【作者】 廖婉琴；

【导师】 梁旭方；

【作者基本信息】 暨南大学 ， 动物遗传工程， 2006， 硕士

【摘要】 随着水体富营养化加剧，藻类所引起的水污染问题已越来越引起人们的关注，藻毒素污染已成为一个全球性的环境卫生问题。研究表明，可溶性谷胱甘肽S-转移酶（soluble glutathione S-transferase，sGST）可催化微囊藻毒素与谷胱甘肽（glutathione，GSH）发生加合反应，起去毒作用，而解偶联蛋白2（uncoupling protein 2，UCP2）则可有效抑制微囊藻毒素诱发活性氧（ROS）导致的肝细胞凋亡。本研究成功克隆鲢鱼（Hypophthalmichthys molitrix）、鳙鱼（Aristichthys nobilis）、草鱼（Ctenopharyngodon idellus）、鲫鱼（Carassius auratu）、鲮鱼（Cirrhinus molitorella）、罗非鱼（Oreochromis nilotica）等淡水鱼类肝脏GST及UCP2基因序列，并对GST、UCP2基因在不同生态习性淡水鱼类肝脏中的表达水平进行比较研究，这将为深入探讨鱼类微囊藻毒素去毒相关基因的表达调控机制，阐述生态系统中微囊藻毒素去毒代谢分子机理提供新信息。 通过逆转录-聚合链式反应（RT-PCR）和快速扩增cDNA末端（RACE）技术分别从鲢鱼、鳙鱼肝脏扩增GST基因cDNA全序列。序列分析表明，鲢鱼、鳙鱼GST基因cDNA序列全长分别为920 bp、978 bp，均编码223个氨基酸。应用简并引物PCR获得草鱼、鲫鱼、鲮鱼、罗非鱼肝脏GST基因cDNA核心序列分别为405 bp、591 bp、405 bp、399 bp。氨基酸序列同源性比较，发现克隆得到的淡水鱼类肝脏GST均属于alpha类GST。应用计算机对鲢鱼GST进行三维结构预测，结果显示，鲢鱼GST三维结构与其它物种GST三维结构非常相似，均包含了N端结构域和C端结构域。应用Genome Walker方法首次在鲢鱼克隆得到淡水鱼类GST基因5’侧翼区序列875 bp，并发现在该调控序列上存在多个脂多糖反应元件（lipopolysaccharide response element，LPSRE），表明来源于毒藻的脂多糖（lipopolysaccharide，LPS）可能对鲢鱼GST表达有潜在调控作用。用半定量PCR方法比较对微囊藻毒素具有不同耐受能力的6种淡水鱼类肝脏GST组成型表达情况。以β-肌动蛋白为外对照，不同淡水鱼类肝脏GST与β-肌动蛋白mRNA（％）的比值分别为：130.7±6.6（草鱼）、103.1±8.9（鳙鱼）、92.6+₁5.0（鲫鱼）、72.3±7.8（鲮鱼）、58.8±11.5（鲢鱼）、33.6±13.7（罗非鱼），表明淡水鱼类肝脏GST组成型表达与这些鱼类对微囊藻毒素的耐受能力存在显著的负相关：耐受能力最强的淡水鱼类（如滤食性鱼类：鲢鱼、罗非鱼）肝脏GST组成型表达量最低，而对微囊藻毒素更多还原

【Abstract】 Cyanotoxins pose a world-wide health threat to animals and humans due to their increasing presence in both drinking and recreational waters. Microcystin detoxification-related liver genes, such as soluble glutathione S-transferase (sGST) and uncoupling protein 2 (UCP2) have been supposed to play an important role in microcystin detoxification in fish body. In this report, molecular cloning and structure characterization of liver GST and UCP2 gene, from major Chinese freshwater fishes with various food habit, including silver carp (Hypophthalmichthys molitrix) (phytoplanktivorous fish), bighead carp (Aristichthys nobilis) (zooplanktivorous fish), grass carp (Ctenopharyngodon idellus) (herbivorous fish), crucian carp (Carassius auratu) (benthos-eating fish), mud carp (Cirrhinus molitorella) (detritus-eating fish), and tilapia (Oreochromis nilotica) (phytoplanktivorous fish), were carried out. In addition, liver GST and UCP2 gene expression levels among these freshwater fishes were also studied, which would shed a new light on the detoxification mechanism of microcystins in freshwater fishes.Two full-length cDNAs encoding GST were cloned and sequenced from the hepatopancreas of planktivorous silver carp and bighead carp. The silver carp and bighead carp GST cDNA were 920 bp and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from grass carp, crucian carp, mud carp and tilapia liver. All these GST could be classified as alpha-class GST on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using computer, and found to fit the classical two-domain GST structure. Using genome walker method, a 875 bp 5’-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish, might be regulate by LPS, released from the toxic blue-green algae producing microcystins.更多还原