二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

Cloning of the heat shock protein 70 gene from Chilo suppressalis and the analysis of its expression characteristics under heat stress

热休克蛋白70是已知热休克蛋白家族中最重要的一种,它在细胞内的大量表达可以明显改善细胞的生存能力,提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressa lis幼虫热休克蛋白70表达的影响,采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2102bp,开放阅读框(open reading frame,ORF)为1959bp,编码652个氨基酸;5′非编码区(untranslated region,UTR)为81bp,3′UTR为62bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性(73%～97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达,幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致,说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。

Heat shock protein 70 （HSP70） is one of the most conservative and important proteins in heat shock protein family. Overexpression of HSP70 may obviously improve the survivability of cells,and enhance their tolerance to stress. To investigate the effects of heat stress on HSP70 expression in Chilo suppressalis larvae,RT-PCR and RACE methods were used for cloning the full length cDNA encoding HSP70 from haemocytes of C.suppressalis in this study. The cDNA obtained is 2 102 bp in length,containing an ORF of 1 959 bp,encoding 652 amino acid residues,with a 5＇UTR of 81 bp and a 3＇UTR of 62 bp.Cluster analysis confirmed that the deduced amino acid sequence shared high identity with the reported homologous sequences from other insects （73%-97%）. Real-time quantitative PCR showed that HSP70 gene expression could be induced by heat stress. The maximal induction of HSP70 gene expression in larval haemocytes of C.suppressalis was achieved at 36℃. HSP70 protein expression was observed by using flow cytometry. The results revealed that the expression profiles of HSP70 at both the mRNA level and the protein level are highly in agreement with each other.