Molecular cloning and characterization of prophenoloxidase gene in swimming crab Portunus trituberculatus.

摘要

Phenoloxidase (PO), a melanin-synthesizing enzyme, found as a zymogen (proPO) in hemolymp, plays an important role in arthropod defence. In this study, a prophenoloxidase (proPO) 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 cDNA clone had a full-length of 3040 bp, with an open reading frame of 2019 bp, a 5'-untranslated region of 138 bp, and a long 3'-untranslated region of 1707 bp. It encoded a protein of 672 amino acids which has a predicted molecular weight of 77.4 kDa and with an estimated pI of 6.19. It is predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues and a proteolytic activation site. Comparison of amino acid sequences showed that the proPO-deduced amino acid of P. trituberculatus has an overall similarity of 56%-87% to that of other crustaceans. Northern blot analysis revealed that the presence of proPO was expressed in haemocytes, hepatopancreas and ovary, but not in eyestalk, gill and muscle. RT-PCR analysis indicated that proPO showed different expression profiles in crab haemocytes and hepatopancreas after Vibrio alginolyticus challenging. These facts indicated that proPO was potentially involved in the acute response against invading bacteria in P. trituberculatus.