Molecular Identification and Expression Analysis of Carbonic Anhydrase VII in Pufferfish (Takifugu rubripes)
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A carbonic anhydrase VII gene, encoding 277 amino acids, was identified in the intestinal tissue of pufferfish (Takifugu rubripes). The translated protein with an 833-bp complete coding sequence derived from the 1378-bp cloned sequence showed 83% identity with swamp eel CA VII, 76% with zebrafish CA VII, and 77% with coho salmon CA VII-like protein. The cloned protein also showed 68–69% identity with mammalian CA VII. The predicted molecular weight and iso-electric point of the protein were 30.84 kDa and 6.07, respectively. Active site analysis of the pufferfish CA VII indicated that most of the important residues involved in catalytic activity were highly conserved, whereas four cysteine residues at positions 55, 103, 184, and 275 differed from those in human CA II, and were related to cell-defense mechanisms against oxidative damage. Phylogenetic analysis showed that the cloned sequence was clustered within the fish CA VII clade and close to the swamp eel CA VII. Structural modeling of the pufferfish CA VII protein revealed the conservation of zinc binding histidine residues (zinc ion and histidine residues). Differential expression patterns of the pufferfish CA VII were determined with semiquantitative reverse transcription (RT)-PCR and quantitative PCR (q-PCR) as well. The results of q-PCR revealed that the pufferfish CA VII was highly expressed in intestinal tissue. The pufferfish CA VII was also detected within intestinal tissue sections using an in situ hybridization assay.
Keywordspufferfish Takifugu rubripes carbonic anhydrase (CA) VII intestinal tissue
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This paper was supported by Jeonnam Sea Grant R&D program of 2018 [Grant no. 20180330]. Thanks are extended to Chungnam Prefectural Fisheries Institute for providing experimental fishes.
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