Constructing method of insect-killing genetically engineered beauveria bassiana

A construction method, technology of Beauveria bassiana, applied in the construction field of insecticidal Beauveria bassiana engineering bacteria, can solve the problem of unsatisfactory progress in the breeding of highly virulent insecticidal fungus engineering bacteria, shortening of engineering strains, and failure to breed To achieve sustainable development, simplify experimental procedures, and promote environmental ecological balance

Active Publication Date: 2020-01-10
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the progress in the breeding of highly virulent insecticidal fungus engineered bacteria is not ideal enough. So far, no engineered strains that have shortened the lethal time by more than 50% have been selected, and no engineered strains have been used for production.

Method used

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  • Constructing method of insect-killing genetically engineered beauveria bassiana

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of recombinant pbarGPEl-AaIT vector

[0035] [1]Amplified AaIT gene in pUC57-AaIT

[0036] Use the pUC57-AaIT plasmid as a template to synthesize primers to amplify the AaIT gene sequence. The primer sequences are as follows:

[0037] AaIT-F (BamH I): 5'-SEQ ID NO: 1-3';

[0038] AaIT-R (EcoRI): 5'-SEQ ID NO: 2-3'.

[0039] The upstream primer (AaIT-F) introduces the BamH I site, and the downstream primer (AaIT-R) introduces the restriction site of EcoRI. Two restriction sites were introduced at the 5' end to protect the bases. Perform PCR amplification. Reaction system (25μl): DNA (plasmid) 1.0μl, 10×buffer (Mg 2+ ) 2.5 μl, dNTP 1.0 μl, upstream and downstream primers 1.0 μl each, Taq enzyme 0.5 μl, ddH 2 O 18.0 μl. PCR amplification program: 94°C for 5min, 94°C for 45s, 64°C for 45s, 72°C for 1min, a total of 32 cycles. 72°C for 10 minutes. After the amplified product was electrophoresed on 1% agarose gel, the target band was excised and recovere...

Embodiment 2

[0052] Construction of pGPS3Ben-bbchit1-AaIT expression vector

[0053] [1] Amplification of the target gene fragment GpdA-AaIT-TrpC

[0054] The plasmid containing pbarGPE1-AaIT was used as a template, and trpCR and gpdAF were used as primers for PCR amplification. PCR amplification program: pre-denaturation at 94°C for 5 min, followed by 32 cycles: denaturation at 94°C for 45 s, annealing at 60.5°C for 45 s, extension at 72°C for 1.5 min; extension at 72°C for 10 min at the end of the cycle. After the amplified product was subjected to 1% agarose gel electrophoresis, the target band was excised and recovered with DNA GelExtraction Kit. Primers used: forward primer (NotI): gpdAF 5'-SEQ ID NO: 3-3'; reverse primer (NotI): trpCR 5'-SEQ ID NO: 4-3'.

[0055] [2] Digestion of pbenGPS3-Bbchit1 plasmid and GpdA-AaIT-TrpC gene fragment

[0056] Prepare the digestion system in a microcentrifuge tube as follows (20μl system): 10× Buffer H 2μl, 0.1% BufferBSA 2μl, DNA (plasmid or ge...

Embodiment 3

[0069] pbenGPS3-Bbchit1-AaIT vector plasmid introduced into Agrobacterium tumefaciens

[0070] The pbenGPS3-Bbchit1-AaIT plasmid DNA was extracted, and the vector was introduced into Agrobacterium tumefaciens by freeze-thawing method, and the vector plasmid DNA was added to 100 μl Agrobacterium tumefaciens competent cells, mixed evenly, and ice-bathed for 30 minutes. After quick-freezing in liquid nitrogen for 5 minutes, incubate at 37°C for 5 minutes, and immediately ice-bath for 2 minutes. Add 1000μl liquid YEB, 200r / min, culture at 28°C for 3-5h, centrifuge at 10000r / min to collect the bacteria, resuspend the bacteria with 100μl liquid YEB and spread evenly on the YEB plate (concentrations of ampicillin and benomyl respectively 12.0 μg / m and 8.0 μg / mL), pick positive colonies.

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Abstract

The present invention discloses a constructing method of insect-killing genetically engineered beauveria bassiana. Primers are designed to amplify an AaIT gene from a pUC57-AaIT vector by PCR, and a pbarGPEl-AaIT vector is constructed by digestion, purification and pbarGPEl; the primers are used to amplify a GpdA-AaIT-TrpC expression cassette; Not1 single-digestion of pGPS3Ben-bbchit1 is conducted, dephosphorylation is conducted, connection with GpdA-AaIT-TrpC digested by the Not1 is conducted, transformation screening is conducted to obtain a pGPS3Ben-bbchit1-AaIT expression vector, and the pGPS3Ben-bbchit1-AaIT expression vector is introduced into agrobacterium tumefaciens competent cells to obtain positive transformant colonies; and a beauveria bassiana spore suspension is mixed with anagrobacterium tumefaciens solution containing the pGPS3Ben-bbchit1-AaIT vector, and the transformed genetically engineered beauveria bassiana is obtained by screening a solid IM culture medium and aCPZ selection culture medium. The constructed genetically engineered beauveria bassiana can prepare a high-efficiency insect-killing biological control agent, has no pollution to the environment and also has a good prevent and cure effect, and is suitable for popularization in the technical field of biological prevention and cure.

Description

technical field [0001] The invention relates to the technical field of microbial pest control, in particular to a method for constructing insecticidal Beauveria bassiana engineering bacteria. Background technique [0002] Fungal insecticides are biological pesticides with entomopathogenic fungi as active ingredients. Currently, there are about 100 genera and more than 800 species of insecticidal fungi recorded in the world, of which about 50% are concentrated in the subphylum Deuteromycotina , mainly distributed in Beauveria, Metarhizium, Paecilomyces, Hirsutella, Nom uraea, Verticillium (Verticillium) etc. In China, Brazil, Russia and European and American countries, fungal pesticides have been widely used in the control of agricultural and forestry pests and urban insects. More than 60 fungal pesticide products have come out in the world at present. Simultaneously with other pesticides Compared with insect microorganisms, entomopathogenic fungi have the advantages of acti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/80
CPCC12N15/66C12N15/80Y02A50/30
Inventor 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
Owner XUZHOU UNIV OF TECH
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