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Construction method of monascus purpureus mokH gene deletion strain

A gene deletion strain, Monascus purple technology, applied in the field of biogenetic engineering

Inactive Publication Date: 2019-10-15
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some people also knocked out the malic enzyme gene in Aspergillus niger by means of replacement homologous recombination to explore its influence on the metabolism of the tricarboxylic acid cycle of Aspergillus niger. After screening, a genetically stable malic enzyme gene deletion strain was obtained. Strain comparison revealed that deletion of the malic enzyme gene had no significant effect on the tricarboxylic acid cycle

Method used

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  • Construction method of monascus purpureus mokH gene deletion strain
  • Construction method of monascus purpureus mokH gene deletion strain
  • Construction method of monascus purpureus mokH gene deletion strain

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Embodiment 1

[0019] This embodiment provides a kind of Monascus purple wxya A method for constructing a gene deletion strain, comprising the following steps:

[0020] (1) Bacteria culture

[0021] Strain cultivation: Activate the M1 strain on the potato solid medium for 2 generations, inoculate an appropriate amount of bacterial liquid into the seed medium, and cultivate it at 30 °C and 200 r / min for 2 days, and inoculate the seed liquid into the fermented In the culture medium, culture at 30 °C, 150 r / min for 2 days, and then at 25 °C, 150 r / min for 13 days.

[0022] (two). wxya Deletion strain construction

[0023] Cloned from the genome of Monascus purple M1 wxya gene, will wxya After the gene was digested and recovered, it was connected to the pUC18 plasmid, and then the CaMV 35S promoter and the hygromycin B resistance gene were cloned using the pCAMBIA302 plasmid as a template, and the hygromycin B resistance gene with the promoter was inserted into pUC18- wxya recombinant plas...

Embodiment 2

[0024] Verification of embodiment 2 deletion strain

[0025] Replacement targeting vector pUC18- wxya The principle of gene knockout of -hph is as follows: figure 1 As shown, the two homologous arms of the targeting vector undergo two homologous recombination with the homologous sequences on the genome of Monascus violaceum M1 respectively, thus using hph The fragment replaces the genomic DNA wxya fragment, which will eventually hph Resistance genes are integrated into the genome, making wxya Gene inactivation, so as to achieve the purpose of knockout.

[0026] The screened transformants were continuously passed on for 5 generations to screen for stable genetic transformants. The genomic DNA of the transformant was extracted and used as a template, respectively, with wxya -F, wxya -R and hph -F, hph -R primer to amplify wxya and hph . If the transformed strain fails to amplify the wxya gene to amplify hph gene; the control strain was able to amplify wxya ...

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Abstract

The invention discloses a construction method of a monascus purpureus mokH gene deletion strain, wherein the method includes the following steps: (1) strain culture; and (2) construction of the mokH deletion strain. The mokH deletion strain is successfully constructed, and the research basis is improved for exploring the function of the mokH gene in monascus purpureus.

Description

Technical field: [0001] The invention relates to a kind of Monascus purple wxya The invention relates to a method for constructing a gene deletion strain, which belongs to the field of biological genetic engineering. Background technique: [0002] Monascus, also known as "Monascus", belongs to the phylum Fungi, Ascomycota, Ascomycetes, Monascus family, and the genus Monascus in fungal taxonomy. Monascus can produce a variety of secondary metabolites, such as Monacolin K, γ-aminobutyric acid and citrinin, many of which are widely used in food pigments, medicine, wine making, etc. MonacolinK Also known as "lovastatin", it is a physiologically active polyketide produced by Monascus. Studies have found that Monacolin K can inhibit the activity of the key enzyme HMG-CoA in cholesterol synthesis, and can effectively inhibit cholesterol synthesis. Therefore, Monacolin K is regarded as an ideal drug for lowering cholesterol at home and abroad. [0003] Gene knockout technology is...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/90C12N15/80C12N15/65C12R1/645
CPCC07K14/37C12N15/65C12N15/80C12N15/902
Inventor 张婵王成涛孙宝国
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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