Molecular Cloning and Tissue-specific Expression of Cu/Zn and Mn-superoxide Dismutase in the Three-keeled Pond Turtle, Chinemys reevesii

Abstract

Both copper/zinc superoxide dismutase(SOD; Cu/Zn-SOD, SOD1) cDNA and manganese SOD(Mn-SOD, SOD2) cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5’-untranslated region(UTR), a 249-bp 3’-UTR, and a 468-bp open reading frame(ORF) encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point(pI). The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5’-UTR, 912-bp 3’-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity(77.4%–87.1%) and identity(65.4%–74.4%) with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity(83.6%–95.6%) and identity(76.1%–88.9%) with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.