Method for knocking out fungus genes

A gene knockout and fungal technology, applied in the field of genetic engineering, can solve the problems of few types and limitations of site-specific DNA recombinases, and achieve the effect of avoiding the effect of SCRaMbLE and improving the stability.

Inactive Publication Date: 2016-02-17
TIANJIN UNIV
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Problems solved by technology

At present, the site-specific DNA recombinase used for gene knockout in fungi is mainly Cre recombinase, and there are too few types of site-specific DNA recombinases, which limits its application, so new ones suitable for gene knockout in fungi are needed. site-specific DNA recombinase

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  • Method for knocking out fungus genes
  • Method for knocking out fungus genes
  • Method for knocking out fungus genes

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Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1 Contains the construction of the recombinant bacterial strain of Vika-vox recombinant system

[0059] Experimental Materials:

[0060] Saccharomyces cerevisiae codon optimization was performed according to the reported amino acid sequence of Vika recombinase. The DNA molecule encoding Vika recombinase after optimization has the nucleotide sequence shown in SEQ ID NO: 3, and the gene was synthesized in Jinweizhi Company to obtain plasmid pUC57-Vika .

[0061] According to the reported CreEBD fusion gene with SCW11 promoter, which has the nucleotide sequence shown in SEQ ID NO: 24, the gene was synthesized in Jinweizhi Company to obtain the plasmid pUC57-pSCW11-CreEBD.

[0062]The strain Saccharomyces cerevisiae synIII, which artificially synthesized the third chromosome of Saccharomyces cerevisiae, was donated by the Jef Boeke laboratory of Johns Hopkins University in the United States, and its chromosome sequence is numbered in GenBank: KC880027.1 (http: / / ...

Embodiment 2

[0079] Example 2: Knockout of Saccharomyces cerevisiae Chromosomal Fragments Using Vika-vox Recombinase System

[0080] The recombinant strain yLQH207 was inoculated into the medium for cultivation. After the glucose in the medium was exhausted, galactose mother solution (20%) was added to make the final concentration of galactose 20g / L to induce the expression of the GAL1 promoter. The bacterial solution after 0h, 2h, 8h, 18h and 24h of induction with galactose was selected and spread on the YPD plate, and cultured at 30°C for 2 days. 49 single colonies were randomly picked and streaked to the YPD plate, cultured at 30°C for 24 hours, and then copied to the SC-Ura plate. Because the length of the fragment between the two vox on the yLQH207 genome is 6.2kb, when the Vika-vox recombinase system is activated for gene knockout, its phenotype is changed by Ura + Trp - becomes Ura - Trp - , leaving a scar sequence of 34bp vox site on the genome. Since there is a Ura3 nutrient ...

Embodiment 3

[0084] Embodiment 3: Utilize Vika-vox recombination system to knock out the application of genome Cre gene

[0085] (1) Construction of Cre recombinant strains

[0086] Using plasmid pRS414 as template and trp1L-vox-F / R as primers, the genome integration fragment trp1L-vox (385bp) was amplified. Using the plasmid pUC57-pSCW11-CreEBD as a template and pSCW11-Cre-F / R as a primer, the genome integration fragment pSCW11-CreEBD-2 (3443bp) was amplified. Using plasmid pRS416 as template and Ura3-Cre-F / Ura-vox-R as primers, PCR was carried out to obtain the genome integration fragment Ura3. Using plasmid pRS414 as template and vox-Trp1R-F / R as primers, PCR was carried out to obtain the genome integration fragment vox-Trp1R (390bp). The above four genome integration fragments with homology arms were obtained by PCR. The nucleotide sequence information of each primer is shown in Table 4. The gene sequence information corresponding to each genome integration fragment is shown in Tab...

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Abstract

The invention belongs to the technical field of gene engineering and discloses a method for knocking out fungus genes. The method includes: respectively introducing nucleotide sequences identified by Vika recombinase into the upstream and downstream of target genes in fungus genomes; identifying protein with Vika recombinase activity, shearing the nucleotide sequences identified by the Vika recombinase, and knocking out the target genes to obtain fungi with the target genes being knocked out. The method has the advantages that a Vika-vox recombinase system is successfully applied to fungus gene knocking out, knocking out of the target genes is achieved, a new method is provided for fungus gene knocking out, and function researches and performance optimization of fungus genes are benefited.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a fungal gene knockout method. Background technique [0002] Gene knockout is a genetic modification technology that aims at a certain genetic gene of interest, through transformation to lose specific gene function, and to study its possible impact on related life phenomena, and then speculate on the biological function of the gene. Through gene knockout, it is possible to knock out mutant genes with normal genes to improve traits and treat genetic diseases, and to knock out normal genes with mutated genes to study the role of genes in development and regulation. The application of gene knockout in fungi mainly includes: research on the structure and function of genes, improvement of industrial production strains, and modification of secondary metabolite synthesis pathways. [0003] At present, the methods for gene knockout mainly include the following methods: (1) g...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/865
Inventor 元英进林秋卉吴毅姜国珍李柄志
Owner TIANJIN UNIV
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