System screening and clone identification method for heat shock protein genes of agasicles hygrophila

Abstract

The invention relates to a system screening and clone identification method for heat shock protein genes of agasicles hygrophila and belongs to the technical field of gene engineering. The method comprises the steps that larvas of one to three ages, pupas of 2-6 days of age and imagoes which are subjected to eclosion in one day are taken, and the treatment samples above are collected and frozen for storage in liquid nitrogen; overall-length cDNA segment of mRNA is synthesized, an overall-length cDNA segment is screened, and cDNA gene banks different in capacity are established; the screened overall-length cDNA is subjected to secondary PCR amplification; the tail end of the overall-length cDNA is repaired; then secondary screening is conducted, and a testing gene bank is obtained; after the gene bank is established, the capacity of the gene banks is detected, after the gene bank quality detection result reaches the demand, on-computer sequencing is conducted, obtained non-redundant transcript sequences are compared with NR, Swissprot, GO, COG, KOG, Pfam and KEGG databases, annotation information of a transcript is obtained, all the heat shock protein genes are screened, EST sequence groups of each piece of heat shock protein are spliced and assembled, the most complete gene sequence is obtained, and finally according to the complete sequence, a primer is designed, and clone sequencing is conducted.

Description

technical field [0001] The invention relates to a method for systematic screening and cloning identification of the heat shock protein gene of A. chinensis, belonging to the technical field of genetic engineering. Background technique [0002] Insects often face cold or hot temperature stress in nature. After long-term evolutionary adaptation, insects have also developed a variety of physiological and biochemical coping strategies. Many genes related to temperature tolerance have been identified in organisms, among which the heat shock protein gene may be one of the genes most closely related to temperature stress, and it participates in many physiological and biochemical processes as a molecular chaperone. Heat shock proteins (HSPs) are a class of genetically highly conserved proteins that are newly synthesized or increased in content when cells or organisms are subjected to heat shock or other stressors. , development, differentiation, gene transcription and other functio...

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Problems solved by technology

[0005] Moreover, the identification of heat shock protein genes is mostly carried out by conservative sequence design of primers for PCR amplification, cloning and sequencing, or cloning and identification based on next-generation transcriptome sequencing information, but the identification of all heat shock protein genes is usually not completed by the above two methods and access to the complete gene sequence

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