阅读说明：本技术 一种杀虫球孢白僵菌工程菌构建方法 (Method for constructing insecticidal beauveria bassiana engineering bacteria ) 是由 李同祥 孙会刚 田林 汤薇 黄天姿 董玉玮 李文 王春艳 高兆建 王陶 于 2019-11-18 设计创作，主要内容包括：本发明公开了一种杀虫球孢白僵菌工程菌构建方法,设计引物从pUC57-AaIT载体中PCR扩增AaIT基因,经酶切、纯化和pbarGPEl构建pbarGPEl-AaIT载体；用引物扩增出GpdA-AaIT-TrpC表达盒；用Not1单酶切pGPS3Ben-bbchit1并去磷酸化,与用Not1酶切的GpdA-AaIT-TrpC连接,转化筛选获得pGPS3Ben-bbchit1-AaIT表达载体,将其导入根癌农杆菌感受态细胞中获得阳性转化子菌落；球孢白僵菌孢子悬液和含pGPS3Ben-bbchit1-AaIT载体的根癌农杆菌液混合,经固体IM培养基、CPZ选择培养基筛选获得转化球孢白僵菌工程菌株。本发明构建的球孢白僵菌工程菌株可制备高效杀虫生物防治剂,对环境无污染且防治效果良好,适合在生物防治技术领域推广。(The invention discloses a method for constructing a beauveria bassiana engineering bacterium, which comprises the steps of designing a primer to amplify an AaIT gene from a pUC57-AaIT carrier through PCR, and constructing a pbarGPEl-AaIT carrier through enzyme digestion, purification and pbarGPEl; amplifying a GpdA-AaIT-TrpC expression cassette by using a primer; singly digesting pGPS3Ben-bbchi 1 by using Not1 enzyme, dephosphorizing, connecting with GpdA-AaIT-TrpC digested by Not1 enzyme, transforming, screening and obtaining a pGPS3Ben-bbchi 1-AaIT expression vector, and introducing the vector into a root cancer agrobacterium infected cell to obtain a positive transformant colony; mixing the beauveria bassiana spore suspension and the agrobacterium tumefaciens liquid containing pGPS3Ben-bbchit1-AaIT carrier, and screening by a solid IM culture medium and a CPZ selective culture medium to obtain the transformed beauveria bassiana engineering strain. The beauveria bassiana engineering strain constructed by the invention can be used for preparing a high-efficiency insecticidal biological control agent, has no pollution to the environment and good control effect, and is suitable for popularization in the technical field of biological control.)

1. A construction method of a pesticidal beauveria bassiana engineering bacterium is characterized by comprising the following specific steps:

firstly, designing and amplifying AaIT gene primers, introducing BamHI enzyme cutting sites and EcoRI enzyme cutting sites during primer design, and amplifying an AaIT gene sequence from pUC57-AaIT by using the designed primers;

step two, after the PCR amplification product of the AaIT gene is purified, carrying out enzyme digestion by using BamHI and EcoRI enzymes, and simultaneously carrying out enzyme digestion on pbarGPEl by using BamHI and EcoRI enzymes; respectively purifying, and establishing a connection reaction system to connect the AaIT gene and the pbarGPEl;

thirdly, transforming the connecting product into an escherichia coli competent cell and verifying to construct a pbarGPEl-AaIT vector;

fourthly, designing a primer containing a Not1 enzyme cutting site, and amplifying a GpdA-AaIT-TrpC expression cassette from a pbarGPEl-AaIT vector;

fifthly, digesting pGPS3 Ben-bbcot 1 by using Not1 enzyme, and carrying out dephosphorylation treatment;

sixthly, connecting the dephosphorylated pGPS3Ben-bbchit1 obtained in the fifth step with a GpdA-AaIT-TrpC expression cassette to obtain a pGPS3Ben-bbchit1-AaIT expression vector, transforming escherichia coli, and extracting a plasmid;

the plasmids extracted in the seventh step and the sixth step are introduced into agrobacterium tumefaciens competent cells by a freeze-thaw method, resistance screening is carried out, and agrobacterium tumefaciens positive colonies are obtained;

and step eight, mixing the beauveria bassiana spore suspension with the agrobacterium tumefaciens bacterial liquid containing the pGPS3Ben-bbchit1-AaIT vector, and performing co-culture on a solid IM culture medium and screening for multiple times on a CPZ selective culture medium to obtain the transformation engineering strain.

2. The method for constructing the insecticidal beauveria bassiana engineering bacteria according to claim 1, which is characterized in that: BamHI and EcoR1 enzyme cutting sites are respectively introduced into the AaIT gene amplification primer in the first step, and the primer sequence is as follows: AaIT-F (BamHI) as shown in 5' -SEQ ID NO: 1-3'; AaIT-R (EcoRI) as shown in 5' -SEQ ID NO: 2-3'.

3. The method for constructing the insecticidal beauveria bassiana engineering bacteria according to claim 1, which is characterized in that: the pbarggpel vector used in the second step contains BamHI and EcoRI cleavage sites.

4. The method for constructing the pesticidal beauveria bassiana engineering bacteria according to claim 1, wherein the concentration molar ratio of AaIT to pbarGPEl in the ligation reaction system in the second step is 3: 1.

5. The method for constructing the insecticidal beauveria bassiana engineering bacteria according to claim 1, which is characterized in that: the E.coli competence used in the third step was DH-5. alpha.

6. The method for constructing the insecticidal beauveria bassiana engineering bacteria according to claim 1, which is characterized in that: in the fourth step, the sequences of forward and reverse primers used for amplifying the GpdA-AaIT-TrpC from the pbarGPEl-AaIT vector contain Not1 enzyme cutting sites.

7. The method for constructing the insecticidal beauveria bassiana engineering bacteria according to claim 1, which is characterized in that: and the suspension thallus in the seventh step is coated on a YEB plate containing aminobenzyl and benomyl, wherein the concentration of the ampicillin and the benomyl are respectively 12.0 mu g/m and 8.0 mu g/mL.

8. The method for constructing the insecticidal beauveria bassiana engineering bacteria according to claim 1, which is characterized in that: the solid IM medium in the eighth step contains: NaCl 0.03% (w/v), MgS0₄.7H₂0 0.03％(w/v)，K₂HP0₄0.03% (w/v), 0.5% (v/v) glycerol, 0.04mmo1/L MES, 3.5mmol/L glucose and 350mmol/L acetosyringone.

Technical Field

The invention relates to the technical field of microbial pest control, in particular to a method for constructing a pesticidal beauveria bassiana engineering bacterium.

Background

The fungus pesticide is a biological pesticide taking entomopathogenic fungi as an effective component, and the currently recorded insecticidal fungi in the world belong to about 100 genera and more than 800 species, about 50% of the fungi are concentrated in Deuteromycotina (Deuteromycotina), and are mainly distributed in Beauveria ((Beau veria), Metarhizium (Metarhizium), Paecilomyces (Paecilomyces), Hirsutella (Hirsutella), mildew (Nom uraea), Verticillium (Verticillium), etc. in China, Brazil, Russia and Europe and America, fungal pesticides have been widely used for controlling agricultural and urban insects.

Among entomopathogenic fungi, Beauveria bassiana (Beauveria bassiana) is the most studied and the best application effect. The distribution range of the beauveria bassiana is wide, the beauveria bassiana exists in mountains with the altitude of several meters to more than 2000 meters, and the beauveria bassiana can invade 6 meshes of 15 families of 200 kinds of insects and mites. Different from the mechanism that bacteria, viruses and the like can exert a poisoning effect after being eaten by insects to the alimentary canal, beauveria bassiana directly penetrates the epidermis through the attachment and germination of conidia to hosts, hypha grows in the blood cavity to generate beauvericin (macrocyclic lipoxin) and calcium oxalate crystallization, insect poisoning is caused, and the insects die. Beauveria bassiana invades the insect body mainly through the insect body wall. It has effects on pests with chewing type and piercing-sucking type mouthparts. The insecticidal composition has good compatibility with the environment, is safe to people and livestock, has no toxicity to beneficial insects, can survive, diffuse and colonize in the environment, and keeps a certain population quantity, so that the pest quantity can be continuously kept in a low-level state, and the adjustable and continuous development of plant protection and environment protection is realized. Therefore, the beauveria bassiana has received more and more attention in the aspect of comprehensive pest control, and becomes one of the ideal choices for the research and development of biopesticides.

Beauveria bassiana has been developed as a widely used microbial insecticide. The united states environmental protection summer heat (EPA) in 1995 allowed the beauveria bassiana preparation Mycotrol RWP produced by Mycotech corporation to be used for controlling aphids and whiteflies. The research and application of beauveria bassiana begin in the 50 th century in China, and beauveria bassiana is registered in China in 2006. For years, beauveria bassiana is used in China to effectively control nearly 40 kinds of agricultural and forestry pests, mainly including pine moth, corn borer, tea lesser leafhopper, grub, locust, peach fruit borer, yellow cutworm, potato beetle, monochamus alternatus, termite, rice leafhopper, wood ephedra moth, camphor leaf beetle, sweet potato weevil, tea lesser leafhopper, soybean pod borer, tryporyza incertulas, broomcorn cutworm, black wing termite, beet weevil, clover moth, white poplar leaf moth and the like. The application of beauveria bassiana to control pine moth and corn borer is one of the most successful cases in the largest application area of insecticidal fungi in the world, and currently, 50 kilohm is still kept²One year, the maximum year of the application area reaches 134 kilohm²Wherein the prevention and treatment effect of the beauveria bassiana on the pine moth can reach more than 90 percent.

However, beauveria bassiana also has the defects of longer infection incubation period, slow pest knocking-down speed, unstable control effect, narrow insecticidal spectrum and the like other biological pesticides, which are important factors influencing the application and popularization of beauveria bassiana, and also cause the occupancy rate of the fungus insecticide in the pesticide market to be low.

Introducing exogenous toxin gene (such as Andropones australis insect-specific neurotoxin polypeptide gene, AaIT) into entomopathogenic fungi to improve insecticidal toxicity of fungi. The lethal dose of the metarhizium anisopliae engineering strain constructed by introducing a scorpion venom gene (AaIT) to the tobacco hornworm and the Aedes aegypti is reduced by at least 20 times. Therefore, the insecticidal efficiency of the entomogenous fungi can be greatly improved by adopting gene engineering technologies such as over-expression of virulence genes or exogenous toxin genes and the like. The safe and efficient fungal engineering bacteria pesticide obtained by modifying entomopathogenic fungi by means of genetic engineering and the like becomes the main direction and trend of fungal biopesticide development. The production strain is genetically improved purposefully, high-efficiency engineering bacteria are obtained, a new high-activity formulation is developed, the fungal pesticide suitable for the market needs is finally created, and the industrial pace of the fungal pesticide is greatly accelerated.

Over the past 10 years, the breeding research of the high-toxicity insecticidal fungus engineering bacteria has made a remarkable progress, the toxicity of the engineering strains is obviously improved, and the application of the insecticidal fungus has shown a good prospect. However, the development of the breeding of the high-toxicity insecticidal fungus engineering bacteria is not ideal enough, so that no engineering strain with the death time shortened by more than 50 percent has been bred, and none of the engineering strains is used for production. Therefore, research and development of safe and high-toxicity insecticidal fungi engineering bacteria are urgently needed.

A genetic transformation method and an experimental operation flow for high-toxicity over-expression of entomogenous fungi containing endogenous virulence gene bbchat 1 and exogenous virulence gene AaIT are established by mediating and transforming beauveria bassiana with agrobacterium tumefaciens, and an experiment and technical platform are provided for developing entomogenous fungi genetic improvement and molecular biology. Provides basic resources for the development of high-efficiency pesticides, overcomes the long-standing problem of the weakness of the fungal pesticides, greatly accelerates the industrialization pace of the fungal pesticides, achieves biological treatment, and has important significance for promoting the development of ecological agriculture.

Disclosure of Invention

The invention aims to provide a high-toxicity engineering bacterium transformation method and an experimental operation flow for establishing entomogenous fungi containing an endogenous toxicity gene bbchat 1 and an exogenous toxin gene AaIT, and the insect pest biological control method has the advantages of no environmental pollution, good control effect and low cost. In order to solve the technical problems, the invention provides a method for constructing an insecticidal beauveria bassiana engineering bacterium, which comprises the following specific steps:

(1) designing primers for amplifying the AaIT gene, introducing BamHI and EcoR1 enzyme cutting sites during primer design, and amplifying the AaIT gene sequence from pUC57-AaIT by using the designed primers.

(2) After the PCR amplification product of the AaIT gene is purified, enzyme digestion is carried out by BamHI and EcoRI enzymes, and meanwhile enzyme digestion is carried out by BamHI and EcoRI enzymes to pbarGPEl (BioVector NTCC Inc.), after purification, a ligation reaction system is established to connect the AaIT gene and the pbarGPEl (the molar ratio is 3: 1).

(3) The ligation product was transformed into E.coli competent cells and verified to construct pbarGPEl-AaIT vector.

(4) A primer containing a Not1 enzyme cutting site is designed, and a GpdA-AaIT-TrpC expression cassette is amplified from a pbarGPEl-AaIT vector.

(5) pGPS3 Ben-bbcot 1(Biovector NTCC Inc.) was digested with Not1 enzyme and dephosphorylated.

(6) Connecting the dephosphorylated pGPS3Ben-bbchi 1 obtained in the fifth step with a GpdA-AaIT-TrpC expression cassette to obtain a pGPS3Ben-bbchi 1-AaIT expression vector, transforming escherichia coli, and extracting a plasmid.

(7) And (3) introducing the plasmids extracted in the sixth step into the agrobacterium tumefaciens competent cells by a freeze-thaw method, resuspending the thalli, and uniformly coating the thalli on a YEB (YEB) plate (containing 12.0 mu g/m of ampicillin and 8.0 mu g/mL of benomyl respectively) for resistance screening to obtain agrobacterium tumefaciens positive colonies.

(8) The beauveria bassiana spore suspension and the agrobacterium tumefaciens bacterial liquid containing pGPS3Ben-bbchit1-AaIT carrier are mixed according to a certain proportion, and the transformation engineering strain is obtained by co-culture on a solid IM culture medium and multiple screening on a CPZ selective culture medium.

In the above method, beauveria bassiana can be obtained by self-made or purchased by conventional preparation method. The beauveria bassiana and the pUC57-AaIT are obtained by self-control.

Further, the YEB medium of the step (7) is sucrose 0.5% (w/v), yeast extract for bacteria 0.1% (w/v), trypsin extract for bacteria 1% (wlv), MgS0₄.7H₂0 0.05％(w/v).

Further, the concentration of ampicillin and benomyl in the plate coating resistance screening in the step (7) is 12.0 μ g/m and 8.0 μ g/mL respectively.

Further, the step (8) of CPZ (Chaudhur's medium) comprises sucrose 3% (w/v), NaNO₃0.2％(w/v),KCl0.05％(w/v),MgSO₄.7H₂O 0.05％(w/v),FeSO₄0.0001％(w/v),K₂HPO₄0.1% (w/v), 500. mu.g/mL of cefuroxime and 60. mu.g/mL of PPT.

Further, the IM culture medium in the step (8) comprises the following components in parts by weight: NaCl 0.03% (w/v), MgS0₄.7H₂00.03％(w/v)，K₂HP0₄0.03% (w/v), 0.5% (v/v) glycerol, 0.04mmo1/L MES.

Compared with the prior art, the invention has the following beneficial effects:

1. in the construction of expression vector, ben (benomyl) and Amp are adopted^rThe (ampicillin resistance) is a resistance mark, so that the transformant is effectively screened out, the experimental procedure is simplified, the working efficiency is improved, and the beauveria bassiana engineering bacteria have new benomyl resistance.

2. The construction of pGPS3 Ben-bbcot 1-AaIT constitutive bivalent expression vector with benomyl (Ben) as resistance marker gene, chitinase (bbcot 1) and northern African scorpion toxin (AaIT) as target gene, combining with the engineering bacteria obtained by Agrobacterium tumefaciens mediated transformation of Beauveria bassiana, not only inserting the target gene into the genome of the engineering bacteria to obtain stable heredity. In the process of infecting pests, the chitinase can be overexpressed to quickly penetrate through the body walls of the insects, and simultaneously, scorpion toxin is generated to poison the pests, so that the knockdown time is greatly shortened, and the insecticidal toxicity is improved.

3. The beauveria bassiana engineering bacteria is adopted for biological control, so that on one hand, harm of chemical agents to people and livestock is avoided, on the other hand, environmental ecological balance is promoted, sustainable development of agriculture is realized, and the beauveria bassiana engineering bacteria is suitable for popularization and use in the field of planting.

Drawings

FIG. 1 is agarose gel electrophoresis picture of PCR verification product of engineering bacteria. Lane 1: PCR products (AaIT and ben amplification products), lane M: maker 1000bp

Detailed Description

The methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are by mass unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

PbarGPEl and pbarGPEl-AaIT were purchased from Biovector plasmid vector strain cell gene collection.