团头鲂谷胱甘肽S-转移酶基因的克隆及其在氨氮胁迫中的表达分析

谷胱甘肽S-转移酶(glutathione S-transferase,GST)是一类多功能蛋白家族,主要参与解毒和抗氧化防御过程。为了研究GST在团头鲂(Megalobrama amblycephala)肝脏解毒过程中的作用,克隆并分析了团头鲂1个谷胱甘肽S-转移酶基因(命名为MaGST)cDNA序列,采用实时荧光定量PCR研究了其在氨氮胁迫下的表达规律。MaGST包含1个长218个氨基酸的完整开放阅读框,具有GST蛋白家族的保守碱基和保守结构域。通过MEGA 5.0软件分析系统进化树发现,团头鲂GST与其他动物mu型GST聚为一簇,表明团头鲂GST属于mu型GST。荧光定量PCR结果显示MaGST基因在团头鲂各组织中均有表达,在肝脏和鳃中表达量最高,肌肉中表达量最低,同时在氨氮胁迫过程中该基因在肝和鳃中的表达规律相似,均在胁迫期间表达量显著上调;氨氮胁迫24 h时鳃和肝组织均存在组织损伤。研究结果提示在团头鲂肝脏和鳃组织中GST基因参与了氨氮胁迫的解毒过程。将该基因的编码区重组到p ET-21(a+)载体后在大肠杆菌中得到诱导表达,重组MaGST的GST活力为(10.36±0.68)U·mg~(-1)蛋白。

Molecular Cloning, Characterization and mRNA Expression of Mu-typ Glutathione S-Transferases from Megalobrama amblycephala

Glutathione S-transferases (GSTs) play an important role in cellular detoxification and may have evolved in protecting cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione S-transferase (MaGST) from Bluntnose black bream Megalobrama amblycephala. The full-length cDNA of MaGST was 1 011 bp in length containing an open reading frame of 654 bp that encoded a 218-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate mu-type GSTs in a phylogenetic tree (NJ). Quantitative real-time RT-PCR analysis showed that mRNA expression of MaGST was detected in all tissues, including the brain, liver, muscles, gills, spleen, and intestines, with the highest level of expression in the liver and gill, the lowest expression in the muscles. Exposure to waterbrone ammonia significantly increased the mRNA expression of MaGST (P < 0.05) in liver and gill, respectively. Fish exposed to waterbrone ammonia also showed liver and gill tissue damage in 24 h. To further characterize the catalytic properties of this enzyme along with MaGST, we constructed the recombinant MaGST plasmid with a 6×His-Tag at the N-terminal of MaGST cDNA. Recombinant MaGST was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The GST activity of the recombinant MaGST was (10.36 ± 0.68) U·mg-1 protein. Overall, the results suggest a potential role for MaGST in detoxification and possibly conferring immune protection.