Rice blast bacterium nontoxic gene Avr-Pib and application

An avirulent gene, rice blast fungus technology, applied in the field of genetic engineering, can solve problems such as toxicity and achieve the effect of preventing infection

Inactive Publication Date: 2008-08-13
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many avirulent genes also play roles in pathogenicity and toxicity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: The establishment of the blast fungus T-DNA mutant library and the acquisition of pathogenicity enhanced mutants

[0012] The present invention screens the existing blast fungus T-DNA mutant library to obtain a mutant strain T940009401 with significantly enhanced pathogenicity. Firstly, the T-DNA mutant library of Magnaporthe grisea was established: Agrobacterium strain AGL-1 (containing pBHt1) was cultured on the basic medium (containing Kan50μg / mL) at 28°C for 48h, the culture was drawn and centrifuged, and washed with induction medium. Twice, resuspended in the induction medium containing Kan and acetosyringone, cultured with shaking at 28°C for 6 h; 6 cells / mL) were mixed, spread on the cellulose membrane on the co-culture medium, and incubated at 28°C for 36h. Wash the membrane with 2 mL basic medium, collect the fungal and bacterial cultures, spread them on the selection medium plate containing hygromycin and cefotaxime sodium, pick a single transforma...

Embodiment 2

[0014] Example 2: Acquisition of mutant T-DNA flanking sequences and location of tagged genes

[0015] The T-DNA flanking sequence of T940001602-2 was obtained by TAIL-PCR, and the primers used were: LB1: 5'-GGGTTCCTATAGGGTTTCGCTCATG-3'; LB2: 5'-CATGTGTTGAGCATATAAGAAACCCT-3'; LB3: 5'-GAATTAATTCGGCGTTAATTCAGT-3' ; AD9: 5'-TCGTTCCGCA-3'. After the products of the second and third steps of TAIL-PCR were subjected to 1.0% agarose electrophoresis, the specific fragments in the products of the third step were recovered. The recovered fragments were connected to pGEM-T Easy Vector for cloning. The cloned positive transformants are sequenced, and the sequence obtained after removing the vector sequence and the T-DNA sequence is compared with the rice blast fungus database. The comparison results showed that the sequence was located in the promoter region of MGG05450. It is speculated that MGG05450 may have ATP-NADK activity.

Embodiment 3

[0016] Example 3: Knockout of tagged genes

[0017]MGG05450 was knocked out in the wild strain Guy11 by homologous recombination. Firstly, a recombinant plasmid containing upstream and downstream homologous fragments of the gene to be knocked out and a fungal selection marker is constructed. Primers for amplifying upstream and downstream homologous fragments were designed separately. The hygromycin transferase gene containing promoter and terminator was excised from the pCSN43 plasmid with restriction endonuclease SalI, and after recovery and purification, the fragment was ligated in vitro with two other homologous fragments with T4 ligase. The ligation product was amplified with a primer for the 5' end of the upstream fragment and a primer for the 3' end of the downstream fragment. The amplified product was connected to a T-vector for cloning. The plasmid DNA of the positive transformants obtained by cloning was extracted, and the protoplast transformation of Magnaporthe g...

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Abstract

A rice blast bacterium innocuity gene Avr-Pib is obtained by separation and appraising in T-DNA insertion mutation. The out-knocking mutant of the gene can cause susceptibility of rice breed variety containing Pib disease-resistant gene. The out-knocking mutant and field susceptibility individual plant can recover non-toxicity by function complementation experiment and the breed variety is disease-resistant. The gene can be used as molecule target of newly pesticides and paddy rice disease-resistant genetic engineering.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to screening of blast pathogenic mutants, knockout of avirulent genes, functional complementation and application. Background technique [0002] Rice blast caused by Magnaporthe grisea (Herbert) Barr (asexual generation Pyricularia grisea) infecting rice is one of the most important diseases on rice. The sexual generation is a haploid heterothallic ascomycete. The fungus can infect more than 50 kinds of grasses, including many weeds and important cereal crops such as wheat, barley, corn, etc., and also cause certain economic losses. At present, planting disease-resistant rice varieties is the most economical and effective measure, but the pathogenicity of blast fungus mutates rapidly, and a highly resistant variety loses resistance after 3-5 years of production and becomes a susceptible variety. Therefore, how to cultivate long-lasting disease-resistant varieties and find mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/09C12N15/82
Inventor 王宗华林艳鲁国东李宏宇潘初沂
Owner FUJIAN AGRI & FORESTRY UNIV
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