Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses

A virulence, piriformis technology, applied in the direction of antifungal, genetic engineering, plant genetic improvement, etc., can solve the problem of reducing the ability of infection nail formation, the inability of piriformis to form a normal form, melanizing appressorium, and infection. Problems such as the reduction of pathogenicity of diseased rice varieties

Inactive Publication Date: 2007-04-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Deletion of this gene resulted in the inability of Pyrosporium to form normal and melanized appresses, and significantl

Method used

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  • Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses
  • Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses
  • Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening and identification of mutants.

[0033] 1. Preparation of conidia

[0034] The hyphae of each REMI transformant of pyrosporium bacterial strain P131 is fully interrupted, and equably spreads to the tomato juice oatmeal substratum (every liter contains 150ml tomato juice, 30-50 gram oatmeal boils 30 minutes and filters and gets filtrate, 20 grams of agar) plate, cultivated at 26°C-28°C, when the new mycelium can be seen growing out of the medium surface, gently wash off the mycelium with a cotton swab, rinse with water, cover with a single layer of gauze, and put it on 26 Cultivate under light at ℃-28℃ for 48 hours, and a large number of pyrosporium spores can be seen on the surface of the medium.

[0035] 2. 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 at 4000rpm for 5 minutes to collect the spores, and then suspend them in 0.25...

Embodiment 2

[0042] Example 2 Co-segregation analysis of mutant phenotype changes and insertion markers

[0043] The method of genetic hybridization was used to analyze the co-segregation of the insertion marker hygromycin resistance gene and the mutant phenotype in the mutant, and the mutant was mated with a pear that did not have the mutant phenotype and hygromycin resistance, and had the opposite mating type S1528 strain S1528 was hybridized, and the appressorium morphology and hygromycin resistance of its ascospore progeny were analyzed. The specific methods are as follows:

[0044] The above-mentioned mutant X3073 was confronted with the Pyrospora strain S1528 without hygromycin resistance and with normal appressorial morphology on 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, squ...

Embodiment 3

[0047] Example 3 Cloning of the gene MgPTH12 controlling appressor maturation and morphological variation of Pyrospora sp.

[0048] 1. Plasmid Rescue

[0049] Through the rescue of the inserted plasmid in the mutant, the genome sequence of the side end of the corresponding insertion site was obtained, and the insertion position of the plasmid in the mutant was determined. The specific method is as follows:

[0050] Select the insert plasmid pUCATPH (Lu, S., Lyngholm, L., Yang, G., Bronson, C., Yoder, O.C. 1994. Tagged mutants at the Toxl locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration. Proc. Natl. Acad .Sci.USA91: 1264-12653), the restriction endonuclease XhoI which has no restriction site in pUC18 completely digested the genome of mutant X3073. After purification by ethanol precipitation, self-ligation was performed with T4 DNA ligase, and the competent cell JM109 of Escherichia coli was transformed. Plasmids of transformants were extracted ...

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Abstract

The invention discloses a new necessary MgPTH12 gene and utility to make pear spore bacterium attach to mature and lead to disease, which is characterized by the following: the gene and its cDNA and coding product possess the sequence in the SEQ ID No.:1, No.:2 and No.:3; the protein of coded gene possesses homeo structural region, which is not similar to known functional protein; the gene removes abnormal and matured attaching cell, which reduces forming frequency of infecting tin and extending infecting hypha without pathogenic ability for rice; the homologous gene of gene lies in the plant pathogenic bacteria widely, which can be applied to design and sieve new drug to prevent fungus.

Description

technical field [0001] The invention belongs to the fields of plant pathology, pesticide science and microbial genetic engineering. The invention proves a MgPth12 gene which controls appressor maturation and pathogenicity from pyrosporium through insertion mutation, gene complementation and gene knockout. This gene may encode a transcription factor, the deletion of which leads to a significant reduction in the pathogenicity of Pyrosporium to rice. Therefore, the expression of the gene and its coded functional protein can be used as a target site in the screening and design of antifungal agents. Background technique [0002] Magnaporthe grisea 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 widespread and serious. In general, rice blast damage c...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/63A61K38/16A61P31/10
Inventor 彭友良张凯赵文生张裕君时涛
Owner CHINA AGRI UNIV
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