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Method for detecting expression of rice blast relevant gene in infected rice leaf tissue

A disease-related gene and rice blast fungus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc.

Inactive Publication Date: 2009-04-29
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, there is no report on the use of real-time fluorescent quantitative PCR to detect the gene expression of rice blast fungus in the initial stage of rice infection

Method used

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  • Method for detecting expression of rice blast relevant gene in infected rice leaf tissue
  • Method for detecting expression of rice blast relevant gene in infected rice leaf tissue
  • Method for detecting expression of rice blast relevant gene in infected rice leaf tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] (1) culturing Magnaporthe grisea in liquid and extracting the total RNA of its mycelia, and reverse-transcribing the mRNA therein into cDNA;

[0021] (2) Inoculate the spores of Magnaporthe grisea on the leaves of rice susceptible variety Lijiang Xintuan Heigu, and extract the total RNA of the infected tissue at different time periods early after inoculation and reverse transcribe the mRNA therein into cDNA;

[0022] (3) Synthesis of cDNA: Take 2.5 μg of each total RNA sample, add 0.5 μg / μl Oligo (dT) 2.5 μl, 10 mM dNTP 2.5 μl, RNase-free water to 30 μl, 65 ° C water bath for 5 minutes, and quickly place in Cool on ice, add 5×First-Strand Buffer 10μl, 0.1M DTT (dithiothreitol) 5μl, 40U / μl RNase inhibitor 2.5μl, 42℃ water bath for 2μmin, then add SuperScript II reverse transcriptase 2.5μl , 42°C water bath for 50 minutes, 70°C water bath for 15 minutes to stop the reaction;

[0023] (4) Select the actin (actin) gene of Magnaporthe grisea MG03982.5 as an internal referen...

Embodiment 2

[0037] (1) culturing Magnaporthe grisea in liquid and extracting the total RNA of its mycelia, and reverse-transcribing the mRNA therein into cDNA;

[0038] (2) Inoculate the spores of Magnaporthe grisea on the leaves of rice susceptible variety Lijiang Xintuan Heigu, and extract the total RNA of the infected tissue at different time periods early after inoculation and reverse transcribe the mRNA therein into cDNA;

[0039](3) Synthesis of cDNA: Take 2.5 μg of each total RNA sample, add 0.5 μg / μl Oligo (dT) 2.5 μl, 10 mM dNTP 2.5 μl, RNase-free water to 30 μl, 65 ° C water bath for 5 minutes, and quickly place in Cool on ice, add 5×First-Strand Buffer 10μl, 0.1M DTT (dithiothreitol) 5μl, 40U / μl RNase inhibitor 2.5μl, 42℃ water bath for 2μmin, then add SuperScript II reverse transcriptase 2.5μl , 42°C water bath for 50 minutes, 70°C water bath for 15 minutes to stop the reaction;

[0040] (4) Select the actin gene of Magnaporthe grisea MG03982.5 as an internal reference gene, ...

Embodiment 3

[0055] (1) culturing Magnaporthe grisea in liquid and extracting the total RNA of its mycelia, and reverse-transcribing the mRNA therein into cDNA;

[0056] (2) Inoculate the spores of Magnaporthe grisea on the leaves of rice susceptible variety Lijiang Xintuan Heigu, and extract the total RNA of the infected tissue at different time periods early after inoculation and reverse transcribe the mRNA therein into cDNA;

[0057] (3) Synthesis of cDNA: take 2.5 μg of each total RNA sample, add 0.5 μg / μl Oligo (dT) 2.5 μl, 10 mM dNTP 2.5 μl, RNase-free water to 30 μl, 65 ° C water bath for 5 minutes, quickly placed in Cool on ice, add 5×First-Strand Buffer 10μl, 0.1M DTT (dithiothreitol) 5μl, 40U / μl RNase inhibitor 2.5μl, 42℃ water bath for 2μmin, then add SuperScript II reverse transcriptase 2.5μl , 42°C water bath for 50 minutes, 70°C water bath for 15 minutes to stop the reaction;

[0058] (4) Select the actin gene of Magnaporthe grisea MG03982.5 as an internal reference gene, an...

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Abstract

The invention relates to a method for detecting the expression quantity of pathogenic genes of rice blast germs in infected rice leaf tissue. The invention provides the method for detecting the expression quantity of the pathogenic genes in different time points after rice is infected by the rice blast germs. The method adopts a technical proposal that the rice blast germs are inoculated on a rice leaf by spraying, sampling is performed and total RNA is extracted; real-time fluorescent quantitative PCR detection is performed on pathogenic genetic fragments of rice blast germs by an SYBR Green I embedded fluorescence method; the C(t) value of a sample to be detected is recorded; and the expression quantity of the related pathogenic genetic fragments is calculated according to the formula. The method can quickly and accurately detect the expression quantity of the pathogenic genes of the rice blast germs on different periods of time in the initial stage of inoculation of the rice blast germs, analyzes the expression quantity of different pathogenic genes in different rice cultivars and the variation of the different pathogenic genes on different periods of time after inoculation, and analyzes the spatiotemporal expression mode of the related pathogenic genes of the rice blast germs and so on. The method has accurate detection result and good repeatability, and is favorable for explaining the functions of the genes.

Description

Technical field: [0001] The invention relates to a method for detecting the expression level of rice blast fungus pathogenicity-related genes in infected rice leaf tissue, belonging to the field of plant protection. Background technique: [0002] People often use RT-PCR, Northern in situ hybridization technology and gene microarray (gene chip) technology when studying gene expression. RT-PCR can only detect gene expression qualitatively but not quantitatively. Y. Takano used the Northern in situ hybridization technique to study the gene expression of rice blast fungus infecting rice. However, this method is suitable for expression studies of a small number of genes and requires at least 5-10 μg of total RNA. The multiples of gene up-regulation or down-regulation can only be roughly estimated from the grayscale of the RNA image. The sensitivity and accuracy are not high, and the genes with small expression levels of pathogenic bacteria in the host cannot be quantitatively d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 刘林李成云朱有勇李华杨静苏源李进斌王云月张悦孔垂思于钦亮梁飞
Owner YUNNAN AGRICULTURAL UNIVERSITY
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