黄斑篮子鱼去毒相关基因的克隆与肝脏组成型表达分析  

Molecular cloning of cDNA core sequences of detoxification-related genes and liver constitutive expression in rabbitfish Siganus oramin

作　　者：王琳[1] 梁旭方[1] 林群[1] 李光照[1] 胡永乐[1] 

机构地区：[1]暨南大学生命科学技术学院,广东广州510632

出　　处：《热带海洋学报》2009年第6期79-87,共9页Journal of Tropical Oceanography

基　　金：国家科技部"863"项目(2007AA09Z437);国家自然科学基金项目(30670367);广东省科技计划项目(2007B020701002;2005B20301005);广东省自然科学基金项目(031886);教育部留学回国人员科研启动基金项目

摘　　要：从基因水平探讨海洋鱼类对海洋藻毒素的去毒分子机理。采用RT-PCR法成功克隆了黄斑篮子鱼Siganus oramin肝脏I时相代谢酶细胞色素P450 1A(CYP1A)、II时相代谢酶alpha型谷胱甘肽S-转移酶(GSTA)和rho型谷胱甘肽S-转移酶(GSTR)、热休克蛋白70(HSP70)、alpha 1型钠钾ATP酶(ATP1A1)及β-肌动蛋白(beta-actin,ACT)基因cDNA核心序列,序列分别长879 bp、582 bp、588 bp、660 bp、749 bp和554 bp。序列同源性分析发现,属鲈形目的黄斑篮子鱼CYP1A、GSTA和GSTR与同属鲈形目的牙鲆Paralichthys olivaceus、欧洲鲽Pleuronectesplatessa、真鲷Pagrus major、鲤形目的斑马鱼Brachydanio rerio相应氨基酸序列同源性较高,CYP1A和GSTA与非洲爪蟾(两栖类)、鸡(鸟类)、小鼠、大鼠和人(哺乳类)相应氨基酸序列同源性低,这可能与鱼类Ⅰ、Ⅱ时相去毒酶基因承担水环境毒素去毒代谢的特殊功能有关;而HSP70、ATP1A和β-肌动蛋白在鱼类、两栖类、鸟类、哺乳类中均有较高的同源性,这可能与这些基因在机体中承担的最基本的生命功能相关。应用半定量RT-PCR的方法,以β-肌动蛋白作为外参照,在指数期增长范围内分别得到了CYP1A、GSTA、GSTR、HSP70和ATP1A1 mR-NA与β-肌动蛋白mRNA(%)的比值,确定黄斑篮子鱼肝脏去毒相关基因的组成型表达水平。其中,黄斑篮子鱼肝脏CYP1A、GSTA和GSTR基因组成型表达相对较高,HSP70和ATP1A1基因组成型表达相对较低,这可能与不同基因在黄斑篮子鱼海洋藻毒素去毒分子机理中承担的作用相关,为海洋藻毒素在海洋鱼类中的积聚及代谢去毒分子机制的研究提供了相关数据。Partial cDNA sequences of hepatic cytochrome P-450 1A （CYP1A）, glutathione S-transferase alpha （GSTA） , glutathione S-transferase rho （GSTR） , heat shock protein 70 （HSP70） , Na＋/K＋-ATPase alpha 1 （ATPIA1） and heta-actin （ACT） genes from rahbitfish （Siganus oramin） were obtained by PCR using degener- ate primers. These sequences were 879 hp, 582 bp, 588 bp, 660 bp, 749 bp and 554 bp in length, respec- tively. Homology of the CYP1A, GSTA and GSTR amino acid sequence are high with bastard halibut （Paralichthys olivoceus）, European plaice （Pleuronectes platessa）, red sea bream （Pagrus major） and zebrafish CYP1A, GSTA and GSTR, and CYP1A and GSTA are low with human, rat, mouse, chicken and African clawed frog. The identities of HSP70, ATP1A and beta-actin amino acid sequences are highly conserved between fish, bird, amphibian and mammals. Using semi-quantitative RT-PCR within the exponential phase, with betaactin as an external control, the constitutive transcriptional expression of genes of phase I （CYP1A）, phase II （GSTA and GSTR） xenobiotic metabolizing enzymes （XMEs）, HSP70 and ATP1A1 were also investigated. The levels of CYP1A, GSTA and GSTR mRNA expression were higher, and the levels of HSP70 and ATP1A1 mRNA ex- pression were lower. The constitutive expression pattern of these genes shows an accordant relationship with their func- tions, which provides data for our understanding of the detoxification mechanism of marine algae toxin in marine fish.

关 键 词：细胞色素P450 1A 谷胱甘肽S-转移酶 热休克蛋白70 钠钾ATP酶 基因克隆 表达分析 黄斑篮 子鱼Sigartus oTolnitt 

分 类 号：Q178.531[生物学—水生生物学]