Molecular characterization and expression analysis of extracellular copper-zinc superoxide dismutase gene from swimming crab Portunus trituberculatus.

An extracellular CuZnSOD cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the ecCuZnSOD full-length cDNA consisted of 965 bp with an open reading frame of 579 bp. It encoded a polypeptide of 192 amino acids which had a predicted molecular weight of 20.0 kDa and with an estimated pI of 6.23. The deduced amino acid sequence contained a putative signal peptide of 31 amino acids. It is predicted to possess all the expected features of CuZnSOD members, including amino acids responsible for binding Cu and Zn, two putative CuZnSOD signatures, two cysteines and one N-linked glycosylation site. Sequence comparison showed that the CuZnSOD deduced amino acid sequence of P. trituberculatus has similarity of 80%, 76%, 55% and 50% to that of blue crab Callinectes sapidus, mud crab Scylla serrata, crayfish Pacifastacus leniusculus and freshwater prawn Macrobrachium rosenbergii, respectively. The ecCuZnSOD transcripts expressed in all examined tissues, including haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. RT-PCR analysis indicated that ecCuZnSOD transcripts both in haemocytes and hepatopancreas increased in the first 3 h after Vibrio alginolyticus challenging, as the bacterial infection progressed, the challenged crabs decreased to levels significantly lower than control by 96 h post-infection. These facts indicated that ecCuZnSOD was potentially involved in the acute response against invading bacteria in P. trituberculatus.