鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特 的关键作用，因而也称为微囊藻毒素去毒酶. 从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因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鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列. 序列分析结果表明，鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920 bp，其中5′-UTR长74 bp，3′-UTR长174 bp，编码区长672 bp，编码223个氨基酸. 应用基因组步行法，在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878 bp序列. 与哺乳动物及海水鱼sGST基因不同，鲢鱼微囊藻毒素去毒酶基因的5′侧翼区，发现存在多个脂多糖反应元件(LPSRE)，表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.

Cloning and Analysis of the Full-length cDNA Sequence and 5'-Flanking Region of Silver Carp Soluble Glutathione S-transferase Gene

Soluble glutathione S-transferase (sGST) of freshwater fish is extremely important to microcystin purification from fish body, and is therefore also named microcystin detoxifizyme. PCR using two degenerated primers, yielded a cDNA fragment of 591 bp from the liver of a phytoplanktivorous freshwater fish, silver carp (Hypophthalmichthys molitrix), which consumed substantial amounts of toxic blue green algae in the food. The full length cDNA fragment was obtained by 5′ and 3′ RACE. The complete silver carp sGST cDNA was 920 bp length, containing an open reading frame of 672 bp (encoding 223 amino acids), flanked by 74 bp 5′UTR and 174 bp 3′UTR. The deduced amino acid sequence from this sGST cDNA, contains two conserved domains, N-terminal domain (glutathione bindind site) and C terminal domain (substrate binding site). Comparison of the N-terminal and C terminal domains of silver carp sGST with rock bream (Oplegnathus fasciatus), red sea bream (Pagrus major), chicken, rat, mouse and human sGST, showed that the N terminal domain is highly conserved (66.3%—96.3%) in the sGSTs of fish, birds and mammals, while the C terminal domain has less similarity (38.3%—55.5%) among different sGSTs, which is consistent with the different function of the two domains. A 878 bp 5′-flanking region of silver carp sGST gene was obtained using genome walker method. The key promoter element TATA box, as well as CAAT box and GC box has been found in the flanking region. Completely different from the sGST genes of marine fish and land vertebrates, several lipopolysaccharide (LPS) responsive elements have been identified in the promoter region of silver carp sGST gene, indicating that the expression of silver carp microcystin detoxifizyme gene, might be regulated by LPS from the toxic blue green algae producing microcystins.