南极独角雪冰鱼HSP90β基因cDNA序列克隆及其原核表达

Cloning and prokaryotic expression of HSP90β cDNA sequence of an Antarctic iecfish Chionodraco hamatus

摘要:

为研究南极鱼类HSP90β蛋白与低温适应的相关性,本研究克隆获得了南极独角雪冰鱼(Chionodraco hamatus,CH)热休克蛋白HSP90β基因的完整开放阅读框,并以尼罗罗非鱼(Oreochromis niloticus,ON)HSP90β蛋白为对照,进行低温胁迫下的原核表达研究。结果表明:克隆获得的独角雪冰鱼HSP90β基因编码区全长为2 184 bp,推定编码728个氨基酸。多重序列比对显示,独角雪冰鱼HSP90β与其他鱼类HSP90β的氨基酸序列同源性在87%以上;蛋白结构域分析发现,独角雪冰鱼HSP90β具有HSP90家族的保守信号区,其重要功能位点的氨基酸残基非常保守。构建pET28-CH-HSP90β和pET28-ON-HSP90β重组表达载体,观察4 ℃低温胁迫8 h和12 h后,HSP90β蛋白对大肠杆菌生存能力的影响。结果显示:C. hamatus-HSP90β组的大肠杆菌存活率显著高于O.niloticus-HSP90β组和空载体pET28a对照组,这表明南极冰鱼HSP90β在低温条件下具有更加显著的细胞保护功能。由此推测HSP90β蛋白参与了南极冰鱼低温适应的生理过程。

Abstract:

In order to testify the correlations between Antarctic fish HSP90β protein and coldness adaptation, we cloned the complete open reading frame of the HSP90β gene from an Antarctic icefish, Chionodraco hamatus and investigated the prokaryotic expression of C. hamatus-HSP90β protein under cold stresses. As a control, the ORF of Oreochromis niloticus HSP90β was also cloned. Results showed that the coding region of C. hamatus HSP90β gene is 2184 bp in length, encoding 728 amino acids. Multiple sequence alignment results showed that C. hamatus HSP90β shared more than 87% identities with other fishes. Protein domain analysis showed that C. hamatus HSP90β possessed the conserved domain of the HSP90 family, and showed highly conserved in the important functional site. Recombinant pET28-CH-HSP90 and pET28-ON-HSP90 prokaryotic expression plasmids were constructed and expressed in Escherichia coli to testify the function of C. hamatus HSP90β under 4℃ stress. Results showed that the survival rate of C. hamatus-HSP90β group was significantly higher than that of the O.niloticus-HSP90β and the control group. Our results indicated that C. hamatus HSP90β protein possessed more remarkable protective effects against cold stress and HSP90β protein might be involved in cold adaptation physiological process in C. hamatus.