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Function and usage of magnaporthe oryzae MoCHS1 gene and coded protein thereof

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

[0005] From the above research reports, it can be seen that the formation and morphological construction of conidia of Magnaporthe oryzae is a very complicated process, which requires the participation of many genes
However, there are still few reports on gene cloning and molecular mechanism in this regard.

Method used

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  • Function and usage of magnaporthe oryzae MoCHS1 gene and coded protein thereof
  • Function and usage of magnaporthe oryzae MoCHS1 gene and coded protein thereof
  • Function and usage of magnaporthe oryzae MoCHS1 gene and coded protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, Cluster analysis of homologous proteins in MoChs1 and other fungi

[0036] Using the amino acid sequence of the rice blast fungus MoChs1 protein in NCBI ( http: / / www.ncbi.nlm.nih.gov / ) to perform a blastp search on different species to obtain its homologous analogues among different species: NCU0311.4 (Neurosporacrassa, XP_961338), FGSG10116.2 (Fusarium graminearum, XP_390292), BC1G04441.1 (Botrytis cinerea, XP_001557191), UM04290.1 (Ustilago maydis, XP_760437), SCRG (Saccharomyces cerevisiae, NP_009594). Wherein, the amino acid sequence of the MoChs1 homologous protein in Fusarium graminearum is shown in SEQ ID No: 4, the amino acid sequence of the MoChs1 homologous protein in Botrytis cinerea is shown in SEQ ID No: 5, and the amino acid sequence of the MoChs1 homologous protein in Ustilago maydis is shown in Shown in SEQ ID No:6. Use clustalx1.83 software to compare and analyze the above amino acid sequences, and construct a phylogenetic tree between Mo...

Embodiment 2

[0037] Embodiment 2, the effect of MoCHS1 gene in blast fungus conidia morphogenesis and conidia production

[0038] 1) Construction of knockout vector

[0039] Gene knockout adopts the method of homologous recombination, and replaces the coding region of the MoCHS1 gene in the wild-type P131 with the hygromycin phosphotransferase gene. The specific strategy is shown in the appendix figure 2 a. Using the genomic DNA of wild-type P131 as a template, primer P1 (5'-TACTAGTCGATGTCCGTCAGCG-3', containing a SpeI restriction site at the 5' end) and primer P2 (5'-AGAATTCCTGAGTGAGATGGCG-3', containing an EcoRI enzyme at the 5' end cleavage site) as the left arm; use primer P3 (5'-AGTCGACGACTAAGGCTTGGTG-3, 5' end containing SalI restriction site) and primer P4 (5'-TGGTACCAGGACACCTTCTTCG-3', 5' end containing KpnI restriction site) amplified fragment was used as the right arm. The left arm was double-digested with SpeI and EcoRI, and the right arm was double-digested with SalI and Kp...

Embodiment 3

[0052] Embodiment 3, the effect of MoCHS1 gene in the formation of rice blast fungus appressorium

[0053] 1) Formation of appresses after conidia attach

[0054] Pipette 20ul of wild-type P131, knockout LA40, and complement GC30 spore suspensions (concentration: 50,000 spores / ml) and drop them on hydrophobic coverslips. After cultivating in the dark at 25°C for 24 hours, observe under a microscope and The formation rate of appressoria was counted. According to the statistics of appressoria formation rate = number of conidia that germinated to form appressoria / number of germinated spores, it was found that the appressoria formation rates of wild-type P131, knockout LA40 and complement were 96%, 61% and 95%, respectively. % (attached Figure 5 ), the appressorial formation rate of the knockout body was significantly lower than that of the wild type, while the appressorial formation rate of the complement could be restored to the wild type level.

[0055] 2) Formation of appr...

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Abstract

The invention discloses the function and the usage of a magnaporthe oryzae MoCHS1 gene and a coded protein thereof. The gene controls the conidium form, the conidium yield, the appressorium formation rate and the pathogenicity of magnaporthe oryzae. The gene as well as the cDNA and the coded protein thereof respectively comprise nucleotide or amino acid sequences SEQ ID No: 1, No: 2 and No: 3 in the sequence list. The knockout of the MoCHS1 gene causes the change of the form establishment of the magnaporthe oryzae conidium; the conidium is reduced and has no diaphragm or has only one diaphragm; the yield of the conidium is decreased to 2% of wild type bacterial strains; the formation rate of the appressorium is decreased to 60% of the wide type bacterial strains; and the infestation capability on rice vanes is obviously decreased. The protein coded by MoCHS1 or / and the expression or the modification of the homologous protein in other pathogenic fungi can be taken as important candidate targets and can be used for designing and screening novel antifungal drugs.

Description

technical field [0001] The invention relates to the application of a gene and its coded protein which affect the morphological establishment and pathogenicity of fungal conidia in the field of microbial genetic engineering and plant protection. Background technique [0002] Magnaporthe oryzae is a fungus belonging to the subphylum Ascomycota, which can infect rice, wheat, barley 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] Magnaporthe oryzae uses conidia as the source of primary infection and re-infection of host plants. The conidia of Magnaporthe oryzae ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N15/63
Inventor 彭友良孔令安张燕杨俊赵文生戚琳璐
Owner CHINA AGRI UNIV
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