Varroa, S. tax : pDS-GFP) after the two bee engineered bacteria were effectively colonized in the bee intestine at a similar rate (ANOVA, P > 0.05).

[0059] The experimental data of colonization stability test showed that after bees were inoculated S. alv. The colonies were able to remain stable in the bee intestines for a long time, and the number of colonized bacteria increased at a similar rate from the first to the fifth day.

[0060] In the above experiment, in the experiment of Varroa mite inhibition, 4 bees died in the sucrose solution before the inoculation of Varroa mites during the actual feeding process. Therefore, except for the sucrose solution group which only had 9 culture dishes, the other experimental groups all had 10 culture dishes, but this did not affect the experimental results. S. alv. : There was no significant difference in the survival rate of Varroa mites between the pDS-GFP and sucrose solution groups (Log-rank test, adjusted P < 0.05). However, when the above experimental groups were compared with the expression strain S. tax : pDS- Varroa When comparing the experimental groups, the engineered bacteria S. alv. : pDS-GFP experimental group (Log-rank test, adjusted P< 0.01) and sucrose solution group (Log-rank test, adjusted P < 0.05) had significantly higher varroa survival and higher bee mortality, but there was no significant difference in bee survival among the three treatments, see Figure 3 Part B of

[0061] See also Figure 4 , compared with the sucrose solution group, the expression strain inoculated S. alv. : pDS- Varroa The relative expression levels of the four genes in the Varroa mite group were significantly decreased (N = 8, Wilcoxon rank sum test, adjusted P < 0.001), and inoculated with engineered bacteria S. alv. : Compared with the experimental group inoculated with pDS-GFP, the expression strain S. alv. :pDS- Varroa The expression levels of three genes of Varroa mite in the experimental group were reduced (N = 8, Wilcoxon rank sum test, adjusted P < 0.05). The box plot shows the highest value, lowest value and median of the data, and the lower and upper edges of each box represent the first quartile and the third quartile, respectively. ns means no significant difference, * means the significance level is 0.05, ** means the significance level is 0.01, and *** means the significance level is 0.001.

[0062] One day after Varroa mites were inoculated into bees, the relative expression levels of the four genes tested were found to be higher in chitin synthase 1, chitin synthase 2, and vacuolar H + -ATPase 1 expression gene, vacuolar H + The relative expression levels of these four genes were significantly different among the three treatment groups (Kruskal-Wallis test, P < 0.001), see Figure 3 Compared with the sucrose solution group, all four genes were expressed in the expression strain S. tax : pDS- Varroa The expression was decreased in the experimental group (Wilcoxon rank sum test, adjusted P < 0.001), in addition to the engineered bacteria S. alv. : Comparison of pDS-GFP experimental group, form strain S. alv. : pDS- Varroa Varroa mites in the experimental group CHS2(Wilcoxon rank sum test, adjusted P < 0.05))、 VATPS1 (Wilcoxon rank sumtest, adjusted P < 0.001)、 VATPS2 (Wilcoxon rank sum test, adjusted P < 0.001) gene expression levels were all reduced. Compared with the sucrose solution group, the engineered bacteria S. alv. : pDS-GFP experimental group Varroa mites CHS1 , CHS2 , VATPS2 The expression of genes was also significantly lower. The primers used to detect the expression of related genes in Varroa mites were Chitin synthases (LOC111254093), Chitin synthases (LOC111246312), Vacuolar type H + -ATPase (LOC111245360), Vacuolar type H + -ATPase (LOC111254335), β-actin (LOC111248310), GAPDH (LOC111244103), whose gene sequences are SEQ ID NO.6 to SEQ ID NO.11 respectively.

[0063] See also Figure 5 In the table shown, the expression levels of each gene were compared pairwise and the FDR was corrected. Sugar represents the sucrose group and GFP represents the engineered bacteria. S. alv. : pDS-GFP experimental group, Varroa Indicates inoculation expression strain S. alv. : pDS- Varroa of the experimental group.

[0064] In summary, the expression strain S. alvi: pDS-Varroa used in the experiment can effectively and stably colonize in the intestines of honey bees, and can deliver dsRNA to the body of Varroa mites, reducing the expression level of corresponding genes and effectively inhibiting the parasitism of Varroa mites.

[0065] Finally, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A biological control method for inhibiting Varroa mite parasitism, characterized in that: The method comprises the following steps: S1, the mRNA fragment shown in the gene sequence SEQ ID NO.1 is used as the expression gene of chitin synthase 1, the mRNA fragment shown in the gene sequence SEQ ID NO.2 is used as the expression gene of chitin synthase 2, and the mRNA fragment shown in the gene sequence SEQ ID NO.3 is used as the expression gene of vacuolar H + -ATPase 1 expression gene, the mRNA fragment shown in the gene sequence SEQ ID NO.4 was used as the vacuolar H + -ATPase 2 expression gene was connected to the plasmid vector pBTK800 by enzyme digestion to obtain the constructed pDS- Varroa Plasmid, inserted into pDS- Varroa The expression gene sequence of the plasmid is shown in SEQ ID NO.5; S2, the pDS- Varroa Plasmid electroporation into bee gut bacteria S. alvi To obtain the expression strain S. alvi : pDS- Varroa ; S3. Expression strain S. alvi : pDS- Varroa Carry out expansion cultivation and colonize the bees after they leave the hive; S4. The expression strain obtained in step 2 S. alvi : pDS- Varroa Conduct one or more of the following: colonization stability test, Varroa mite inhibition effect test, and expression effect test.

2. A biological control method for inhibiting Varroa mite parasitism according to claim 1, characterized in that: The expression gene of chitin synthase 1, the expression gene of chitin synthase 2, the vacuolar H + -ATPase 1 expression gene, vacuolar H + -ATPase 2 expression gene combination was added to both ends of the gene insertion sequence to synthesize a double-stranded DNA fragment, and a vector ligation kit was used to achieve restriction enzyme digestion and ligation with the plasmid vector pBTK800 to complete the pDS- Varroa Construction of plasmids.

3. A biological control method for inhibiting Varroa mite parasitism according to claim 2, characterized in that: Step S2 pDS- Varroa Plasmid electroporation into bee gut bacteria S. alvi Before, the bee intestinal bacteria S. alvi Transformed into competent cells, competent cells were prepared by the following method: S21. Honey bee intestinal bacteria S. alvi Columbia blood agar plates were used to isolate the intestinal symbiotic bacteria of honey bees in an incubator at 35°C and 5% CO2. The strain was wkB2. S22, the obtained bee intestinal bacteria S. alvi A single colony was added to 50 mL of Columbia liquid medium and incubated in an incubator at 35 °C and 5% CO for 48 h; S23, after the incubation in step S22 is completed, the incubation solution is centrifuged at 7000 rpm for 10 min to collect the bacterial precipitate; S24. Wash the separated bacterial precipitate three times with autoclaved double distilled water, and finally resuspend the bacterial pellet with 1 mL of autoclaved 10% glycerol, dispense 100 μL into a centrifuge tube, and store in a -80°C refrigerator.

4. A biological control method for inhibiting Varroa mite parasitism according to claim 2, characterized in that: Step S2 pDS- Varroa Plasmid electroporation into bee gut bacteria S. alvi The electrotransformation method is as follows: the electrotransformation process includes taking out 100 μL of bacterial solution from a -80°C refrigerator, adding 500 ng of plasmid and mixing thoroughly, incubating on ice for 20 minutes, adding 75 μL of the mixed solution to a 1 mm electrotransfer cup after the incubation is completed, using a Bio-Rad instrument, after electrical stimulation according to the parameters of voltage 1800 V, capacitance 25 μF, and resistance 1 Ω, the bacterial cells are transferred to 50 mL of Columbia liquid culture medium, and incubated overnight in an incubator at 35°C and 5% CO2; after incubation overnight, the bacterial cell sample is cultured on Columbia blood agar plates with spectinomycin for 3 days to obtain the expression strain. S. alvi : pDS- Varroa , and stored it in glycerol and stored in a -80°C freezer.

5. A biological control method for inhibiting Varroa mite parasitism according to claim 1, characterized in that: The colonization stability test in step S4 includes obtaining a plasmid pDS-GFP that has been verified in the early stage and can stably express dsRNA but does not target host and parasite genes, and electroporating the pDS-GFP plasmid into the bee intestinal bacteria. S. alvi To obtain engineered bacteria S. alvi : pDS-GFP; Two experimental groups were set up using bees that had been treated with antibiotics. S. alvi : pDS- Varroa Vaccination group, S. alvi : In the pDS-GFP inoculation group, dead bees were cleaned up every day, and on the 1st, 3rd, and 5th day after bacterial colonization, the hindgut tissue of the same number of bees was dissected from each feeding cup of each experimental group, ground with sterile PBS solution, and then gradiently diluted and smeared on Columbia blood agar plates containing spectinomycin at a concentration of 60 μg / mL. The number of CFU was counted after 3 days, and the experimental data were statistically analyzed to determine the colonization effect.

6. A biological control method for inhibiting Varroa mite parasitism according to claim 1, characterized in that: The Varroa mite inhibition experiment in step S4 includes obtaining a plasmid pDS-GFP that has been verified in the early stage and can stably express dsRNA but does not target host and parasite genes, and electroporating the pDS-GFP plasmid into the bee intestinal bacteria. S. alvi To obtain engineered bacteria S. alvi : pDS-GFP; The experiment used bees that had been treated with antibiotics and were divided into three treatment groups: S. alvi : pDS- Varroa Vaccination group, S. alvi : The pDS-GFP inoculated group and the sucrose solution treated group, the inoculated bees were transferred to the feeding cups, after 2 days of feeding, the bees in the three treatment groups were anesthetized by CO2 and the feeding cups were redistributed within the group, and mites were placed on the body surface of each bee with a fine-pointed brush; dead bees were cleaned up every day during the experiment, and the number of dead bees and varroa mites was recorded until the 8th day of the experiment, and the experimental data were counted and analyzed to determine the varroa mite inhibition effect.

7. A biological control method for inhibiting Varroa mite parasitism according to claim 1, characterized in that: The expression effect test in step S4 includes colonizing the expression strain on the first day after the bees leave the hive S. alvi : pDS- Varroa , inoculated with Varroa mites on the 3rd day after the bees left the hive; on the 4th day after inoculation, the surviving Varroa mites on the living bees were obtained, the RNA of a single Varroa mite was extracted, and the RNA was reverse transcribed, and the relative expression levels of the 4 target genes of Varroa mites were tested by a fluorescence quantitative kit, and the experimental data were counted and analyzed to determine the expression effect.

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