Molecular Cloning And Characterization Of Microsystin Detoxification-related SGST And GPx In Taiwan Snakehead (Channa Maculate)

Microcystins are being increasingly recognised as a potent stress factor in aquatic ecosystems and a health hazard to our drinking water and freshwater aquaculture products. Microsystin detoxification-related genes such as soluble glutathione S-transferase (sGST) and glutathione peroxides (GPx) play an important role in microcystin detoxification in fishes. Taiwan snakehead (Channa maculatelies) stands on the upper level of freshwater ecosystem, and can accumulate high densities of microcystins through food chain. Taiwan snakehead can survive and reproduce in highly polluted waters and show a very strong resistance to microcystins and many other toxins. According to the deduced amino acid sequences of sGST and GPx from other vertebrates, two degenerate primers were designed and synthesized for cloning the cDNA of each gene. A 210bp cDNA fragment of sGST and a 263bp cDNA fragment of GPx were successfully amplified by RT-PCR from the liver of Taiwan snakehead. The full sGST cDNA sequence of Taiwan snakehead was further obtained by using the method of 3'- and 5'-rapid amplification of cDNA ends (RACE). Nucleotide sequence analysis revealed that the cloned sGST cDNA was 898bp in length , and the nucleotide sequence was composed of an open reading frame (ORF) of 678bp encoding a protein of 225 amino acid residues. A search of the BLAST protein database revealed that, the deduced amino acid sequence of Taiwan snakehead sGST was most similar to the sGST of other fishes, such as largemouth bass (Micropterus salmoides) (74% identity) and red seabream (Pagrus major) (74% identity), which were recently classified as a new class of sGST, the Rho-class sGST, now found to exsist only in fish and other aquatic organisms. Taiwan snakehead sGST also showed high sequence identity to several other fishes, such as gilthead seabream (Sparus aurata), plaice (Pleuronectes platessa), green spotted puffer (Tetraodon nigroviridis) and European flounder (Platichthys flesus) (73%, 72%, 69% and 67% respectively), and even shared 45% identity to amphioxus (Branchiostoma belcheri tsingtaunese) and 41% identity to green algae (Coccomyxa sp.). A neighbor-joining tree revealed that, all of them clustered together with high bootstrap values, and...