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引用本文:丁雪芬,梁旭方,汪祖昊,雷腊梅,韩博平.鳙鱼微囊藻毒素去毒酶基因cDNA全序列的克隆与序列分析.湖泊科学,2007,19(3):326-332. DOI:10.18307/2007.0315
DING Xuefen,LIANG Xufang,WANG Zuhao,LEI Lamei,HAN Boping.Cloning and analysis of the full-length cDNA sequence of bighead carp (Aristichthys nobilis) microcystin-detoxifizyme gene. J. Lake Sci.2007,19(3):326-332. DOI:10.18307/2007.0315
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鳙鱼微囊藻毒素去毒酶基因cDNA全序列的克隆与序列分析
丁雪芬, 梁旭方, 汪祖昊, 雷腊梅, 韩博平
暨南大学生命科学技术学院, 广州 510632
摘要:
淡水鱼类可溶性谷胱甘肽S-转移酶(soluble glutathione S-transferase, sGST)在微囊藻毒素去毒代谢过程中起着关键作用, 又被称为微囊藻毒素去毒酶。通过简并引物克隆鳙鱼肝脏sGST基因cDNA核心序列, 应用5’-RACE和3’-RACE技术分别扩增该序列的5’末端和3’末端序列, 最后通过序列拼接获得鳙鱼肝脏sGST基因cDNA全序列。序列分析结果表明, 鳙鱼肝脏sGST基因cDNA全长934bp, 其中5’-UTR长104bp, 3’-UTR长158bp, 编码区672bp, 编码223个氨基酸。鳙鱼与海水鱼类、鸟类、哺乳类等不同类群sGST的N-末端功能域氨基酸同源性较高, 达75%左右, 而C-末端功能域氨基酸同源性较低, 仅为43.6%-55.9%, 这与脊椎动物sGST的N-末端功能域均存在相同的GSH结合位点, 而C-末端功能域存在不同的底物结合位点相一致。鳙鱼sGST的C-末端功能域的低同源性可能与其承担微囊藻毒素去毒代谢之特殊功能有关。
关键词:  微囊藻毒素去毒酶基因  cDNA序列  分子克隆  鳙鱼
DOI:10.18307/2007.0315
分类号:
基金项目:国家自然科学基金(30670367);广东省水文局蓝藻重点项目;广东省科技计划项目(2005820301005);广东省自然科学基金(031886);广州市科技计划项目(06A1207088);教育部留学回国人员科研启动资金联合资助
Cloning and analysis of the full-length cDNA sequence of bighead carp (Aristichthys nobilis) microcystin-detoxifizyme gene
DING Xuefen, LIANG Xufang, WANG Zuhao, LEI Lamei, HAN Boping
Technology, Jinan University, Guangzhou 510632, P. R. China
Abstract:
Soluble Glutathione S-transferase (sGST) of freshwater fish is extremely important to microcystin purification from fish body, and therefore is also named microcystin-detoxifizyme.PCR using two degenerated primers, yielded a cDNA fragment of 405 bp from the liver of bighead carp(Aristichthys nobilis).This cDNA fragment was completed by 5- and 3-RACE.The complete sGST cDNA sequence of bighead carp was 934 bp in length, containing an open reading frame of 672 bp (encoding 223 amino acids), flanked by 104 bp 51UTR and 158 bp 3-UTR.The deduced amino acid sequence from this sGST cDNA fragment contains two conserved domains, N-terminal domain (glutathione-binding site) and C-terminal domain (substrate-binding site).Comparison of the N-terminal domain and C-terminal domain of bighead carp sGST with rock bream (Oplegnathus fasciatus), red sea bream (Pagrus major), chicken, mouse, rat and human sGST showed that, the N-terminal domain is highly conserved (75%) in the sGST of fishes, Aves and mammals, while the C-terminal domain has less similarity (43.6% -55.9%) among different sGST, which is consistent with the different function of the two domains.From the homologous analysis result, we might conclude that, there is a big difference in the C-terminal substrate-binding site between the microcystin-detoxifizyme gene of freshwater fish and the glutathione S-transferase gene of mammals, which seems to be consistent to the special function of this gene in freshwater fish for microcystin detoxification.
Key words:  Microcystin-detoxifizyme gene  cDNA sequence  molecular cloning  bighead carp
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