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Magnaporthe grisea MoPPF3 gene and function and application of coding protein of magnaporthe grisea MoPPF3 gene

A rice blast fungus and protein technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problems of gene cloning and few molecular mechanisms

Inactive Publication Date: 2011-04-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few reports on gene cloning and molecular mechanisms in this regard

Method used

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  • Magnaporthe grisea MoPPF3 gene and function and application of coding protein of magnaporthe grisea MoPPF3 gene
  • Magnaporthe grisea MoPPF3 gene and function and application of coding protein of magnaporthe grisea MoPPF3 gene
  • Magnaporthe grisea MoPPF3 gene and function and application of coding protein of magnaporthe grisea MoPPF3 gene

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Experimental program
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Embodiment 1

[0034] Embodiment 1, the isolation and identification of gene MoPPF3

[0035] 1) Screening for mutants with reduced infection spike formation rate. The conidia (2 × 10 5 ) were respectively inoculated into onion epidermal cells, and after 24 hours of dark moisturizing at 25° C., observed and counted the formed infection nails under a microscope with a magnification of 10×40, and screened out a reduction in the formation rate of infection nails (about 27% ) mutant MS5087. See figure 1 .

[0036] 2) Genetic analysis of mutant MS5087. The mutant MS5087 was sexually crossed with the wild-type strain S1528, which has no hygromycin resistance and has the opposite mating type, and the ascospores of the hybrid offspring were selected for analysis of hygromycin resistance and infection spike formation rate. Among the 35 ascospores tested, 18 hygromycin-sensitive ascospores showed wild phenotypes, and 17 hygromycin-resistant ascospores showed mutant phenotypes (decreased infection ...

Embodiment 2

[0039] Embodiment 2, the effect of gene MoPPF3 in the formation process of blast fungus infection spike

[0040] 1) MoPPF3 gene complementation mutant MS5087

[0041] a. Construction of complementary vector

[0042] The neomycin phosphotransferase gene was excised from plasmid pSM331 with XbaI, and ligated with pBlueScriptKS(+) digested with XbaI to obtain plasmid pKN. According to the nucleotide sequence of the gene MoPPF3, the forward primer hbup (5'-AGGTACCTGACAGCAGTCCGTG-3', KpnI) and the reverse primer hbdown (5'-ACTCGAGAGTTTGTGGCGTCTG-3', XhoI) were designed respectively. Using the genomic DNA of the wild-type strain P131 as a template, a 3118bp DNA fragment was amplified with fidelity LA-Taq enzyme, which contained the MoPPF3 gene and its upstream 1.5kb promoter sequence, and its sequence corresponds to SEQ in the sequence listing ID №: 1-3118bp in 1. After the fragment was digested with KpnI and XhoI, it was connected to the vector pKN digested with the same enzymes...

Embodiment 3

[0056] Embodiment 3, the effect of MoPPF3 gene in blast fungus pathogenicity

[0057] The conidia suspension (concentration of 5×10 4 per milliliter) evenly spray and inoculate on the front of rice leaves at the four-leaf and one-heart stage, or the front of barley leaves at the one-leaf and one-heart stage, or connect the mycelium blocks to the front of rice leaves at the four-leaf and one-heart stage, at 26°C and 100% relative humidity Incubate in the dark for 36 hours, and continue to moisturize and cultivate for 3 days after exposure to light, and observe the incidence. The results showed that the knockout CD5 of the MoPPF3 gene could not form diseased spots on the host plants rice and barley, and could not form diseased spots on the scratched rice leaves, while wild-type P131 could not form diseased spots on the rice and barley leaves can form typical rice blast lesions ( Figure 5 ). This indicated that, compared with wild-type P131, the pathogenicity of MoPPF3 knocko...

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Abstract

The invention discloses a magnaporthe grisea MoPPF3 gene and function and application of coding protein of the magnaporthe grisea MoPPF3 gene. The gene can control formation and pathogenicity of a magnaporthe grisea infection nail, the gene and cDNA (complementary deoxyribonucleic acid) thereof and protein coded by the gene respectively have the nucleotide or amino acid sequence shown as SEQ ID No. 1, No. 2 and No. 3 in a sequence list. The gene can be expressed in conidium, appressorium and infected hypha of magnaporthe grisea and is necessary for fatty acid metabolism. The MoPPF3 gene is knocked out, so that formation rate of magnaporthe grisea is reduced to be 1 / 3 that of wild type, osmotic pressure endurance capacity is obviously reduced, and capability to infect rice leaves is completely lost. Expression or modification of the protein coded by the MoPPF3 gene or / and homogenous protein thereof in other pathogenic fungi can be taken as important candidate targets and can be used for designing and screening antifungal novel medicament. The invention discloses a magnaporthe grisea gene and function and application of the coding protein of the magnaporthe grisea MoPPF3 gene, wherein the gene can control formation and pathogenicity of the magnaporthe grisea infection nail.

Description

technical field [0001] The invention relates to the application of a gene and its coded protein for controlling the formation and pathogenicity of fungal infection spikes in the field of microbial genetic engineering and plant protection. Background technique [0002] Magnaporthe oryzae belongs to Ascomycota subphylum fungus, which can infect rice, wheat, barley and other gramineous plants, causing blast. In particular, the rice blast caused by the fungus infecting rice occurs every year in various rice cultivation areas in the world, and the damage is extensive and serious. In general, rice blast damage can reduce rice production by 5-10%, and severely diseased fields can lead to failure of rice harvest. Rice blast has been popular in my country for many times, and it is also one of the main diseases of rice in my country. [0003] The infection of plants by Magnaporthe oryzae begins with conidia. The conidia attached to the plant leaves germinate to form germ tubes, and...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N15/63
Inventor 彭友良杨俊张燕孔令安王大伟赵文生陈小林戚琳璐
Owner CHINA AGRI UNIV
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