Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof

A technology of pathogenic fungi and trehalase, applied in the field of transgenic strains and their preparation, can solve problems such as difficult research and achieve the effects of good repeatability, high efficiency and accelerated utilization

Active Publication Date: 2010-11-17
CHONGQING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, due to the difficulty of research, there are few studies on the period from the time when the pathogenic fungus enters the insect hemocoel to the death of the host (post-invasion), and this period is exactly the critical period of the life-and-death struggle between the pathogenic fungus and the host. It accounts for most of the time in the pathogenic process of fungal infection, shortening the infection time of pathogenic fungi in insects can effectively improve the insecticidal activity of entomopathogenic fungi

Method used

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  • Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof
  • Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof
  • Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof

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Embodiment 1

[0061] A preparation method of entomopathogenic fungus transgenic strain:

[0062] 1. Construct the constitutive expression vector of acid trehalase gene:

[0063] First, according to pBGFP (Cao YQ, Peng GX, He ZB, Wang ZK, Yin YP, Xia YX. Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains. Biotechnology Letters, 2007, 29: 907 -911) construct filamentous fungal expression vector pBarEx: design primer 1 (5'-atta gacgtc gcaggtcgacagaagaatgac-3', the underline is the Aat II restriction site) and primer 2 (5'-gtcggccgggcgtcgttctgggctcatggtagatccactagagcggccgc-3') to amplify the tryptophan synthase promoter (pTryC) sequence from Aspergillus nidulans genomic DNA; design primer 3 (5'-gcggccgctctagtggatctacc atg agcccagaacgacgcccggccgac-3', reverse complementary series with primer 2; where atg is the start codon of the Bar gene) and primer 4 (5'-cgc ggatcc agcttttattagatctcggtgac...

Embodiment 2

[0096] Preparation of Entomopathogenic Fungus Transgenic Engineering Strain MaOE017 Spore Powder Fungal Pesticide:

[0097] 1ml concentration is 10 7 The water suspension of mature conidia of Metarhizium anisopliae transgenic engineering strain MaOE017 per ml was inoculated in 100ml 1 / 4 strength Sabouraud's liquid medium (1 / 4SDA) (10g / l glucose, 2.5g / l peptone, 5.0 g / l yeast extract, initial pH 6.5) in a 250ml Erlenmeyer flask, 28°C, 150rpm shaking culture for 3 days to prepare liquid strains; soak rice at room temperature (25°C) for 4 hours and then put it into a mushroom bag In the medium, sterilize at 121°C for 30 minutes and then cool to 25°C; inoculate the liquid strain of the genetically engineered strain MaOE017 at a ratio of 1:10 by weight, mix evenly, cultivate at 28°C for 15 days, and then dry it with discolored silica gel in a desiccator. Use an 80-mesh sieve to collect the dried spore powder fungal pesticides of Metarhizium anisopliae transgenic strain MaOE017, an...

Embodiment 3

[0099] Preparation of entomopathogenic fungal transgenic engineering strain MaOE017 oil suspension fungal pesticide:

[0100] Put 800g of rapeseed oil and 200g dry spore powder containing Metarhizium anisopliae transgenic strain MaOE017 in a blender (1000-1500 rpm) and mix for 20 minutes evenly, and configure it into 20% Metarhizium anisopliae after filtering with an 80-mesh filter screen Oil suspension fungal pesticide, diluted 3-5 times with oil and then sprayed with ultra-low volume technology.

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Abstract

The invention provides an entomopathogenic fungi transgenic strain which contains a homologous or heterologous acidic trehalase constitutive exogenous gene. The preparation method of the entomopathogenic fungi transgenic strain comprises the following steps: firstly building an acidic trehalase full-length cDNA gene constitutive expression vector; establishing a submerged conidia transformation system; and finally selectively screening to obtain the entomopathogenic fungi transgenic strain. The invention also provides application of Entomopathogenic fungi acidic trehalase transgenic strain in preparing fungal pesticide. The entomopathogenic fungi transgenic strain has high efficiency of expressing trehalase; and the preparation method has the advantages of easy operation, good repeatability and good reproducibility. The entomopathogenic fungi transgenic strain can be applied to preparing the fungal pesticide. When the fungal pesticide acts on insects, through the overexpression of the acidic trehalase gene via the transgenic strain, the activity of acidic trehalase in an insect host is improved, utilization of the trehalase is accelerated and growth of entomopathogenic fungi in the insect host is promoted, thus effectively improving the insecticidal activity of the fungal pesticide such as metarhizium anisopliae, beauveria bassiana and the like.

Description

technical field [0001] The invention relates to a transgenic bacterial strain and its preparation method and application, in particular to an entomopathogenic fungus transgenic bacterial strain and its preparation method and application. Background technique [0002] Pest control is an important part of agricultural production. Chemical pesticides have played an active role in the prevention and control of harmful organisms and in increasing agricultural production. However, the extensive and long-term use of chemical pesticides has caused a series of environmental and food safety problems. Due to the advantages of safety and sustainable control, microbial biological control has been widely concerned by people. Among them, entomopathogenic fungi are the largest group of entomopathogenic microorganisms, which cause 60% of the natural death of insects, and are one of the important factors controlling the population of insects in nature. Entomopathogenic fungi mainly infect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/56C12N15/63A01N63/04A01P7/04C12R1/645C12R1/79
Inventor 夏玉先彭国雄
Owner CHONGQING UNIV
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