关键词: 绿豆象, 全长转录组测序, 热激蛋白, 基因家族, 生物信息学分析, 表达分析

Abstract:

【Objective】The purpose of this study is to identify the gene members of the Callosobruchus chinensis heat shock protein (HSP) superfamily, and to clarify the expression changes of HSP genes in C. chinensis after high and low temperature stress, so as to provide a theoretical basis for further exploration of HSP gene function.【Method】The CDS and protein sequences of HSP genes of different insects were downloaded from Insect Base 2.0 and used as a reference for local BLASTp and tBLASTn comparison search in the full-length transcriptome sequencing database of C. chinensis. At the same time, target sequences were screened again by combining HMMER and key words to complete the summary of search results. Bioinformatics analysis of HSP superfamily genes in C. chinensis was performed using CDD, MEGA, ProtParam, and other online analytical tools. Seven candidate HSP genes were screened out based on high and low temperature transcriptome sequencing data of C. chinensis adults and the expression characteristics of 7 CcHsps were compared and analyzed by qRT-PCR technique under different developmental stages and temperature stresses of C. chinensis.【Result】A total of 31 HSP genes were identified, including 3 HSP90s, 8 HSP70s, 8 HSP60s, and 12 sHSPs (small HSP). Physicochemical analysis showed that the proteins encoded by CcHsps contain 159-776 amino acid residues (aa), the molecular weights are about 18.4-88.9 kDa, and the theoretical isoelectric points are 4.95-9.17. Subcellular localization results showed that most CcHsps were located in the cytoplasm, while a few genes were located in the mitochondrial matrix, endoplasmic reticulum and nucleus. Phylogenetic analysis showed that different family members of HSPs in C. chinensis could integrate well with HSP in other insects, which indicating their evolutionary conservation. The results of qRT-PCR showed that the 7 candidate CcHsps were differentially expressed under different temperature stresses. After high temperature stress, the expression level of CcHsp20.102 in male and female adults was up-regulated by 1 000 and 500 times, respectively, and the expression level of CcHsp70-5 in male and female adults was up-regulated by 500 and 450 times. After the larvae undergoing high and low temperature stress, the expression level of CcHsp19.855 and CcHsp70-5 was significantly different.【Conclusion】A total of 31 complete HSP superfamily gene members were identified by the full-length transcriptome sequencing data of C. chinensis, which were divided into 4 subfamilies. Different HSP families had different gene structures, protein conserved domains and gene expression characteristics. The differential expression of 7 candidate CcHsps in different developmental stages and under different temperature stresses indicated that they played different functions and roles. It is speculated that CcHsp20.102 and CcHsp70-5 may perform important functions in the adult resistance to high temperature stress, and the high temperature tolerance of larvae may be related to the differential expression of CcHsp19.855 and CcHsp70-5.

Key words: Callosobruchus chinensis, full-length transcriptome sequencing, heat shock protein, gene family, bioinformatics analysis, expression analysis