Molecular cloning and characterization of a novel c-type lysozyme gene in swimming crab Portunus trituberculatus

Abstract

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. In this study, the full-length cDNA of a novel c-type lysozyme was cloned from the haemocytes of swimming crab Portunus trituberculatus using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of lysozyme was of 984 bp, containing a 5′ untranslated region (UTR) of 90 bp, a 3′ UTR of 222 bp with a poly (A) tail, and an open reading frame (ORF) of 672 bp encoding an 18 amino acids signal peptide and a mature polypeptide of 205 amino acids with the predicted molecular weight of 23.76 kDa. The conserved catalytic sites Glu51 and Asp69, and the eight conserved cysteine residues identified in lysozyme suggesting it belonged to c-type lysozyme. Also, the swimming crab c-type lysozyme may belong to calcium-binding c-type lysozymes because of containing the three Asp residues (Asp98, Asp103 and Asp104), which are conserved in calcium-binding c-type lysozymes. Quantitative real-time RT-PCR analysis revealed that the mRNA transcripts of lysozyme were expressed at the highest level in haemocytes, with higher level in gill, moderately in hepatopancreas and muscle while low in stomach and heart. The mRNA expression of lysozyme in haemocytes was down-regulated significantly after challenge with Vibrio alginolyticus at 3 h, and then kept at the low expression level until 24 h. As time progressed, the mRNA expression of c-type lysozyme was recovered to the control level after 24 h injection. The results indicate swimming crab c-type lysozyme may be an inducible acute-phase protein and bacterial challenge could decline its mRNA expression.