Verticillium dahlia delta VdKu70 defective mutant strain and application thereof

A technology of Verticillium dahliae and mutants, applied to fungi, using vectors to introduce foreign genetic material, peptides, etc., can solve the problem of precise gene targeting, hindering the study of the host coexistence mechanism of the invasion mechanism of Verticillium dahliae, The low efficiency of gene targeting in the wild-type strain of Verticillium dahliae achieves the effect of increasing the frequency

Inactive Publication Date: 2015-04-08
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The gene targeting efficiency of the wild-type strain of Verticillium dahliae is very low, and it is difficult to achieve precise gene targeting, which seriously hinders the research on the invasion mechanism and coexistence mechanism of Verticillium dahliae

Method used

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  • Verticillium dahlia delta VdKu70 defective mutant strain and application thereof
  • Verticillium dahlia delta VdKu70 defective mutant strain and application thereof
  • Verticillium dahlia delta VdKu70 defective mutant strain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Obtaining and identification of ΔVdKu70-deficient mutant of Verticillium dahliae

[0039] 1. Experimental method

[0040] 1.1 Construction of gene knockout plasmid pGKO-Hyg-Ku70

[0041] The homologous recombination DNA fragment of Ku70 gene was constructed using In-Fusion (Frandsen R., Frandsen M, Giese H. Targeted gene replacement in fungal pathogens via Agrobacterium tumefaciens-mediated transformation[M] / / Plant Fungal Pathogens.Humana Press, 2012: 17-45.) Cloning technology. Using the plasmid pUC-Hyg as a template and Hyg-F / Hyg-R as primers (see Table 1 for primer names and sequences), a hygromycin resistance gene cassette with a length of 1.8 kb was amplified. The genome of Verticillium dahliae was used as a template, and Ku70-5F / Ku70-5R and Ku70-3F / Ku70-3R were used as primers respectively, and the 16 bp at the 5' end of the primers Ku70-5R / Ku70-3F contained an interaction with Hyg-F / R The 16 bp at the 5' end of the primer Ku70-5F / Ku70-3R contains two...

Embodiment 2

[0077] Example 2 Evaluation of gene targeting efficiency of ΔVdKu70-deficient mutant strain Vd991ΔKu70-1

[0078] 1. Experimental method

[0079] 1.1 Construction of gene knockout plasmids pGKO-neo-2-OdE2, pGKO-neo-Fre6, pGKO-neo-FreB

[0080] The present invention replaces the 1.8kb hygromycin gene expression cassette with the geneticin gene expression cassette 2.6 kb, uses the plasmid pCAM-neo as a template, and uses neo-F / neo-R as primers (see Table 1 for primer names and sequences) , to amplify a geneticin resistance gene expression cassette of 2.6 kb in length.

[0081] Utilize the same method as Example 1 to construct gene knockout plasmids pGKO-neo-2-OdE2 (VDAG_10018.), pGKO-neo-Fre6 (VDAG_09283.1), pGKO-neo-FreB (VDAG_06616.1) (for primer sequences see Table 1).

[0082] 1.2 Agrobacterium-mediated transformation of Verticillium dahliae

[0083] Transform gene knockout plasmids pGKO-neo-2-OdE2, pGKO-neo-Fre6, pGKO-neo-FreB into Agrobacterium AGL-1 by electric shock ...

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Abstract

The invention discloses a verticillium dahlia delta VdKu70 defective mutant strain and application thereof. The verticillium dahlia delta VdKu70 defective mutant strain is characterized in that a gene knock-out vector carrying a Ku70 gene homologous recombination DNA fragment is constructed, then the agrobacterium-mediated verticillium dahlia is subjected to genetic transformation, and a Ku70 gene-deleted VdKu70 defective mutant strain can be screened from mass transformants; the microbe preservation number of the Ku70 gene-deleted VdKu70 defective mutant strain is CGMCC No.10107. The obtained verticillium dahlia delta VdKu70 defective mutant strain has the morphological characteristics and virulence which are basically the same as those of a wild strain and is more sensitive to UV treatment; with the adoption of the verticillium dahlia delta VdKu70 defective mutant strain for gene targeting as a background strain, the targeting efficiency is increased by 40 to 60% compared with the wild strain. The background strain is provided for the study on verticillium dahlia infection mechanisms, coexistence mechanism with a host, and key pathogenic target gene.

Description

technical field [0001] The present invention relates to a mutant strain of Verticillium dahliae, in particular to a mutant strain of Verticillium dahliae ΔVdKu70 deficiency, and the present invention also relates to the mutant strain of Verticillium dahliae ΔVdKu70 as a background bacterial strain in Verticillium dahliae The application in the research of the infection mechanism, the coexistence mechanism with the host, and the key target gene of pathogenicity belongs to the field of construction and application of Verticillium dahliae mutant strains. Background technique [0002] Verticillium dahliae (Verticillium dahliae) is a fungal disease with a wide range of hosts. It can infect more than 200 economic crops such as cotton, tomato, flax, potato, tomato, cucumber, tobacco, pepper and eggplant, and is extremely harmful. Serious and difficult to control with chemicals, it has become a bottleneck in the development of crop production. With the successful sequencing of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12R1/645
CPCC07K14/37C12N15/80
Inventor 程红梅齐希梁苏晓峰郭惠明
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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