绿豆象热激蛋白超家族基因的鉴定及表达分析

Identification and Expression Analysis of Heat Shock Protein Superfamily Genes in Callosobruchus chinensis

【目的】鉴定绿豆象(Callosobruchus chinensis)热激蛋白(heat shock protein,HSP)超家族基因成员,明确高、低温胁迫后HSP基因在绿豆象中的表达变化,为深入挖掘HSP基因功能提供理论依据。【方法】从Insect Base 2.0下载不同昆虫HSP基因的CDS和蛋白序列,并以此为参考在绿豆象全长转录组测序数据库中进行本地BLASTp和tBLASTn比对搜索,同时结合HMMER和关键词两种方法再次筛选目标序列,最终完成搜索结果的汇总。利用CDD、MEGA、ProtParam等在线分析工具对绿豆象HSP超家族基因进行生物信息学分析。根据绿豆象成虫高、低温转录组测序数据筛选出7个候选CcHsp,采用实时荧光定量PCR(qRT-PCR)技术比较分析其在绿豆象不同虫态(幼虫、蛹、成虫)及不同温度胁迫下的表达特性。【结果】共鉴定出31个HSP基因,其中包括3个HSP90、8个HSP70、8个HSP60和12个sHSP(small HSP)。理化性质分析显示CcHsp编码的蛋白质包含159—776个氨基酸残基(aa),分子量介于18.4—88.9 kDa,理论等电点为4.95—9.17。亚细胞定位结果显示多数CcHsp定位于细胞质中,少数基因定位于线粒体基质、内质网和细胞核。系统发育分析表明绿豆象热激蛋白不同家族成员与其他昆虫的热激蛋白进化关系较近,显示了其在进化上的保守性。qRT-PCR分析发现,不同虫态在不同温度胁迫后7个候选CcHsp差异表达。CcHsp20.102经高温胁迫后在雌、雄成虫体内的表达量分别上调1000和500倍;CcHsp70-5经高温胁迫后在雌、雄成虫体内的表达量分别上调500和450倍;幼虫经高、低温胁迫后,CcHsp19.855和CcHsp70-5表达差异显著。【结论】通过绿豆象全长转录组测序数据共鉴定出31个完整的热激蛋白超家族基因成员,分为4个亚家族,家族间蛋白结构、保守结构域和基因表达特征存在差异。CcHsp20.102和CcHsp70-5可能在成虫抵御高温胁迫中行使重要功能,而幼虫的高温耐受性可能与CcHsp19.855和CcHsp70-5的表达差异有关。

【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 1000 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.