条斑紫菜PyMGST3基因克隆、表达及功能分析

EXPRESSION AND FUNCTIONAL ANALYSIS OF MICROSOMAL GLUTATHIONE S-TRANSFERASE GENE IN PYROPIA YEZOENSIS UNDER HEAVY METAL STRESSES

微粒体谷光甘肽硫转移酶(microsomal glutathione S-transferases,MGST)作为膜结合蛋白之一,具有谷光甘肽转移酶和过氧化物酶活性,在细胞及细胞器的抗氧化胁迫中扮演着重要角色,但MGST基因在紫菜(Pyropia yezoensis)中的鉴定与分析还未见报道。本文采用RACE-PCR方法首次克隆了条斑紫菜的微粒体GST基因的全长,命名为PyMGST3,同时利用生物信息学及实时定量PCR方法对该基因的序列及诱导表达特征进行了分析,并通过原核表达进一步验证了该基因的酶活性及在抗氧化胁迫中的作用。结果表明,PyMGST3基因的c DNA序列全长为681bp,其中开放阅读框长度417bp,5'-UTR长度80bp,3'-UTR长度184bp,在受到Cd^(2+)和Cu^(2+)胁迫时,上调表达;PyMGST3蛋白具有三个跨膜结构域及跨膜疏水区,与皱波角叉菜的MGST蛋白相似性最高为60%,与其它藻类的MGST蛋白的相似性较低;在大肠杆菌中表达及纯化后的PyMGST3蛋白具有谷光甘肽转移酶活性;此外,超表达PyMGST3蛋白的重组菌株提高了对抗Cd^(2+)和Cu^(2+)毒害的能力。这些结果暗示PyMGST3基因很可能在条斑紫菜遭遇重金属等引起的氧化胁迫时起到重要的保护作用。

Microsomal glutathione s-transferase is one of membrane-bound enzymes. It has activities of glutathione transferase and peroxidase for playing important protection roles in cell and organelle under oxidative stress. However, this gene has never been studied in Pyropia yezoensis. In this study, a novel microsomal glutathione S-transferase 3（designated as PyMGST3） gene was cloned from P. yezoensis. The full length of PyMGST3 gene was 681 bp with a poly A tail. The gene had a 417 bp ORF, 80 bp 5′-UTR, and 184 bp 3′-UTR. The PyMGST3 protein contained three transmembrane and hydrophobic domains and the sequence of PyMGST3 shared higher similarities with the MGST proteins of other species. The results of the real-time PCR show that the PyMGST3 gene was up-regulated under Cd^（2＋） and Cu^（2＋） stresses. The purified PyMGST3 protein showed the activity of glutathione transferase after expressed in E. coli cells. The analysis results of transformed E. coli cells with PyMGST protein indicate that the abilities to resist Cd^（2＋）and Cu^（2＋） stresses were improved. Therefore, the PyMGST3 gene may have defense mechanisms associated with oxidative stress in P. yezoensis.