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Pyricularia grisea mitophagy related pathogenic factor, gene and application thereof

A technology of mitophagy and rice blast fungus, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as incomplete mechanism

Active Publication Date: 2020-01-10
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies in recent years have shown that the regulation of mitochondrial dynamic balance is of great significance to the growth, differentiation and pathogenic infection of Magnaporthe grisea, but the mechanism is not yet comprehensive.

Method used

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  • Pyricularia grisea mitophagy related pathogenic factor, gene and application thereof
  • Pyricularia grisea mitophagy related pathogenic factor, gene and application thereof
  • Pyricularia grisea mitophagy related pathogenic factor, gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Isolation and cloning of the MoUTH1 gene.

[0030] The amino acid sequence of the rice blast fungus pathogenic factor MoUTH1 protein is shown in SEQ ID No.1. The nucleotide sequence (coding region) of the Magnaporthe grisea MoUTH1 gene is shown in SEQ ID No.2.

[0031] In order to elucidate the biological function of MoUTH1 in the growth, development and pathogenicity of Magnaporthe grisea, we constructed a knockout mutant of Magnaporthe grisea MoUTH1 gene. Follow the steps below to construct the knockout vector:

[0032] The MoUTH1 gene knockout vector was constructed by homologous recombination gene knockout method. The length of the MoUTH1 gene was about 1.5kb, and it was replaced by the hygromycin B gene by homologous recombination method to obtain the MoUTH1 knockout mutant. Specific steps are as follows:

[0033] Using the genome of the wild strain B157 of Magnaporthe grisea (a gift from Naqvi Laboratory, Temasek Academy of Life Sciences, Singapore) as a templa...

Embodiment 2

[0046] Identification of a MoUTH1 knockout mutant of Magnaporthe grisea.

[0047] Design a forward primer MoUth1-5tF (MoSun4-5tF: 5'-TGGGACTAGACCAGAAGAGAGGC-3') in front of the upstream flanking sequence, and design a reverse primer pFGL820-5R (5'-TCGACCAAGTCCCTCCACACA-3') on the resistance gene, Use this pair of primers to detect knockout positive transformants, and the detection system is:

[0048]

[0049] The reaction conditions are:

[0050] Pre-denaturation at 95°C for 30s;

[0051] Denaturation at 95°C for 7s, annealing at 63°C for 20s, extension at 72°C, the extension time was determined according to the size of the amplified fragment (30s / kb), a total of 34 cycles, and the annealing temperature decreased by 0.2°C for each cycle;

[0052] Extend at 72°C for 2 min.

[0053] A total of 7 mutant strains were identified, and one knockout mutant was selected for subsequent analysis and named ΔMouth1.

Embodiment 3

[0055] Construction of MoUTH1 gene complementation vector and acquisition of complementation strain.

[0056] In order to verify that the phenotype of the mutant is caused by the deletion of MoUTH1, we constructed the complementary vector pMouth1-C, used the wild-type B157 genome as a template, and used primers MoUTH1-cF / cR to amplify the MoUTH1 gene (sequence as SEQ ID No. 5), using T4 ligase to connect to the EcoRI and KpnI restriction sites on the linearized vector pFGL822 (addgene ID: 58225).

[0057] MoUTH1-cF: 5'-AGAAACTCGAGAATTCATGGCGTTCAGGAGGTGGTTCG-3';

[0058] MoUTH1-cR: 5'-TCGCCCTTGCTCACGGTACCGCTGCCCTTGCTGAAAACAATG-3'.

[0059] The completed complementation vector was transformed into the knockout mutant ΔMouth1 strain, which was named Mouth1-C.

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Abstract

The invention discloses a pyricularia grisea mitophagy related pathogenic factor, a gene and an application thereof. The gene deletion mutation caused by homologous recombination causes slow growth ofpyricularia grisea, reduction of sporulation quantity, reduction of formation of infected hyphae and great reduction of pathogenicity on susceptible rice varieties. Therefore, the most important purpose of the pathogenic factor is to design and screen a compound capable of destroying the expression and shearing of the gene and the expression of the encoded protein, or design and screen a compoundcapable of modifying an amino acid sequence of the protein by applying the achievements, thereby developing a new antifungal drug. Meanwhile, the gene family where the gene is located does not have ahomologous gene in a plant, so that an antifungal drug developed by taking the gene as a target has relatively small influence on a host plant.

Description

technical field [0001] The invention relates to the technical field of crop fungal infection prevention and control, in particular to a pathogenic factor, gene and application related to mitophagy of rice blast fungus. Background technique [0002] Rice blast is a rice disease that seriously threatens rice production in the global rice-growing areas. The disease is a fungal disease caused by the ascomycete Magnaporthe oryzae. Rice blast, rice sheath blight and rice bacterial blight are listed as the three major diseases of rice, among which rice blast is the most serious. At present, Magnaporthe grisea has become a model organism to reveal the interaction of plant pathogenic fungi. The loss of rice production caused by rice blast accounts for 10%-30% of the total production every year in the world, and some fields even fail. There are epidemics and outbreaks of rice blast almost every year in my country. In addition to harming rice, blast fungus can also infect other gras...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12Q1/6895C12N15/80
CPCC07K14/37C12N15/80C12Q1/6895C12Q2600/136C12Q2600/158
Inventor 寇艳君孟帅时焕斌邱结华
Owner CHINA NAT RICE RES INST
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