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Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes

A technology of internal reference gene and Chilo suppressalis, which is applied in the direction of biochemical equipment and methods, microbe determination/inspection, etc., can solve the problem of non-existence of internal reference gene

Active Publication Date: 2016-01-27
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

Internal reference genes commonly used in insects include Rps3, Actin, GAPDH, Tublin, Rpl10, 18SrRNA, 28SrRNA, etc., but in fact, the mRNA expression level of any internal reference gene is constant under all external or internal conditions, ideal internal reference genes don't exist

Method used

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  • Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes
  • Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes
  • Method for screening reference genes for chilo suppressalis under temperature stress and application of reference genes

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Embodiment Construction

[0047] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. The use of the internal reference gene stability analysis software was carried out according to its instructions.

[0048] 1. Feeding of Chilo borer.

[0049] Chilo borer larvae collected from paddy fields were reared to the 2nd generation with artificial feed in an artificial climate chamber: temperature 25°C, humidity 75%, light: darkness = 14 hours: 10 hours, and then the 3rd instar larvae were used for breeding. temperature stress treatment.

[0050] 2. Temperature stress treatment

[0051] Set the light incubator (15°C, 20°C, 25°C, 30°C, 35°C, 40°C) or refrigerator (5°C, 10°C) to the treatment temperature, and place the 3rd instar larvae of Chilo borer in a glass containing artificial feed. The tube was sealed with a cotton plug, and after 3 hours of treatment, the samples were collected to extract total RNA; 3 biological replicates were set ...

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Abstract

The invention discloses a method for screening reference genes for chilo suppressalis under different temperature stress conditions and application of the reference genes. The method includes utilizing the chilo suppressalis cultivated under room-temperature conditions as a control material; carrying out fluorescence quantitative PCR (polymerase chain reaction) detection by the aid of the chilo suppressalis as an experimental material after the chilo suppressalis is under high-temperature and low-temperature stress; inputting Ct values of all alternative reference genes into delta Ct, RefFinder, BestKeeper, GeNorm, NormFinder reference gene stability analysis software and analyzing the Ct values of all the alternative reference genes; screening the most stable in-vivo reference genes TUB for the chilo suppressalis under the temperature stress conditions. The method and the application have the advantage that expression of genes related to the temperature stress conditions can be accurately studied by the aid of genes, namely the reference genes.

Description

technical field [0001] The invention belongs to the technical field of expression stability of real-time fluorescent quantitative PCR internal reference genes in molecular biology, and in particular relates to a screening method and application of internal reference genes of Chilo suppressalis under different temperature stresses. Background technique [0002] Chilosuppressalis belongs to the family Lepidoptera and is an important pest of rice in my country. It causes symptoms such as withered sheath, dead heart, white ear, withered booting ear and insect damage to rice, causing huge economic losses. With the renewal of rice cultivation system and the change of rice varieties, the population of Chilo suppressalis increases year by year. [0003] Under low temperature conditions in winter, Chilo borer survives the winter in rice stumps, straw, and other crop residues such as grasses in the field. It begins to emerge in early May, and its damage runs through the entire growing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/158C12Q2600/166
Inventor 鲁艳辉李小荣李阳钟列权徐启强徐红星郑许松吕仲贤
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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