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Knockout method for verticillium dahliae gene

A technology of Verticillium dahliae and gene knockout, which can be applied in the direction of biochemical equipment and methods, fungi, and microorganism determination/inspection. cumbersome issues

Inactive Publication Date: 2012-09-05
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This is because after cloning homologous recombination DNA fragments into T-DNA (transfer DNA), although the transformation efficiency of homologous recombination fragments has been greatly improved by means of Agrobacterium-mediated transformation methods, due to the nature of T-DNA itself, It will be randomly integrated into a certain segment of the bacterial genome, resulting in the random insertion of T-DNA transformants and the positive transformants of gene knockout produced by homologous double crossover all have the same antibiotic selection tag and are difficult to distinguish. However, the efficiency of homologous recombination is much lower than that of random insertion. Therefore, the number of gene knockout transformants is far less than that of random insertion transformants, which makes the screening method of positive transformants cumbersome, time-consuming, and wastes a lot of time. human, material and financial resources

Method used

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  • Knockout method for verticillium dahliae gene
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  • Knockout method for verticillium dahliae gene

Examples

Experimental program
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Effect test

Embodiment 1

[0031] (1) Construct a recombinant plasmid for homologous recombination, the process diagram is as follows figure 1 shown, including three rounds of PCR process:

[0032] The first round of PCR: using the genomic DNA of Verticillium dahliae (see the database website http: / / www.broadinstitute.org / for the complete gene sequence) as a template, and using 5F-ADE4 and 5R-ADE4 as primers, obtained by PCR The DNA fragment of the upstream flanking sequence of the gene ADE4 (gene sequence number is VDAG_00128, Broad Institute), about 1000 bp, and using 3F-ADE4 and 3R-ADE4 as primers, the DNA fragment of the downstream flanking sequence of the gene ADE4 was obtained by PCR, about 1000 bp; Take pUCHyg as a template, and use Hyg-F and Hyg-R as primers, carry out PCR reaction, and amplify the DNA fragment of the hygromycin resistance gene cassette with a length of 1.8kb; the primer sequences are as shown in Table 1. The conditions of the PCR are shown in Table 2; the PCR products of the ...

Embodiment 2

[0058] (1) construct a recombinant plasmid for homologous recombination, and the process is the same as in Example 1, including three rounds of PCR processes:

[0059] The first round of PCR: Using the genomic DNA of Verticillium dahliae as the template and 5F-ChsV and 5R-ChsV as primers, the DNA fragment of the upstream flanking sequence of the ChsV gene (VDAG 00420, Broad Institute) was obtained by PCR, which is approximately 1000bp, using 3F-ChsV and 3R-ChsV as primers, the DNA fragment of the downstream flanking sequence of ChsV gene was obtained by PCR, about 1000bp; using pUCHyg as template, Hyg-F and Hyg-R as primers, PCR reaction was performed, and amplification was performed. The DNA fragment of the hygromycin resistance gene cassette with a length of 1.8 kb was added; the primer sequences were shown in Table 3, and the conditions of the PCR were the same as those in Example 1, as shown in Table 4;

[0060] The second round of PCR: using the fusion PCR method, the DNA...

Embodiment 3

[0074] Knockout of Verticillium dahliae ADE4 gene was carried out according to the method of Example 1.

[0075] The difference is that the final concentration of F2dU used for screening is 0.5 μM, and 53 candidate transformants with knockout of ADE4 gene were selected from the plate for verification, and only 2 were positive transformants. Only 3.8% of the children are.

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Abstract

The invention provides a knockout method for a verticillium dahliae gene. The knockout method is characterized by comprising the following steps that: a recombinant plasmid used for homologous recombination is constructed, wherein the recombinant plasmid consists of a carrier and a homologous recombinant DNA fragment, the homologous recombinant DNA fragment contains an exogenous resistance gene used for substituting the gene to be knocked out and flanking sequences which are positioned at two sides of the exogenous resistance gene and meet the requirements of homologous recombination, and the carrier contains an exogenous conditional lethal gene; and the recombinant plasmid is transformed into the verticillium dahliae for homologous recombination through an agrobacterium-mediated method so as to obtain transformants and screen out positive transformants. ADE4 and ChsV genes of verticillium dahliae are respectively knocked out by the knockout method, and proportions of gene knockout transformants in the total transformants are 87% and 44% respectively. Therefore, compared with the prior art, the efficiency of the knockout method for the verticillium dahliae gene is greatly improved.

Description

technical field [0001] The invention relates to a method for gene knockout of Verticillium dahliae. Background technique [0002] my country is a large agricultural country. As the second largest crop after food, cotton is a strategic material related to the national economy and the people's livelihood. It is also a commodity involving the two major industries of agriculture and textile industry. It is the main source of income for more than 100 million cotton farmers nationwide. The main raw material of cotton is also the daily necessities of the people. Cotton yarn and cotton cloth are also important commodities for export. Therefore, the production, circulation, processing and consumption of cotton are closely related to the lives of the people and the interests of the vast number of cotton farmers, and also have an important impact on the development of the national economy. [0003] Verticillium dahliae caused by Verticillium dahliae is the main disease of cotton and se...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/10C12N15/63C12N1/15C12Q1/68C12Q1/04
Inventor 戴小枫田李陈捷胤汪佳妮
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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