Method for detecting magnaporthe grisea genes for enhancing magnaporthe grisea strain pathogenicity

A pathogenicity and gene technology, which is applied in the field of detecting the prokaryotic expression of rice blast genes in vitro and in-strain overexpression to improve the infectivity of the strain, can solve the problems of detecting the effector protein gene of pathogenic bacteria and treating plants with the prokaryotic expression product of a single gene, etc. To achieve the effect of increasing the infectivity of pathogenic bacteria

Inactive Publication Date: 2015-12-23
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
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Problems solved by technology

[0004] At present, the research on the effector protein genes of pathogenic bacteria is focused on treating plants only by using the prokaryotic expression products of the genes to clarify its influence on the...

Method used

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  • Method for detecting magnaporthe grisea genes for enhancing magnaporthe grisea strain pathogenicity
  • Method for detecting magnaporthe grisea genes for enhancing magnaporthe grisea strain pathogenicity
  • Method for detecting magnaporthe grisea genes for enhancing magnaporthe grisea strain pathogenicity

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Experimental program
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Embodiment 1

[0014] The prokaryotic expression product of the gene was centrifuged at 4°C to collect the bacteria, and the bacteria were suspended with freshly prepared buffer solution (20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; 10mM β-mercaptoethanol). The supernatant was collected by centrifugation after ultrasonography to disrupt the cells in an ice-water bath. The treatment is ① spray inoculate the prokaryotic expression product of the gene on the leaves of rice variety Lijiang Xintuan Heigu at a concentration of 1.0 μg / ml for 24 hours, then inoculate strong / weak pathogenic strains, and investigate the anti-sensitivity response, real-timeRT- The expression of rice defense-related genes was analyzed by PCR; ②The overexpressed strain of the gene was used to inoculate the leaves of rice variety Lijiang Xintuan Heigu, and the lesion area was measured for 7 days, and the relative growth of fungi on the lesion was analyzed by qPCR. The control is ① Inoculate the prokaryotic expression produ...

Embodiment 2

[0021] The prokaryotic expression product of the gene was centrifuged at 4°C to collect the bacteria, and the bacteria were suspended with freshly prepared buffer solution (20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; 10mM β-mercaptoethanol). The supernatant was collected by centrifugation after ultrasonography to disrupt the cells in an ice-water bath. The treatment is ① spray inoculate the prokaryotic expression product of the gene on the leaves of rice variety Lijiang Xintuan Heigu at a concentration of 1.0 μg / ml for 24 hours, then inoculate strong / weak pathogenic strains, and investigate the anti-sensitivity response, real-timeRT- The expression of rice defense-related genes was analyzed by PCR; ②The overexpressed strain of the gene was used to inoculate the leaves of rice variety Lijiang Xintuan Heigu, and the lesion area was measured for 7 days, and the relative growth of fungi on the lesion was analyzed by qPCR. The control is ① Inoculate the prokaryotic expression produ...

Embodiment 3

[0028] The prokaryotic expression product of the gene was centrifuged at 4°C to collect the bacteria, and the bacteria were suspended with freshly prepared buffer solution (20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; 10mM β-mercaptoethanol). The supernatant was collected by centrifugation after ultrasonography to disrupt the cells in an ice-water bath. The treatment is ① spray inoculate the prokaryotic expression product of the gene on the leaves of rice variety Lijiang Xintuan Heigu at a concentration of 1.0 μg / ml for 24 hours, then inoculate strong / weak pathogenic strains, and investigate the anti-sensitivity response, real-timeRT- The expression of rice defense-related genes was analyzed by PCR; ②The overexpressed strain of the gene was used to inoculate the leaves of rice variety Lijiang Xintuan Heigu, and the lesion area was measured for 7 days, and the relative growth of fungi on the lesion was analyzed by qPCR. The control is ① Inoculate the prokaryotic expression produ...

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Abstract

The invention discloses a method for detecting magnaporthe grisea genes for enhancing magnaporthe grisea strain pathogenicity, and relates to the field of plant protection and biological technology. The method comprises the steps that a reaction of resistance or susceptibility and a real-time RT-PCR are inspected for analyzing defense related gene expression of rice early stage response and a qPCR is adopted for detecting the relative fungus growth amount of disease spots; a prokaryotic expression product of the genes is sprayed to leaves for 24 h on the basis of the concentration being 1.0 microgram/ml-6.0 microgram/ml, and then strong/weak pathogenic strains are inoculated; the reaction of resistance or susceptibility and the real-time RT-PCR are inspected for analyzing rice defense related gene expression; overexpression strains of the genes are utilized for being inoculated on rice blades in an in-vitro mode, and the reaction of resistance or susceptibility and the real-time RT-PCR are inspected for analyzing the rice defense related gene expression and the qPCR is inspected for analyzing relative fungus growth amount on disease spots. The method can comprehensively and accurately clear pathogen effect protein genes and improve the pathogen infective ability, and the accurate and comprehensive method is provided for determining the pathogen effect protein for improving the pathogen infective ability in the future.

Description

Technical field: [0001] The invention relates to a method for detecting prokaryotic expression of rice blast fungus gene in vitro and overexpression in the strain to improve the invasiveness of the strain, belonging to the fields of plant protection and biotechnology. Background technique: [0002] When plants are infected by pathogenic bacteria, they trigger an immune response by recognizing type III effector proteins. Type III effector proteins are secreted into host cells through the type III secretion pathway (T3SE). T3SE has multiple functions and can transport effector proteins to host cells. It is involved in multiple metabolic pathways, such as induction of defense-related gene expression, activation of downstream defense signals, specific protein modification, and production of SA, JA, and Et signaling molecules. It is well known that the vast majority of type III effector proteins are non-toxic proteins, which are usually related to the HR response of the host plan...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6895
Inventor 杨静李成云鄢进陆杨亚琼
Owner YUNNAN AGRICULTURAL UNIVERSITY
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