CN117587043A

CN117587043A - Solanum nigrum phenol oxidizing zymogen gene and application thereof

Info

Publication number: CN117587043A
Application number: CN202210992285.6A
Authority: CN
Inventors: 汤方; 罗健; 王志强
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-08-18
Filing date: 2022-08-18
Publication date: 2024-02-23

Abstract

The invention discloses a black wing soil termite phenol oxidizing zymogen geneOfPPOAnd dsRNA (ds)OfPPO) It was verified that it plays an important role in combating SM1 and Bt infestations, and at the same time it was demonstrated that ds was usedOfPPOAfter the black wing termites are treated, the mortality rate of the workers can be obviously improved by using bifenthrin, imidacloprid or chlorfenapyr, which indicates that the black wing termites dsOfPPOImproving the toxicity of the medicament and deepening understanding of the black wing soil termitesOfPPOIs a physiological function of (a)And the function, can also be used as a new target, develop corresponding immunosuppressant, and provide theoretical basis for further improving the biological control or chemical control effect of controlling the black wing soil termite.

Description

Solanum nigrum phenol oxidizing zymogen gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a phenol oxidase gene of black wing soil termite and application thereof.

Background

The black wing soil termite Odontotermes formosanus (Shiraki) belongs to soil termites, is very miscellaneous in feeding habit, can be more than 100 plants such as fir, tulip tree, camphor tree, masson pine, poplar and the like, is mainly the rootstock of the harmful plants, causes dead seedlings, causes poor growth and even death of adult trees, has wide distribution range, is distributed in 21 provinces in China, is in north latitude of 35 degrees (Luoyang) and is in south to north latitude of 20 degrees (sea amperes). The black wing subterranean termites can also harm reservoirs and river dikes, so that the dikes leak water, collapse and breach, and great economic loss is caused. Because the black wing subterranean termites are hidden underground and the damage caused by the black wing subterranean termites is hidden, the black wing subterranean termites are not easy to detect by people, and great difficulty is brought to control. The chemical agent is used for preventing and controlling the black wing soil termites, which is the most common prevention and control means at present, but with the importance of people on ecological civilization construction, the development of environment-friendly biological prevention and control agents for achieving pest prevention and control with little or no chemical agent becomes important content of ecological civilization construction.

Phenol oxidizing enzymes are usually present in the form of their precursor pro-phenol oxidizing enzymes (PPO) in their inactive state, PPO and hemocyanin, catechol oxidase and tyrosinase all belonging to the family of copper-containing proteins type 3, type 3 copper ion proteins having two copper ions and three histidines at each active site. Phenol Oxidase (PO), also known as tyrosinase, is a type of multi-subunit copper-containing oxidoreductase that oxidizes phenols to quinone compounds in the presence of oxygen molecules, and is widely found in animals, plants, and microorganisms in nature. It is normally synthesized in the insect's blood cells in the form of its precursor pro-phenoloxidase in its inactive state, released into the haemolymph and then activated by specific proteases to become active PO, a process which is achieved by a complex phenol oxidase cascade. PO oxidizes monophenols to catechol, and then oxidizes to produce p-benzoquinone or o-benzoquinone, which ultimately produces cytotoxic substances and melanin, which deposits on the surface of pathogenic microorganisms to form encapsulation and blackening to isolate them, and quinone and other toxic intermediates such as 5, 6-dihydroxyindole-can also directly kill pathogenic microorganisms. The existence of the characteristics makes the PO get close attention of academia in the years, and related fields include biology, medicine, chemistry, agriculture and the like, and hundreds of POs are reported in research. For PO research of insects, the existing research mainly aims at basic characteristics, tissue distribution, functions, PO inhibitor application and the like, and meanwhile, a plurality of results are obtained in research on microscopic molecular level. In insects, PO catalyzes the hydroxylation of L-tyrosine to form ortho-dihydroxyphenylalanine (L-dopa), followed by oxidation of L-dopa to dopaquinone, which is then subjected to a series of non-enzymatic reactions to form the end product melanin. Melanin has an important physiological function during normal development of insects, while PO plays a key role in its formation. In general, PO is involved in hardening and darkening of the epidermis, immunization, wound healing, etc. in insects. PO has been studied in some other insects but less in insects of the sub-order Isoptera, particularly in the important pest, blackwing subterranean termites, has not been reported.

RNA interference (RNAi) is a mechanism that utilizes exogenous double-stranded RNA to target degradation of specific mRNA and silence the target mRNA. RNAi has great prospect in pest control due to high specificity. The dsRNA needs to exert RNA interference effect in cells, and methods for allowing in vitro dsRNA to enter cells mainly comprise microinjection, feeding and soaking transfection methods, and methods such as virus infection, symbiont mediation, transgenic plant mediation, nanoparticle coating, liposome modification and the like are derived on the basis of the methods. RNAi is a valuable reverse genetics tool, can be used for researching gene functions of various organisms, has the characteristics of specificity, high efficiency, stability, easiness in operation, low cost and the like, and is the most likely novel technology applied to pest control at present. The application prospect of biological control by RNAi is wide, and the technology is predicted to be further perfected and developed in future, and finally, the technology is well applied.

Disclosure of Invention

The invention aims to: the invention provides a black wing soil termite phenol oxidizing zymogen gene PPO (OfPPO), and researches the effect of the OfPPO in resisting infection of Serratia marcescens (Serratia marcescens, SM 1) and bacillus thuringiensis (Bacillus thuringiensis, bt) and the application of the OfPPO in medicament control through RNAi technology, and the result shows that dsOfPPO treated black wing soil termite reduces the immunity of termites, so that the capability of resisting SM1 and Bt is reduced, and simultaneously, the dsOfPPO improves the toxicity of the medicament.

Specifically, the invention obtains a black-wing soil termite phenol oxidizing zymogen gene by utilizing a molecular cloning technology, and the nucleotide sequence of the gene is shown as SEQ ID No: 1. The NCBI ORF Finder and BlastP prove that the complete open reading frame of the OfPPO is 2085bp, the predicted polypeptide sequence consists of 694 amino acid residues, as shown in SEQ ID No. 2, and the sequence contains two conserved hydrolysis sites and two typical copper ion binding sites (CuA and CuB), wherein the two copper binding sites respectively contain 3 conserved histidines, and are confirmed to be PPO genes of the black wing soil termites. The Neighbor-training phylogenetic tree construction and the multiple sequence comparison of the OfPPO gene are carried out by utilizing MEGA-X and DNAMAN, which show that the OfPPO gene and the PO gene from the Taiwan lactis termite and the Nemadaio termite are in the same branch and have obvious homology.

The invention further provides application of the black wing termite phenol oxidizing zymogen gene in prevention and control of the black wing termite.

Furthermore, the application provides dsRNA for preventing and controlling the black wing termites based on the phenoloxidase zymogen gene, which is transcribed from the nucleotide sequence of the black wing termite phenoloxidase zymogen gene.

RNAi technical studies indicate that dsOfPPO treatment of subterranean termites reduces termite immunity, so that the capacity of resisting SM1 and Bt is reduced, and the OfPPO plays an important role in resisting SM1 and Bt infection. Meanwhile, after the black wing termites are treated by dsofPPO, the rate of death of the workers is obviously improved by using bifenthrin, imidacloprid or chlorfenapyr. This suggests that the subterranean termite dsOfPPO increases the virulence of the agent.

The invention also provides DNA for encoding the phenol oxidase gene dsRNA.

Further, the invention also provides a recombinant expression vector comprising the DNA encoding dsRNA.

The invention also provides a host bacterium which converts the recombinant expression vector.

The invention also provides application of the dsRNA of the black wing subterranean termite pro-phenol oxidizing enzyme gene in improving the toxicity of an insect-resistant medicament.

Wherein the anti-insect agent is any one of bifenthrin, imidacloprid and chlorfenapyr, and specifically when the anti-insect agent is applied, the black wing termite is firstly treated by the black wing termite phenol oxidation zymogen gene dsRNA for 3-24 hours, and then is treated by the anti-insect agent. Preferably, the subterranean termites are treated with the subterranean termite phenol oxidizing zymogen gene dsRNA for 6 hours and then treated with an insect-resistant agent.

The invention also provides an application of the dsRNA of the black wing soil termite phenol oxidizing zymogen gene in enhancing insecticidal toxicity of Serratia marcescens SM1 or Bacillus thuringiensis Bt.

Specifically, when in use, the black wing subterranean termite is treated with dsRNA for 3-24 hours, and then treated with Serratia marcescens SM1 or Bacillus thuringiensis Bt. Preferably, the subterranean termites are treated with the subterranean termite phenol oxidizing zymogen gene dsRNA for 6 hours and then with Serratia marcescens SM1 or Bacillus thuringiensis Bt.

The beneficial effects are that: aiming at the obtained black wing subterranean termite phenol oxidizing zymogen gene ofPPO, the invention is designed dsRNA (dsOfPPO), which verifies that the gene plays an important role in resisting SM1 and Bt infection, and simultaneously proves that after the black wing subterranean termite is treated by dsOfPPO, the mortality of workers can be obviously improved by using bifenthrin, imidacloprid or chlorfenapyr for treatment. The results show that the dsOfPPO of the black wing subterranean termite improves the toxicity of the medicament, deepens the physiological function and effect of the OfPPO of the black wing subterranean termite, can be used as a new target, develops a corresponding immunosuppressant, and provides a theoretical basis for further improving the biological control or chemical control effect of the black wing subterranean termite.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 shows the results of an OfPPO electrophoresis detection pattern;

FIG. 2 shows the result of dsOfPPO electrophoresis detection pattern;

FIG. 3 is a graph of the results of the interference efficiency of dsOfPPO on the Alternaria alternata OfPPO;

FIG. 4 is a graph showing the effect of dsOfPPO on SM1 virulence after treatment with subterranean termites;

FIG. 5 is a graph showing the effect on Bt virulence of dsOfPPO treated subterranean termites;

FIG. 6 is a graph showing the effect on imidacloprid virulence of dsOfPPO treated subterranean termites;

FIG. 7 is a graph showing the effect of dsOfPPO on chlorfenapyr virulence after treatment of subterranean termites;

fig. 8 is a graph showing the effect on the toxicity of bifenthrin after dsOfPPO treatment of black-fin termites.

Detailed Description

The test materials involved in the following examples are as follows:

the tested insects are black wing soil termites, and are fed into a feeding device with a fungus nursery area and a feeding area separated, the feeding condition is a dark environment, the temperature is 25+/-1 ℃, and the relative humidity is 90+/-5%. The test subjects adopt individuals with consistent and robust feeding workers.

Adding 1mL of SM1 strain stored at-80deg.C into 100mL fermentation medium, culturing at 30deg.C and 200rpm in shaking table to OD value of about 0.4, and diluting SM1 bacterial solution to concentration of 8.75X10 ¹⁰ CFU/mL. Adding 1mL of Bt bacterial liquid preserved in a laboratory at-80deg.C into 100mL LB culture medium, culturing at 30deg.C and 200rpm in shaking table to OD value of about 0.2, and diluting the Bt bacterial liquid to 2.14X10 ¹⁰ CFU/mL。

Tetracyclines: 1g of tetracycline was weighed, dissolved in 50mL of ultra-pure water (20 mg/mL), filtered through a 0.22 μm filter, and packaged into 1 mL/tube, and stored in a refrigerator at-20 ℃.

IPTG: 0.2383g of IPTG was weighed, dissolved in 10mL of ultrapure water (100 mM), filtered through a 0.22 μm filter, and then packaged into 1 mL/tube, and stored in a refrigerator at-20 ℃.

Ampicillin; 500mg of ampicillin was weighed, dissolved in 5mL of ultrapure water (100 mg/mL), filtered through a 0.22 μm filter, and then packaged into 1 mL/tube, and stored in a refrigerator at-20 ℃.

LB solid medium: agar powder for bacteria is added into LB liquid medium to a final concentration of 1.2%, and the mixture is autoclaved and stored at 4 ℃ after being poured into a flat plate.

Example 1 cloning of the subterranean termite phenol oxidizing zymogen gene.

(1) Cloning of the OfPPO gene of the termite and the termite with black wing soil.

Total RNA of the black wing termites is extracted, reverse transcription is respectively carried out by referring to the instruction book of PrimeScript 1st Strand cDNA Synthesis Kit kit, and the first step system is as follows:

after the above systems were mixed, the procedure was as follows:

65℃ 5min

rapidly cooling on ice, and performing a second reaction.

The second step system is as follows:

after the above systems were mixed, the reaction procedure was as follows:

42℃ 60min

95℃ 5s

after the reaction was completed, the mixture was stored in a refrigerator at-20 ℃.

The upstream and downstream primers were designed based on the pro-phenol gene sequence (OfPPO) in the subterranean termite transcriptome using Primer Premier 5.0 (Premier company, canada) software (table 1).

Table 1 primers used in the experiments

PCR amplification of the target Gene:

the cDNA obtained by the reverse transcription was amplified by PCR, and the following was placed on ice:

after the above systems were mixed, the reaction procedure was as follows:

94℃5min

94℃30s,58℃30s,72℃1min (30 cycles)

92℃10min

4℃∞

After the reaction, the PCR product was subjected to 1% gel electrophoresis, and photographed under a gel imaging system, and the electrophoresis result is shown in FIG. 1. And (5) purifying and recycling the PCR products identified to be correct through a gel recycling kit. The gel recovery product was ligated with pClone007 at room temperature for 1-5min, the ligation product transformed into E.coli DH 5. Alpha. Competent cells, the ligation product transformation step:

(1) e.coli DH5 alpha competent cells are taken out from a refrigerator at the temperature of minus 80 ℃ and dissolved in ice water;

(2) 10. Mu.L of the ligation product was added to 100ul E.coli DH5α competent cells with a pipette and allowed to incubate for 30min;

(3) heat shock is carried out for 45s in a water bath at 42 ℃, and the obtained product is immediately placed in ice water for standing for 2min after being taken out;

(4) adding a liquid culture medium of SOC or LB, and carrying out shaking culture at 37 ℃ and 200rpm for 1h;

(5) 200. Mu.L of the bacterial liquid was spread on LB solid medium containing Amp penicillin (final concentration 50. Mu.g/ml), and cultured in an incubator at 37℃overnight in an inverted manner.

The transformation product is cultivated in LB (amp+) solid medium for 12-16 h. Positive clones were selected, and after bacterial liquid PCR identification, the recombinant plasmid containing the correct insert was sent to the product for sequencing by the company of south kyoto biotechnology.

(2) Sequence analysis of the OfPPO from the subterranean termite

The obtained OfPPO of the black wing soil termite is subjected to coding sequence prediction and NCBI BlastP on-line comparison by sequencing, the amino acid sequences of the OfPPO are found to contain specific hydrolysis sites, 6 structure-conserved histidines of 2 typical copper binding sites CuA and CuB, and the amino acid sequence characteristics of the OfPPO indicate that the OfPPO belongs to members of the insect PPO family. The complete open reading frame of the obtained black wing soil termite worker ant OfPPO is 2085bp, and the predicted polypeptide sequence consists of 694 amino acid residues. Specifically, 100 sequences with similar homology are obtained by using NCBI BlastP program for homology search, and meanwhile, the OfPPO sequence is found to contain a conserved structure peculiar to the PPO gene.

The result of the Neighbor-training phylogenetic tree and the multiple sequence ratio of the PPO gene of the black wing termite shows that the OfPPO and the Taiwan ivory-white ants Coptotermes formosanus, the wet wood termite Zootermopsis nevadensis (Hagen) and the German cockroach are in the same branch and have obvious homology, wherein the amino acid sequence homology of the OfPPO and the Taiwan ivory-white termite reaches 92.80 percent, and the amino acid sequence homology of the wet wood termite reaches 87.90 percent.

Example 2 synthesis of dsOfPPO from black-fin soil termites.

RNAi site prediction was performed on the nucleic acid sequence corresponding to the open reading frame of the OfPPO gene using siDirect version 2.0 on-line software (http:// siDirect2.RNAi. Jp /). The fragments containing RNAi sequences in the OfPPO gene were designated as dsOfPPO, respectively, and the fragments used for the control were dsGFP, and specific amplification primers were designed using Primer premier 5.0 software (Table 1).

The cDNA of the target sample was amplified by PCR, and the system was prepared on ice as follows:

after the above systems were mixed, the reaction procedure was as follows:

94℃5min

94℃30s,58℃30s,72℃1min (30 cycles)

92℃10min

4℃∞

After the reaction, the PCR product was subjected to 1% gel electrophoresis, and photographed under a gel imaging system, and the electrophoresis result is shown in FIG. 2. The purified target fragment and L4440 plasmid were double digested with restriction endonucleases SacI and SmaI, respectively, while the control plasmid containing green fluorescent protein (Green fluorescent protein, GFP) was double digested with restriction endonucleases SacI and HindIII, as follows:

double enzyme digestion is carried out at 37 ℃ overnight, detection and identification are carried out through 1% agarose gel electrophoresis, and the target fragment and the target vector are purified by using a DNA gel recovery kit respectively. Then, the target fragment and the target vector are connected by using T4 DNA ligase at 16 ℃ overnight, and the connection system is as follows:

ligation was completed to obtain a recombinant plasmid containing the fragment of interest, which was transformed into HT115 (DE 3) competent cells, as follows:

(1) HT115 (DE 3) competent cells were removed from-80℃and rapidly inserted into ice, and after the pellet melted, the plasmid of interest was added and gently mixed by hand dialing the bottom of the EP tube, and allowed to stand in ice for 25min.

(2) The ice was quickly returned to the water bath at 42℃for 45s and allowed to stand for 2 minutes.

(3) To the centrifuge tube, 700. Mu.L of LB sterile medium without antibiotics was added, and resuscitated at 37℃for 60 minutes at 200 rpm.

(4) Centrifuge at 5000rpm for 1min, leave about 50. Mu.L of supernatant gently blow the resuspended pellet and spread on LB solid medium containing tetracycline (concentration 12.5. Mu.g/mL) and ampicillin (concentration 50. Mu.g/mL).

(5) The plates were placed in an incubator at 37℃overnight.

Selecting a monoclonal antibody which is cultured overnight in a culture medium, adding the monoclonal antibody into LB sterile culture solution containing tetracycline and ampicillin, culturing at 37 ℃ and 200rpm until the monoclonal antibody is turbid, and carrying out double digestion and sequencing identification on a connection product according to HT115 bacterial liquid and 50% glycerol according to the following ratio of 1:1 are stored in a refrigerator at-80 ℃.

Mu.l of HT115 bacteria solution was added to 50mL of sterile LB medium, and tetracycline and ampicillin were added to the medium at final concentrations of 12.5. Mu.g/mL and 75. Mu.g/mL, respectively. After culturing at 37 ℃ for 12 hours at 200rpm, 50mL of sterile LB culture solution is added again, simultaneously, the same amount of tetracycline and ampicillin as the previous step is added, IPTG is added when the culture is carried out at 37 ℃ at 200rpm until the OD600 value is 0.4-0.5, the final concentration is 0.8mM, the culture is continued for 4 hours at 37 ℃ at 200rpm, at this time, dsRNA is expressed in a large amount in bacterial solution, and RNA is extracted.

Adding proper amount of RNase enzyme and DNase enzyme to digest DNA and RNA, the system is as follows:

dsOfPPO 1000μL

RNase(1mg/mL) 1μL

DNase(1mg/mL) 1μL

treating at 20-25deg.C for 20min. The dsRNA was purified and diluted to 1. Mu.g/. Mu.L for further use.

Example 3 effect of dsOfPPO treatment on the virulence of SM1 and Bt in black wing subterranean termites.

(1) determination of optimal time for dsOfPPO treatment of termite-black wing soil workers.

Mu.g/. Mu.L of dsOfPPO and dsGFP (positive control) and RNase free ddH ₂ O (negative control), each of which was added with the Nalge dye to a final concentration of 1% (m/v). The above solutions were added to petri dish filters, 400 μl of each petri dish was added, 20 workers per petri dish, 3 replicates per treatment, and the test workers from 3 different populations. Test workers lick and eat filter paper mutually to ingest dsRNA, collect samples of workers with dyed intestinal tracts and abdomen at treatment time of 3, 6, 12 and 24 hours respectively, store the samples into a refrigerator with the temperature of-80 ℃, detect the interference efficiency of the dsRNA by using qRT-PCR technology, and select a time period with high interference efficiency according to the result to carry out subsequent bioassay tests. As a result, as shown in FIG. 3, the expression of the OfPPO gene was disturbed at the disturbance times of 3, 6, 12 and 24 hours, wherein the disturbance efficiency was the highest at the disturbance time of 6 hours.

(2) Effect of dsOfPPO treatment on SM1 and Bt virulence by subterranean termites

Using 1. Mu.g/. Mu.L of dsOfPPO and dsGFP and RNase free ddH ₂ O is respectively treated with the black wing soil termite for 6 hours, and then 1 mu L of Serratia marcescens SM1 bacterial liquid (the concentration is 8.75X10) ¹⁰ CFU/mL) and the chest backboard, the mortality rate was continuously counted until the mortality rate of the control group reached 20% or all the treated workers died. Each treatment was repeated 3 times and the test workers were from 3 different populations.

Using 1. Mu.g/. Mu.L of dsOfPPO and dsGFP and RNase free ddH ₂ O is respectively treated with the black wing soil termite for 6 hours, and then 1 mu L of Bt bacterial liquid (the concentration is 2.14X10) ¹⁰ CFU/mL) at the chest backboard, the mortality rate was continuously counted until the control mortality rate reached 20% or the treated workers all died. Each treatment was repeated 3 times and the test workers were from 3 different populations.

The biological assay results of interfering with the expression of the termite OfPPO in the termite body of the black wing soil by using the RNA interference technology and then treating with SM1 after 6 hours of the expression of the termite body of the black wing soil are shown in fig. 4. For OfPPO, the mortality rate of dsOfPPO-SM1 group of black-wing termites was around 50% and differed from each other at 12 and 24 hours, the mortality rate of dsOfPPO-SM1 group of black-wing termites was nearly 100% and differed from each other at 48 hours, the mortality rate of CK-SM1 group of black-wing termites differed from dsGFP-SM1 group of black-wing termites was around 50% and differed from each other, and the mortality rate of control group was lower than 20%.

The biological assay results of interfering with the expression of the black wing termite OfPPO in the insect body for 6h and then treating with Bt by using the RNA interference technology are shown in fig. 5. The mortality rate of the black-wing termites in the dsOfPPO-Bt group is increased from about 20% to about 40% and different from the other groups at 24 and 48 hours, the mortality rate of the black-wing termites in the CK-Bt group and the dsGFP-Bt group is 50% -60% and different from the other groups at 72 hours, the mortality rate of the black-wing termites in the dsOfPPO-Bt group and different from the other groups at 96 hours is 100%, the mortality rate of the black-wing termites in the CK-Bt group and the dsGFP-Bt group is about 60% and different from the other groups, and the mortality rate of the black-wing termites in the control group is about 20%. From the survival curve, it can be seen that interfering with the aftostermes nigromaculatus OfPPO can increase Bt virulence to the aftostermes nigromaculatus.

In summary, by interfering with the expression of aftostermes formosanus OfPPO by RNAi technology and then treating with SM1 and Bt respectively, mortality of aftostermes formosanus workers was improved, which suggests that when PPO genes were silenced, the aftostermes formosanus immune system was insufficient to clear pathogenic bacteria, confirming that pro-phenol oxidation enzymes are involved in the immune function against bacteria in aftostermes formosanus, suggesting that aftostermes formosanus OfPPO plays an important role in combating SM1 and Bt infestations.

Example 4 improvement of drug toxicity by subterranean termite dsOfPPO

In the embodiment, after the RNAi technology interferes with the expression of the ofPPO, a reagent with proper concentration is added for treatment, and the mortality of the termite workers of the black wing soil termites is observed and statistically analyzed.

And (3) preparation of a reagent:

bifenthrin: 0.0044g bifenthrin was weighed out in 1mL acetone (0.01M), stored in a refrigerator at 4℃and diluted 8100-fold for use.

Chlorfenapyr: 0.0041g chlorfenapyr was weighed into 1mL acetone (0.01M), stored in a refrigerator at 4℃and diluted 81-fold for use.

Imidacloprid: 0.0026g imidacloprid is weighed into 1mL acetone (0.01M), stored in a refrigerator at 4 ℃ and diluted 81 times for use.

dsOfPPO, dsGFP and RNase free ddH were used at 1. Mu.g/. Mu.L ₂ O is respectively treated with the black wing soil termite for 6 hours, and then respectively inoculated with 1 mu L of diluted bifenthrin, chlorfenapyr and imidacloprid, and the death rate is continuously counted until the death rate of a control group exceeds 20% or the treated termite is dead. Each treatment was repeated 3 times and the test workers were from 3 different populations.

The biological assay results of interfering with the expression of the black wing termite OfPPO in the insect body for 6h and then treating with imidacloprid by using the RNA interference technology are shown in fig. 6. dsOfPPO 1-imidacloprid with a black-wing termite mortality rate higher than and different from the other groups from 6h, a black-wing termite mortality rate of approximately 100% by 48h, dsGFP-imidacloprid with a mortality rate higher than and different from 3 groups treated without the agent from 12h, a black-wing termite mortality rate of approximately 50% by 48h, CK-imidacloprid and dsGFP-imidacloprid with a mortality rate higher than CK, dsGFP and dsOfPPO groups from 24h (the 3 control groups have a mortality rate lower than 20%) and a black-wing termite mortality rate of approximately 50% by 48 h.

The biological assay results of interfering with the expression of the termite OfPPO in the termite body for 6h by using the RNA interference technology and then treating the termite with chlorfenapyr are shown in fig. 7. The mortality rate of the black wing termites of the dsOfPPO-chlorfenapyr group is higher than that of other groups and is different when the chlorfenapyr is treated for 6h and 12h, the mortality rate of the black wing termites of the dsOfPPO-chlorfenapyr group is higher than that of the black wing termites of the CK-chlorfenapyr group and the dsGFP-chlorfenapyr group when the chlorfenapyr is treated for 24h and 48h, and the mortality rates of the black wing termites of the CK-chlorfenapyr group, the dsGFP group and the dsOfPPO group are all lower than 20%.

The biological assay results of interfering with the expression of the black wing termite OfPPO in the body for 6h and then treating with bifenthrin by using the RNA interference technology are shown in fig. 8. At 16, 28 and 40h of bifenthrin treatment, dsOfPPO-bifenthrin black-wing termite mortality (from about 50% to 100%) was higher than that of both CK-bifenthrin and dsGFP-bifenthrin groups (from about 10% to about 30%), while the CK, dsGFP and dsOfPPO groups were all lower than 20%.

In conclusion, after interfering with the expression of OfPPO, the treatment of the termite with the right concentrations of bifenthrin, imidacloprid and chlorfenapyr significantly increased the mortality of the termite, which indicates that OfPPO increased the virulence of these 3 agents.

Claims

1. The black wing soil termite phenol oxidizing zymogen gene is characterized in that the nucleotide sequence is shown in SEQ ID No: 1.

2. The use of the prothrombin gene of black wing termites in the prevention and treatment of black wing termites according to claim 1.

3. A dsRNA for controlling a prothrombin gene of a subterranean termite, which is transcribed from the nucleotide sequence of the prothrombin gene of a subterranean termite according to claim 1.

4. A DNA encoding the dsRNA of claim 3.

5. A recombinant expression vector comprising the DNA encoding the dsRNA of claim 4.

6. A host bacterium transformed with the recombinant expression vector of claim 5.

7. Use of the dsRNA of the prothrombin gene of subterranean termite in claim 3 for increasing the virulence of an insect repellent.

8. The use according to claim 6, wherein the anti-insect agent is any one of bifenthrin, imidacloprid and chlorfenapyr, and when applied, the subterranean termite is treated with dsRNA for 3-24 hours before treatment with an insecticide.

9. Use of the dsRNA of the prothrombin gene of subterranean termite phenol according to claim 3 for enhancing insecticidal virulence of serratia marcescens SM1 or bacillus thuringiensis Bt.

10. The use according to claim 9, wherein, at the time of application, the subterranean termite is treated with dsRNA for 3-24 hours before treatment with serratia marcescens SM1 or bacillus thuringiensis Bt.