Double-fluorescence screening method for fungal gene knockout

A gene knockout and screening method technology, applied in the field of fungal gene knockout, can solve the problems of distinguishing difficult-to-transformants, etc., and achieve the effect of omitting the verification process and reducing the workload

Active Publication Date: 2019-08-13
ANHUI AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art, and provide a fungal gene knockout efficient screening method to solve the technical problem in the prior art that it is difficult to distinguish target transformants from heterotopically integrated transformants

Method used

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  • Double-fluorescence screening method for fungal gene knockout
  • Double-fluorescence screening method for fungal gene knockout
  • Double-fluorescence screening method for fungal gene knockout

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Embodiment Construction

[0073] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[0074] This embodiment is applicable to fungi sensitive to bleomycin. Aspergillus flavus NRRL 3357 is a whole-genome sequenced bacterium and is sensitive to bleomycin, so Aspergillus flavus is selected as the implementation object.

[0075] pUM ​​vector construction

[0076] 1) Using the pYES2 vector DNA as a template, use Primerdesign software to design primers:

[0077] Primer ① ccgcggGGAACAACACTCAACCCTA;

[0078] Primer ② ccgcggTTCGATGTAACCCACTCG;

[0079] The lowercase letter part in the above primers is the restriction enzyme cutting site SacII.

[0080] 2) Using the above primers ① and ②, amplify the 2.9-kb URA3-2micro2_origin fragment from the template in step 1), and then insert pCAMBIA1300 into the SacII site of the backbone vector. The constructed vector was named pUM.

[0081] Among them, the amplification...

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Abstract

The invention discloses a double-fluorescence screening method for fungal gene knockout, and relates to the technical field of fungal gene knockout. The method comprises the steps that a 2.9-kb URA3-2micro2_origin fragment in a pYES2 carrier DNA template is amplified, the fragment is inserted into a pCAMBIA1300 carrier, and a pUM carrier is obtained; then, a tef1 promoter, an eGFP gene, a PgpdA promoter, a ble resistance gene, an RFP gene, a TtrpC terminator, an upstream homologous arm of a target gene x and a downstream homologous arm of the target gene x are amplified respectively; finally,a knockout carrier pKO-x of the target gene x is established; the pKO-x is transformed into agrobacterium competent cells, fungi is transformed through an agrobacterium mediated method, antibiotic screening is carried out to obtain a resistance transformant, then fluorescence microscope detection is carried out on the resistance transformant, the resistance transformant is a target transformant ifonly red fluorescence is emitted, the resistance transformant is an ectopic integration transformant if both green fluorescence and red fluorescence exist, and the transformant is a wild type if no fluorescence is emitted. The method can be used for effectively distinguishing the target transformant from the ectopic integration transformant.

Description

technical field [0001] The invention relates to the technical field of fungal gene knockout, in particular to a high-efficiency screening method for fungal gene knockout. Background technique [0002] Phytopathogenic fungi often infect rice, wheat, corn and other important crops, causing serious harm to my country's agricultural output and crop quality. At present, the exploration of the pathogenic molecular mechanism of plant pathogenic fungi mostly involves gene function analysis, among which gene knockout is an important means for gene function analysis, and there are mainly two genetic transformation methods involved: one is the protoplast PEG transformation method, This method has the disadvantages of complex protoplast preparation process, poor repeatability, unstable transformants, and low transformation efficiency. The second is based on the Agrobacterium-mediated genetic transformation method, which has the characteristics of high efficiency, low cost, convenient o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/90C12N15/65C12R1/67
CPCC12N15/80C12N15/902C12N15/65Y02A50/30
Inventor 陶芳赵凯项芳芝赵倩倩
Owner ANHUI AGRICULTURAL UNIVERSITY
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