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Target gene for controlling black rot of cabbage

A black rot and genetic technology, applied in the direction of chemicals for biological control, methods based on microorganisms, and the use of vectors to introduce foreign genetic materials, etc., can solve problems such as aggravating environmental pollution, drug residues, and increasing agricultural costs

Inactive Publication Date: 2008-11-26
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the enhancement of pathogenic bacteria's resistance to drugs, the amount of pesticides is increasing, which not only aggravates environmental pollution and drug residues, but also greatly increases agricultural costs. Therefore, it is urgent to develop new strategies for disease prevention and treatment and new pollution-free drugs

Method used

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  • Target gene for controlling black rot of cabbage
  • Target gene for controlling black rot of cabbage
  • Target gene for controlling black rot of cabbage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Cloning of embodiment 1, wxcB gene (lipopolysaccharide O-antigen synthesis kinase gene)

[0022] According to the gene sequence of wxcB, design primers:

[0023] Forward primer: WF1, 5′-TAC GAATTC ATCTCGCTCGGCATCTCCAG-3',

[0024] Reverse primer: WR1, 5′-AAC GGATCC CACCGATAAGGCAGCTGTAC-3'.

[0025] Using the total DNA of Xanthomonas cabbage black rot as a template, the full-length sequence of the gene was amplified by PCR, cloned into pLAFR3, and the gene clone pLWXCB was constructed and verified by enzyme digestion (see figure 1 ). Sequences were subcloned into pGEM-3Zf(+) and end-sequenced.

Embodiment 2

[0026] Embodiment 2, construction of wxcB gene single point integration mutant

[0027] According to the research method provided by Windgassen et al. (2000), the wxcB gene was mutated.

[0028] According to the internal sequence of wxcB gene, design primers:

[0029] Forward primer: WF2, 5′-ATC GGATCC TGAGACAGAGCATCATCG-3′,

[0030] Reverse primer: WR2, 5′-ATC AAGCTT ACGCTTTGGGTGTCGAGG-3'.

[0031] A 420bp homologous DNA fragment in the ORF of the wxcB gene was amplified by PCR, and cloned into the suicide plasmid pK18mob (double digestion with BamH I and HindIII), and the obtained recombinant plasmid was introduced into the cabbage black rot yellow single Bacillus wild-type ATCC33913 strain, antibiotics (rifampicin 50 micrograms / ml, kanamycin 25 micrograms / ml) to screen for mutants with non-polar integration mutation of wxcB gene. Three mutants were randomly selected for standard PCR verification (see figure 2 ).

Embodiment 3

[0032] Embodiment 3, the growth status of wxcB gene mutant on solid medium

[0033] On solid medium (containing 5.0 g of peptone per liter, 3 g of yeast extract powder, 20 g of glycerol, pH 7.0) plates, use toothpicks to spot wild-type Xanthomonas and wxcB gene single-point integration mutant strains, and culture them at 28°C for 72 Hour. The results showed that the three repeated colonies of the mutant of wxcB gene showed irregular edges and rough surface, while the wild-type strain grew normally and had more extracellular polysaccharides (see image 3 ).

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Abstract

The invention discloses a phathopoiesis related gene (lipopolysaccharide o- antigen synthesis kinase gene) from Xanthomonas causing cabbage black rot. The gene contains one of the following nucleotide sequence: 1) DNA sequence shown in SEQ ID No.1, 2) DNA sequence containing over 80% homology with DNA sequence shown in SEQ ID No. 1. The nucleotides of No. 197-2488 from 5'end of SEQ ID No.1 DNA is an open reading frame containing 2292 nucleotides, nucleotides of No. 197-199 is the initiator ATG of the gene, nucleotides of No. 2489-2491 is the terminator TAA of the gene.

Description

technical field [0001] The invention relates to a new pathogenicity-related gene of plant pathogenic bacteria, especially a pathogenicity-related gene of cabbage black rot fungus. Background technique [0002] Plant diseases have always been one of the main factors that reduce crop yield and quality. With the enhancement of pathogenic bacteria's resistance to drugs, the amount of pesticides is increasing, which not only aggravates environmental pollution and drug residues, but also greatly increases agricultural costs. Therefore, it is urgent to develop new strategies for disease prevention and treatment and new pollution-free drugs . Xanthomonas campestris pv. campestris, also known as Xanthomonas campestris pv. Gram-negative bacteria of black rot. The fungus can invade plants through water pores, stomata or wounds in any growth period of the host, causing diseases. The hosts include cabbage, cauliflower, Chinese cabbage, radish, rapeseed, etc. The typical symptom of bl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N15/82C12R1/01A01P3/00
Inventor 何永强姜伯乐姚子颖危广宁唐东阶冯家勋唐纪良
Owner GUANGXI UNIV