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Method for constructing Ustilago esculenta T-DNA mutant library and analyzing insertion sites

A technology for inserting sites and analysis methods, which is applied in the field of genetic engineering and can solve problems such as increasing the difficulty of screening mutant libraries

Pending Publication Date: 2022-07-12
CHINA JILIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This greatly increases the difficulty of screening mutant libraries

Method used

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  • Method for constructing Ustilago esculenta T-DNA mutant library and analyzing insertion sites
  • Method for constructing Ustilago esculenta T-DNA mutant library and analyzing insertion sites
  • Method for constructing Ustilago esculenta T-DNA mutant library and analyzing insertion sites

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Experimental program
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Effect test

Embodiment

[0033] 1 Materials and methods

[0034] 1.1 Materials

[0035] 1.1.1 Test strains

[0036]The self-fusion strain TSP of wild black powder fungus was constructed and preserved by the Key Laboratory of Biometrics and Inspection and Quarantine Technology of China Jiliang University. Agrobacterium strain EHA105 with the binary vector pNeo3300 carrying the Geneticin G418 resistance gene.

[0037] 1.1.2 Chemical reagents

[0038] Acetosy-ringone (AS), 2-(N-morpholine)ethanesulfonic acid (MES), rifampicin, streptomycin sulfate, kanamycin sulfate, cefotaxime sodium, and genetic Mycin (G418) was purchased from Beijing Soleibao Technology Co., Ltd.; Taq enzyme, agarose and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0039] 1.1.3 Drugs and culture medium

[0040] 0.1M acetosyringone: AS 196.2g, dilute to 10mL with dimethyl sulfoxide (DMSO); 100mg / mL MES: MES 1.5g, dilute to 15mL with distilled water; 100mg / mL cephalosporin: cephalosporin 1g, di...

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Abstract

The invention discloses a construction and insertion site analysis method of an Ustilago esculenta T-DNA mutant library, and belongs to the technical field of gene engineering. The method comprises the following steps: by taking a constructed Ustilago esculenta self-fusion strain TSP as a starting strain and a plasmid containing a geneticin resistance gene as a carrier, constructing a Ustilago esculenta T-DNA mutant library through ATMT, carrying out genome re-sequencing on the constructed Ustilago esculenta T-DNA, and analyzing a T-DNA insertion site of the Ustilago esculenta T-DNA. The method lays a foundation for subsequent regulation and control mechanism research of Ustilago esculenta two-form conversion.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a T-DNA mutant library of wild black powder and for analyzing an insertion site. Background technique [0002] Wild black powder fungus ( Ustilago esculenta ) belongs to the Basidiomycota Solitaria family Solitaceae, which infect wild grass ( Zizania latifolia ) led to the massive proliferation and expansion of stem tissue cells, and finally formed edible water bamboo. Water jasmine is the second largest aquatic vegetable in my country. It is not only delicious, but also rich in protein, vitamins, crude fiber, etc., and has high nutritional value and medicinal value. However, due to improper cultivation and management measures, species degradation and the influence of external environmental conditions, infertile wild jasmine often appeared in the field during the planting process of jasmine, which seriously affected the yield o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/80C40B40/06C40B50/06C12Q1/6869
CPCC12N15/1093C12N15/80C40B40/06C40B50/06C12Q1/6869C12Q2535/122
Inventor 汤近天叶子弘杨芙容张雅芬夏文强崔海峰俞晓平
Owner CHINA JILIANG UNIV
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