3种不同食性淡水鱼类谷胱甘肽S-转移酶Pi、Mu、Theta型cDNA序列的克隆分析及表达

Cloning,sequence analysis and expression of Pi、Mu and Theta classes of glutathione S-transferases genes in three freshwater fishes

鱼类谷胱甘肽S-转移酶（glutathione S-transferase，GST）是鱼类一种重要的Ⅱ相去毒酶，在催化毒素与还原谷胱甘肽（GSH）加合去毒代谢过程中具有关键作用。采用RT-PCR及RACE法，分离、克隆得到草鱼、尼罗罗非鱼pi、mu、theta型GST（GSTpi、GSTmu、GSTtheta）基因、鲢鱼GSTmu、GSTtheta基因的cDNA部分序列并推测各自对应的氨基酸序列。氨基酸序列同源性比较和系统进化分析均表明，鲢鱼、草鱼、尼罗罗非鱼与鱼类GST同源性较高，与哺乳类、鸟类、两栖类GST同源性较低，可能与鱼类GST基因在水环境毒素去毒代谢中承担的特殊功能有关。而不同种鱼类GSTtheta的同源性明显要较GSTpi、GSTmu的同源性低，可能与不同淡水鱼类食性及对毒素耐受性不同有关。用实时荧光定量PCR（RT-PCR）检测三种鱼肝脏中三型GST基因组成型表达水平，发现三种鱼各型之间皆有一定差异，尼罗罗非鱼肝脏整体GSTs基因表达很低，GSTtheta显著低于草鱼（P〈0．05），GSTmu显著低于鲢鱼（P〈0．05）。本研究为从分子水平上研究不同型谷胱甘肽S-转移酶基因在不同食性淡水鱼类体内代谢去毒过程中的作用提供了基础。

Glutathione S-transferase （GST） of freshwater fish is a kind of Phase Ⅱ detoxification enzymes and is important to detoxification from fish body, by catalyzing the conjugation of GSH with toxin. In this study, the partial cDNAs encoding pi-class, mu-class, theta-class GST （GSTpi, GSTmu,GSTtheta） were cloned and sequenced from the grass carp （Ctenopharyngodon idella） and the Nile tilapia （Oreochrornis nilotica）, and the GSTmu, GSTtheta were cloned and sequenced from the sliver carp （Hypophthalmichthys molitrix） by RT-PCR and RACE method. Relative amino acids were deduced. The phylogenetic tree constructed on the basis of GST sequences showed that the GSTs amino acid sequences of sliver carp, grass carp and nile tilapia were highly conserved in fish, which had lower homology with that of amphibian, bird and human. This finding was consistent with the special function of these genes in freshwater fish in toxin detoxification. In the different kinds of fishes, the homology of GSTtheta is lower than GSTpi and GSTmu, it seems to be conserved with different tolerance for toxin in different fishes. Meanwhile, the constitutive expression of liver GSTs genes were determined by real-time RT-PCR. The mRNA expression level of Nile tilapia GSTs genes were the lowest, GSTtheta expression was significantly lower than that of grass carp （P〈0.05）, GSTmu expression was significantly lower than that of sliver carp （P〈0.05）. This study helps to understand the functions of different classes of glutathione S-transferases （GSTs） genes in the metabolism oftoxics in limnetic fishes.