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Applications of verticillium dahlia pathogenicity related gene VdPR1 as anti-verticillium dahlia target gene

A technology for cotton Verticillium wilt bacteria and disease-related genes, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of limited research reports on the molecular mechanism of disease, disease control methods and drugs are difficult to achieve control effects, no control methods, etc.

Active Publication Date: 2015-08-05
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It causes huge losses to cotton production every year, and there is no effective control method so far
Verticillium wilt of cotton is a soil-borne vascular disease, and it is difficult to control and find good resistant materials. Therefore, new cotton varieties with high resistance to Verticillium wilt have not been bred for many years
Research and development of disease control methods and agents are also difficult to achieve the desired control effect
Although the economic loss caused by Verticillium wilt to cotton production cannot be underestimated, the research reports on the molecular mechanism of pathogenicity are still very limited

Method used

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  • Applications of verticillium dahlia pathogenicity related gene VdPR1 as anti-verticillium dahlia target gene
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  • Applications of verticillium dahlia pathogenicity related gene VdPR1 as anti-verticillium dahlia target gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Isolation and cloning of the VdPR1 gene

[0024] Through the pathogenicity determination of the T-DNA insertion mutant library of the strong pathogenicity cotton Verticillium dahliae strain Vd080 (preservation number: CGMCC No.5904), the mutant vdpr1 with significantly reduced pathogenicity was screened. Southern hybridization confirmed The mutant is a single-copy insertion of T-DNA, and the disease-related gene VdPR1 is obtained by using TAIL-PCR and the VdLs.17 genome database.

[0025] 1.1 Southern hybridization to determine the copy number of mutant vdpr1 T-DNA insertion

[0026] The Southern hybridization probe primer HygP-F / R was designed according to the hygromycin fragment sequence with a size of about 500 bp on T-DNA. Genomic DNA of low pathogenicity mutant vdpr1 5ng, NEB buffer 10μL, BamHI 3μL, water up to 100μL. Digest for 5 hours at 37°C, and then bathe in water at 65°C for 10 minutes to terminate the digestion reaction. The methods of probe pr...

Embodiment 2

[0034] Embodiment 2: Construction of VdPR1 gene knockout vector and complementary vector

[0035] 2.1 Construction of knockout vector

[0036] Firstly, the conventional PCR method was used to amplify the upstream 1.2 kb sequence and the downstream 1.2 kb sequence of the VdPR1 gene from the genome of wild-type Vd080 with primers P1 / P3 and P4 / P6 respectively, and the plasmid pUC- A 1.8kb hygromycin resistance gene cassette sequence was obtained from Hyg, wherein primers P3, P4 and HPH-F, HPH-R have reverse complementary linkers. Then combined with the fusion PCR technology, the upstream fragment, the hygromycin resistance gene cassette and the downstream fragment were fused according to 1:3:1 to obtain the fusion product of the upstream-hygromycin-downstream fragment. Secondly, the nested PCR method was used to amplify the three-fragment fusion product with specific primers P2 and P5 with Gateway BP reaction adapters. Finally, this fragment was integrated into the pGKO2-Gatewa...

Embodiment 3

[0039] Example 3: Obtaining of VdPR1 Gene Knockout Mutants and Complementary Mutants

[0040] 3.1 Obtaining knockout mutants

[0041] The knockout vector pGKO-VdPR1 was transformed into Agrobacterium AGL-1 by freeze-thaw method for the genetic transformation of the wild-type strain Vd080, and the concentration of the spore liquid of Verticillium dahliae Vd080 was adjusted to 5×10 6 Mix CFU / mL and Agrobacterium AGL-1 (OD=0.3–0.4) in equal volumes, take 200ul of the mixture and spread it on a microporous membrane that is spread on IM medium (200μmol / L AS), and incubate at 25°C cultured in the box for 2 days. Transfer the above microporous filter to PDA selection medium (50 μg / mL Cef, 50 μg / mL Spe, 50 μg / mL Hyg and 50 μmol / L F 2 dU) and cultured at 25°C for more than 5 days until colonies of transformants appeared. Pick a small amount of mycelium with a toothpick and add it to 200ul sterile water to prepare a spore suspension, and apply WA (Cef, Spe, Hyg, F 2 dU) plate, culti...

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Abstract

The present invention relates to the field of plant diseases, particularly to applications of verticillium dahlia pathogenicity related gene VdPR1 as the anti-verticillium dahlia target gene. According to the present invention, the colony morphology, the growth rate, the extracellular enzyme activity and the pathogenicity of the knockout mutant [delta]VdPR1 and the complementary mutant [delta]VdPR1-C are determined, and are subjected to compared analysis with various indexes of the wild type, such that it is determined that the VdPR1 gene is the verticillium dahlia pathogenicity related gene, is associated with microsclerotium production and melanin accumulation, and provides promotion effects for activities of cellulase and protease.

Description

technical field [0001] The invention relates to the field of plant diseases, in particular to the application of the pathogenicity-related gene VdPR1 of the cotton Verticillium dahliae as a target gene for resisting the cotton Verticillium dahliae. Background technique [0002] Plant diseases are important factors affecting crop production and global food security, and fungi are one of the most important pathogen groups. It has become an important task and focus of scientific research to strengthen the research on the pathogenic mechanism of pathogenic fungi and the mechanism of host disease resistance, and to seek new ways to control fungal diseases. The research on the gene function of pathogenic fungi is one of the keys to solve the above problems. [0003] Cotton verticillium wilt caused by Verticillium dahliae Kleb is a worldwide fungal disease, and it is one of the most common and serious diseases in major cotton-producing countries and regions in the world. It cause...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N15/80C12R1/645
Inventor 张亚林朱荷琴李志芳冯自力冯鸿杰师勇强赵丽红
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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