Fungus expression vector and construction and screening method thereof

A technology for expressing vectors and constructing methods, which is applied in the biological field and can solve problems such as environmental and ecosystem impacts

Active Publication Date: 2011-09-14
CHONGQING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, at present, the screening markers for constructing engineering strains of insecticidal fungi at home and abroad use fungicide resistance markers including Benomyl resistance markers ( β-tubulin genes), herbicide resistance markers ( bar gene), hygromycin resistance marker ( hph Genes), genetically engineered bacteria containing these resistance selection markers (such as herbicide resistance markers bar Genetic engineering bacteria) enter the natural environment as biological pesticides. Once gene d

Method used

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  • Fungus expression vector and construction and screening method thereof
  • Fungus expression vector and construction and screening method thereof
  • Fungus expression vector and construction and screening method thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0027] Example 1 Transformation of Metarhizium anisopliae using Metarhizium anisopliae acid trehalase as a screening marker

[0028] 1. Strains

[0029] Metarhizium anisopliae locust-specific strain CQMa102 ( Metarhizium acridum ), has been disclosed in the authorized text of application number 200310110901.8, and the strain is now available for purchase at the Genetic Engineering Center of Chongqing University. Escherichia coli JM109 was purchased from Broad Parker.

[0030] 2. Construction of the fungal expression vector pEX-ATM using Metarhizium anisopliae acid trehalase gene (ATM) as a selection marker

[0031] First, construct the filamentous fungal expression vector pBarEx according to pAN52-1 (purchased from Tuofei Biotechnology Co., Ltd.): design primer 1 as SEQ ID NO: 1 (5'-atta gacgtc gcaggtcgacagaagaatgac-3', the underline is the Aat II restriction site) and primer 2 is SEQ ID NO: 2 (5'-gtcggccgggcgt cgttctgggctcatggtagatccactagagcggccgc-3') from Aspergillus ni...

Embodiment 2

[0041] Example 2: Transformation of Beauveria bassiana using Aspergillus niger acid trehalase gene (ATA) as a selection marker

[0042] 1. Strains and Vectors

[0043] Beauveria bassiana ( Beauveria bassiana ) strain, the strain number is 3.2055, which was purchased from the Institute of Microbiology, Chinese Academy of Sciences. Escherichia coli JM109 was purchased from Broad Parker.

[0044] 2. Aspergillus niger ( Aspergillus niger ) Construction of fungal expression vector pEX-ATA with acid trehalase gene as selection marker

[0045] The fungal expression vector pEX was prepared in Example 1. Then use the ATA gene cDNA cloning vector pMD18-ATA (the cloning vector is constructed by connecting pMD18-T with ATA, where the pMD18-T vector can be purchased, and the accession number of ATA in Genbank is XM1388557) as a template, according to the black The primer ATA-F designed for the reverse transcription DNA (cDNA) sequence of Aspergillus acid trehalase gene (GenBank acce...

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Abstract

The invention discloses a fungus expression vector. The expression vector is a constructive expression vector which consists of a plasmid expression vector pEX and an acidic trehalase gene serving as a screening marker, wherein the plasmid expression vector pEX is formed by removing a Bar gene in an expression vector pBarEx; and the screening marker gene belongs to acidic trehalase genes such as an ataxia telangiectasia mutated (ATM) gene or an ancient technology activation (ATA) gene. In the invention, a safe and environment-friendly screening marker is used for the fungus expression vector,and insect pathogen project fungi constructed by the fungus expression vector are released to the environment and cannot adversely influence the environment due to the screening marker. The fungus expression vector has a unique design principle, an ingenious construction method, a simple program, a simple screening program for an insect pathogen fungus transgenic bacterial strain of the fungus expression vector and high screening efficiency, does not influence the metabolic balance of the fungi, and has a potential and wide application prospect in the transgenic fungus research and marketization process.

Description

technical field [0001] The invention relates to a fungal expression vector and its construction method and screening method, in particular to a fungal expression vector using acid trehalase gene (Acid trehalase gene) as a screening marker and its construction and screening method, belonging to the field of biotechnology. Background technique [0002] Pest control is an important part of agricultural production. Chemical pesticides play an active role in controlling pests and increasing agricultural production. However, the 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. Insecticidal microbial pesticides are a large class of biological pesticides developed by using insect pathogenic microorganisms. Fungi are the largest group of insect pathogenic microorganisms. Under natural conditions, insects caused b...

Claims

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Application Information

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IPC IPC(8): C12N15/80
Inventor 夏玉先金凯彭国雄
Owner CHONGQING UNIV
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