三角帆蚌热休克蛋白60基因克隆及其表达分析

CLONING AND EXPRESSION ANALYSIS OF HEAT SHOCK PROTEIN 60 GENE FROM HYRIOPSIS CUMINGII

利用3′RACE技术,对高通量转录组测序所得三角帆蚌HSP60基因(hcHSP60)长片段(2629 bp)进行了3?末端克隆,经拼接得到hcHSP60 cDNA全长序列。采用多种分子生物学软件对hcHSP60 cDNA全长序列进行了特征分析,并采用RT-PCR技术检测了其组织分布及经冷热应激后的表达变化。结果显示,hcHSP60cDNA全长为2807 bp,其中开放阅读框为1707 bp,编码568个氨基酸,预测分子量大小为61.04 ku,pH 7.0时的理论等电点为5.63。序列中不存在信号肽与跨膜结构。氨基酸序列保守性分析表明,hcHSP60氨基酸序列与光滑双脐螺HSP60同源性最高(达82%),而与牙鲆、白云金丝鱼HSP60的同源性最低(为75%)。RT-PCR检测结果表明,hcHSP60在肝胰腺中的表达水平最高,而在血液中的表达水平最低。30℃与35℃水温处理三角帆蚌4h后,各组织中hcHSP60表达水平明显上升,表明hcHSP60可能在三角帆蚌耐热应激反应中起着重要作用。

Heat shock protein 60(HSP60) is an important molecular chaperone protein that mainly exists in the mitochondria of organism. In the present study, the cDNA sequence of Hyriopsis cumingii HSP60(hcHSP60) was cloned by 3′ rapid amplification of cDNA ends methods(3′-RACE) based on a long chain sequence of hcHSP60(2629 bp) and was determined by high flux sequencing for transcriptome of H. cumingii blood cells, and its expression in the different tissues was detected by using reverse transcription-polymerase chain reaction(RT-PCR). Results showed that the full-length cDNA of hcHSP60 was 2807 bp that contains an open reading frame of 1707 bp, encoding a protein of 568 amino acid residues with 61.04 ku of predicted molecular weight and 5.63 of the theoretical isoelectric point, which was predicted to have no signal peptide and transmembrane helices. The deduced amino acid sequence of hcHSP60 shares the highest identity(82%) with HSP60 of Biomphalaria glabrata, and the phylogenetic analysis demonstrated that they were clustered in a same clade. The results of RT-PCR indicated that hcHSP60 was constitutively expressed in all 5 examined tissues of H. cumingii with the highest expression in hepatopancreas. The expression of hcHSP60 in all detected tissues was up-regulated obviously by higher water temperature, suggesting that it may play an important role in the stress response against heat.