Screening method for filamentous fungus gene knockout mutant

A gene knockout, filamentous fungus technology, applied in the field of genetic engineering, can solve the problems of high false positive rate and low success rate of gene knockout screening, and achieve the effect of reducing workload and improving screening efficiency

Inactive Publication Date: 2018-09-18
GUIZHOU UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a screening method for gene knockout mutants of filamentous fungi to solve the technical problems of low success rate of gene knockout screening and high false positive rate in the prior art

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  • Screening method for filamentous fungus gene knockout mutant
  • Screening method for filamentous fungus gene knockout mutant
  • Screening method for filamentous fungus gene knockout mutant

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Embodiment Construction

[0018] Below in conjunction with accompanying drawing and specific embodiment the invention is further introduced:

[0019] Take the knockout and screening of the NACα gene of Magnaporthe grisea as an example:

[0020] Search the NAC gene from the rice blast fungus database (http: / / fungidb.org / fungidb / ), find a NACα gene (MGG_02660), named MoNACα, download its CDS and genome sequence, and the nucleotide of the rice blast fungus MoNACα CDS The sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the rice blast fungus MoNACα genome is shown in SEQ ID NO: 2.

[0021] 1. Construct the HPH-GFP fusion expression vector and clone the HPH-GFP fragment

[0022] Use TRP-F / HYG-GFP-R and HYG-GFP-F / GFP-T primer pair to amplify the hygromycin HPH fragment (1352bp, its nucleotide series shown in SEQ ID NO: 19) and GFP fragment (720bp, its nucleotide sequence is shown in SEQ ID NO: 20).

[0023] Wherein, the TRP-F primer nucleotide series is shown in SEQ ID NO: 3, namely: GCTG...

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Abstract

The invention discloses a screening method for a filamentous fungus gene knockout mutant. The method comprises the following steps: (1) constructing an HPH-GFP fusion expression vector, and cloning anHPH-GFP fragment; (2) using a primer with a linker sequence to respectively clone the upstream fragment and the downstream fragment of a gene to be knocked out; (3) forming a chimeric fragment by using the cloned upstream and downstream fragments of the gene to be knocked out and the cloned HPH-GFP; (4) transforming a fungal protoplast by the chimeric fragment to obtain a transformant for later use; and (5) culturing the transformant in a medium containing hygromycin, observing normally grown hyphae by a fluorescence microscope, and selecting the transformant having GFP fluorescence to obtainthe knockout mutant. The method is used to solve the technical problems of low successful rate and high false positive rate of gene knockout in the prior art.

Description

technical field [0001] The invention relates to a screening method for gene knockout mutants of filamentous fungi, belonging to the field of genetic engineering. Background technique [0002] Fungi are widely distributed in nature and are inextricably linked with all aspects of human life. Therefore, the research on fungi has become particularly important, and the research on the gene function of fungi is one of the research hotspots. Genetic modification of fungal DNA is an important technology for gene function research. At present, the methods for genetic modification of filamentous fungi mainly include: (1) PEG-mediated protoplast transformation; (2) Agrobacterium-mediated transformation ( A. tumeafiens mediated transformation, ATMT); (3) electric shock transformation; (4) gene gun transformation; and other methods. [0003] Gene knockout is one of the means to study gene function. The principle of homologous recombination is used to knock out the gene of interest. By ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/65
CPCC12N15/65C12N15/80
Inventor 谢鑫王勇蒋君梅
Owner GUIZHOU UNIV
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