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Construction method for high-gene-knockout-efficiency Aspergillus chevalieri var. intermedius mutant engineering bacterial strain

An Aspergillus shevatii and gene knockout technology is applied in the field of construction of an Aspergillus shevatii intergenic variant engineering strain, which can solve problems such as low gene knockout efficiency, and achieve high gene knockout efficiency, stable genetics, and easy gene knockout. The effect of knockout mutant strains

Inactive Publication Date: 2014-01-22
贵州省生物技术研究所
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Problems solved by technology

Filamentous fungi favor the NHEJ pathway, resulting in lower gene knockout efficiency

Method used

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  • Construction method for high-gene-knockout-efficiency Aspergillus chevalieri var. intermedius mutant engineering bacterial strain

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Embodiment Construction

[0024] Embodiments of the present invention: a method for constructing an engineering strain of Aspergillus shevalley intertype variety with high gene knockout efficiency, specifically comprising the following steps:

[0025] Step (1): using Aspergillus intertype var. intertype genomic DNA as a template, using primers P12 and P13 to amplify the DNA fragment D at the 3' end of the Ku70 gene, and using primers P10 and P11 to amplify the DNA fragment U at the 5' end of the Ku70 gene; The sequence of primer P12 is 5'-CTAGTCTAGACCCGACAAGCCTGAGGACAA-3'; the sequence of primer P13 is 5'-TCCTGAGCTCACGCTCTCTTCTATCAGACCCT-3'; the sequence of primer P10 is 5'-CCCAAGCTTTCAACCACCACCATCCGTCTC-3'; the sequence of primer P11 is 5'-CGGAATTCAATGTGACCTGCTCGCCTCC -3';

[0026] Step (2): DNA fragment D and plasmid pDHt / sknt were double digested with XbaⅠ and SacⅠ, purified and ligated to obtain recombinant plasmid pDHt / sknt-D; DNA fragment U and plasmid pDHt / sknt-D were double digested with HindⅢ ...

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Abstract

The invention discloses a construction method for a high-gene-knockout-efficiency Aspergillus chevalieri var. intermedius mutant engineering bacterial strain. The construction method includes cloning the 3' end DNA segment and the 5'end DNA segment of the ku70 gene, constructing a knockout vector for the ku70 gene, performing agrobacterium tumefaciens-mediated transformation, and indentifying a ku70 gene knockout mutant strain. Compared with the parent Aspergillus chevalieri var. intermedius mutant, the mutant engineering bacterial strain obtained by the invention has a slightly slow grow rate, no significant change in other phenotypes and genetic stability, and can be used as a receptor strain to perform knockout verification of gene functions. When the mutant engineering bacterial strain obtained by the invention is used as the receptor strain, the gene knockout efficiency is high, the gene knockout mutant strain can be obtained easily and heavy screening work is reduced.

Description

technical field [0001] The invention relates to a biological technology, in particular to a method for constructing an engineering strain of Aspergillus shevalley intertype variety with high gene knockout efficiency. Background technique [0002] The Aspergillus intertype varietal, commonly known as "Golden Flower", is the dominant strain in the flowering process of Fuzhuan tea. Cunxi, Liu Shiquan, Zhou Xiaomei, Dong Meng, Hu Zhiyuan 2011). At present, there is still controversy over the nomenclature of the intertype varietal of Aspergillus chevalieri, which may be due to differences in materials and identification methods (Ding Ting, Lv Jiating 2012; Hu Zhiyuan, Zhao Yunlin, Liu Shiquan, Li Yanzi, Xu Yongli 2012), among which 1990 In 1990, Liu Zuoyi and others identified the "Golden Flower" bacteria as Aspergillus shevalley intertype by electron microscope (Liu Zuoyi, Qin Jing, Wang Nailiang 1990). Fuzhuan tea is an important "border sales tea", which is very popular in t...

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/66
Inventor 刘作易曹旸刘永翔
Owner 贵州省生物技术研究所
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