斑鳢(Channa maculata)微囊藻毒素去毒相关基因sGST的克隆与序列分析

MOLECULAR CLONING AND CHARACTERIZATION OF MICROSYSTIN DETOXIFICATION-RELATED sGST IN SNAKEHEAD FISH CHANNA MACULATA

采用RT-PCR技术和RACE技术成功克隆淡水鱼类斑鳢sGST基因cDNA全序列,推测得到氨基酸序列,初步分析其结构功能域及系统进化关系。结果表明,斑鳢sGST基因cDNA序列全长为898bp,编码225个氨基酸。斑鳢sGST与真鲷、金头鲷、鲽、黑头鲦、川鲽、大口黑鲈等最新定名为ρ型的sGST氨基酸同源性较高,ρ型sGST为水生生物所特有并共同占据进化树上独立的分枝;与大鼠、小鼠、人等哺乳动物sGST现有所有类型同源性均很低,并且在进化树上距离也较远,表明本研究成功克隆的sGST基因应属于ρ型,可能在鱼类等水生生物对水栖环境的适应上有重要作用。

Microcystins are being increasingly recognized as a potantial hazard to aquatic ecosystems and human health, as it can sustain in freshwater aquaculture products. Microsystin detoxification-related genes such as soluble glutathione S-transferase （sGST） play an important role in microcystin detoxification in various fishes. The Channa maculata, or commonly called Taiwan snakehead is an upstream animal member of freshwater food chain, and it can survive and reproduce in highly polluted waters and show a very strong resistance to microcystins and many other toxins. Therefore, they were chosen for study on the mechanism of endurance in the fish, and further on human protection against the toxin. The fish samples were collected in a local street market. After deprivation of the life and separation of liver, the sGST was analyzed. A 210bp cDNA fragment of sGST was successfully amplified by RT-PCR from the liver. The full sGST cDNA sequence of the fish was further obtained by 3＇-and 5＇-rapid amplification of cDNA ends （RACE）. Nucleotide sequence analysis revealed that the cloned sGST cDNA was 898bp in length, having 225 amino acid residues encoded. A search in the BLAST protein database revealed that, the deduced amino acid sequence of the sGST is very similar to the sGST of other fishes, such as largemouth bass （Micropterus salmoides） （74% identity） and red sea bream （Pagrus major） （74% identity）, which were recently classified as the Rho-class sGST, a new class of sGST, now found only in fish and other aquatic organisms. Results of the BLAST search and phylogenetic tree building revealed a big difference in sGST between aquatic organisms and land mammals, implying that the new class sGST in fish and other aquatic organisms may have special function for the detoxification of aquatic toxins.