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Magnaporthe oryzae PalH protein and function and application of coding gene thereof

A technology of coding gene, pyrosporium, applied in the direction of genetic engineering, plant gene improvement, application, etc., can solve the problem of few reports of specific gene cloning

Inactive Publication Date: 2011-05-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on specific gene cloning related to fungal growth.

Method used

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  • Magnaporthe oryzae PalH protein and function and application of coding gene thereof
  • Magnaporthe oryzae PalH protein and function and application of coding gene thereof
  • Magnaporthe oryzae PalH protein and function and application of coding gene thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the separation of MoPalH gene

[0043] 1) Screening and genetic analysis of mutants

[0044] By screening the ATMT transformant library of Magnaporthe oryzae P131 strain constructed in our laboratory, a mutant CD9848 with slow colony growth and abnormal colony morphology was obtained. The mutant CD9848 was sexually crossed with the M. oryzae S1528 strain of the opposite mating type, and the hybrid offspring were selected for phenotype and hygromycin resistance analysis. As a result (Table 1), it was found that all the non-hygromycin-resistant offspring were wild-type, and one of the hygromycin-resistant offspring was wild-type, and the rest were mutant phenotypes (slow growth), indicating that the mutant phenotype The insertions of type and T-DNA were co-segregated, and T-DNA was inserted in multiple copies.

[0045] Table 1 Co-segregation analysis of mutant phenotypes and hygromycin resistance

[0046]

[0047] 2) TAIL-PCR and gene sequence analysis...

Embodiment 2

[0049] Embodiment 2, the effect of MoPalH gene in mycelia growth and conidia production

[0050] 1) Construction of knockout vector

[0051]The gene knockout strategy uses homologous recombination to replace the coding region of the MoPalH gene with the hygromycin phosphotransferase gene. Using P131 genomic DNA as a template, use 06440upF (5'-CGTACTAGTTGCGGTAGACCTCC-3'), 06440upR (5'-TAGGAATTCCTTACCCGTATGACAC-3') to amplify fragments as the left arm, 06440dF (5'-GCCAAGCTTGCTAACTCTACTTGC-3'), 06440dR( The 5'-ACGGGTACCTGTCAGCTACAAAG-3') amplified segment was used as the right arm, digested with SpeI+EcoRI and HindIIII+KpnI respectively, and connected to both sides of the hygromycin phosphotransferase gene of the pKOV21 vector according to the upstream and downstream sequence to form a gene replacement vector pKOV06440.

[0052] 2) Construction of complementary vector

[0053] According to the predicted gene, PCR primers 06440CF (5'-CCTGAATTCCTGTTCTAGTCCAAC-3', including the E...

Embodiment 3

[0065] Embodiment 3, the effect of MoPalH gene in blast fungus pathogenicity

[0066] Use the spore suspensions of wild-type P131 and knockout ΔmopalH (at a concentration of 1×10 5 per ml) were evenly sprayed and inoculated on the front of barley leaves grown for about 7 days. Incubate in the dark at 26°C and 100% relative humidity for 36 hours, and then continue to moisturize and cultivate for 3 days after exposure to light, and investigate the incidence of the disease. The inoculation results showed that, compared with wild-type P131, the virulence of the knockout ΔmopalH was significantly reduced, while the virulence of the complement CΔmopalH was restored to the wild-type level.

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Abstract

The invention discloses a magnaporthe oryzae MoPalH gene and the function and application of coding protein thereof. The gene controls the growth of magnaporthe oryzae mycelia, sporogenous amount and a new pathogenic gene MoPalH; and the gene and complementary deoxyribonucleic acid (cDNA) thereof and protein coded thereby have nucleotide or amino acid sequences which are sequences SEQ ID No.2, No.3 and No.1 in a sequence table respectively. Due to the mutation or deletion of the MoPalH gene, compared with wild type P131, the growth rate of the magnaporthe oryzae mycelia is reduced by 11.4 percent, the growth rate of the mycelia is more sensitive to peracid or peralkaline environment, the yield of conidiospores of the magnaporthe oryzae mycelia is reduced to 15 percent of the wild type P131, and the infestation capacity of the magnaporthe oryzae mycelia on paddy is reduced obviously. A FgPalH gene in fusarium graminear can restore the growth rate of mycelia of a magnaporthe oryzae knock-out body delta mopalH, the yield of the conidiospores and pathogenicity to the wild type level, which indicates the FgPalH gene has the similar function with the MoPalH gene. The expression or modification of the MoPalH-coded protein or / and homologous protein of the MoPalH-coded protein in other pathogenic fungi can be used as an important candidate target and is used for designing and screening a novel antifungal medicament.

Description

technical field [0001] The invention relates to the function and application of a gene for controlling fungal mycelium growth, conidia production and pathogenicity and its coded protein in the fields of plant protection and fungal genetic engineering. Background technique [0002] Magnaporthe oryzae is a fungus belonging to the subphylum Ascomycota, which can infect rice, wheat, barley, millet and other grasses, 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 pyrosporium to plants begins with the formation of conidia. The process includes conidia attached to pl...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/63
Inventor 彭友良陈小林赵文生王大伟杨俊张燕
Owner CHINA AGRI UNIV
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