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Fungus virulence new gene MgKIN17 coming from pyricularia gisea and its use

A virulence, piriformis technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problems of unclear differentiation and formation mechanism of appressorium, and uncloned genes.

Inactive Publication Date: 2006-08-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, some genetic loci affected by appressorium formation have been identified, but the genes have not been cloned, such as CON1 and CON7 (Shi and Leung 1995); APF1 (Silue et.al.1998); APP1, APP2 and APP3 (Zhuet .al.1996); APP5 (Chun and Lee 1999)
Nevertheless, the mechanism of appressorial differentiation and formation remains unclear

Method used

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  • Fungus virulence new gene MgKIN17 coming from pyricularia gisea and its use
  • Fungus virulence new gene MgKIN17 coming from pyricularia gisea and its use
  • Fungus virulence new gene MgKIN17 coming from pyricularia gisea and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Screening and identification of mutants.

[0034] a. Preparation of conidia: spread the fully interrupted Pyrospora mycelium evenly on the oatmeal medium plate, and cultivate it at 26°C-28°C. When the new mycelium can be seen growing out of the surface of the medium, Gently wash off the mycelium with a cotton swab, rinse it with water, cover it with a single layer of gauze and culture it under light at 26°C-28°C for about 48 hours.

[0035] b. Preparation of spore suspension: wash the spores described in (1) and filter them in a 50ml centrifuge tube with double-layer lens paper, centrifuge at room temperature for 5 minutes at 4000rpm to collect the spores, and then suspend them in 0.25‰ Tween 20 . Using a hemocytometer and microscope, adjust the concentration of conidia to 10 per mL 4 spore.

[0036] c. Determination of pathogenicity: Spray and inoculate the spore suspension of the wild-type bacterial strain and the mutant evenly on the front of the leaves...

Embodiment 2

[0038] Example 2. Co-segregation analysis of mutant phenotypic changes and insertion markers.

[0039] The above-mentioned mutant H680 and the non-hygromycin-resistant strain S1528 with normal pathogenicity were cultured in confrontation on the tomato oatmeal medium. First cultivate at 25°C until the edges of the colony are about to join together, then move it to 20°C for light culture, and about 20 days form a black protruding ascus at the junction of the colony. Pick the mature ascus shell, squeeze it gently in sterile water, release the ascus in the shell, spread the ascus suspension on the water agar plate, and pick the single hyphae germinated by ascospores in the ascus 24 hours later on the tomato cultured on oatmeal medium. The hygromycin resistance of the progeny of these ascospores was counted, and the germination rate of the spores and the formation rate of appresses, infection nails and infection hyphae were observed and recorded on the onion epidermis. The result...

Embodiment 3

[0042] Example 3 Cloning of genes involved in appressorium formation of Pyricularia spp.

[0043] 1. Plasmid rescue. The genome of the mutant was completely digested with a restriction endonuclease without a restriction site in the inserted plasmid pUCATPH, refined by ethanol precipitation, and then self-ligated, and transformed into competent cells of Escherichia coli. The plasmid of the recombinant transformant was identified by enzyme digestion, in addition to the sequence of pUCATPH, the plasmid also contained part of the genome sequence on both sides. The sequence of these two segments adjacent to the insertion site was compared with the pyrosporium database to determine the disrupted gene ( FIG. 6 ).

[0044] 2. Screening of the genome TAC library: use the partial genome sequence contained in the rescued plasmid as a probe to screen the genome TAC library of Pyrosporium to obtain 8 positive clones, all of which include the full length of the desired gene, and its sequen...

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Abstract

The present invention provides one new gene MgKIN17 from Pyricularia gisea, and the gene features that it has the amino acid sequence from the site 1 to the site 3544 in the SEQ ID No. 1, the cDNA with the nucleotide sequence of SEQ ID No. 2, the promoter with the nucleotide sequence of SEQ ID No. 4, and coded protein with the amino acid sequence of SEQ ID No. 3. The insertion mutation and gene complementation of the present invention shows that the destruction of the gene leads the formation of Pyricularia gisea infecting organ and reduced lesion capacity of rice. Wheat bakanae fungus, corn stalk rot and other pathogenic fungus have homogenous protein with homogeneity over 40 % to MgKIN17 and maybe similar biological function. The gene of the present invention and its protein expression and modification may be used as the target site for designing and screening antifungal medicines.

Description

field of invention [0001] The present invention belongs to the fields of plant pathology, pesticide science and microbial genetic engineering, and provides a promoter and coding region nucleus of a new gene MgKIN17 derived from Pyrospora, which plays an important role in the formation of infected organs and pathogenicity. Nucleotide sequence and the amino acid sequence of the encoded protein. The nucleotide sequence and amino acid sequence provided by the present invention can be used as a target site in the screening and design of antifungal agents, and a certain segment of the nucleotide sequence can also be used as a probe to be used in Pyrospora and other In the clone of the fungal gene with certain homology to this sequence. technical background [0002] Rice blast caused by Magnaporthe grisea is a worldwide rice disease and one of the main diseases of rice in my country. Severely diseased fields can lead to failure of rice harvest. The disease occurs almost every yea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/37A01N63/02
Inventor 彭友良丁胜利黄俊丽魏士平赵文生
Owner CHINA AGRI UNIV
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