CDNA CLONING, CHARACTERIZATION AND MRNA EXPRESSION OF CRYPTOCYANIN FROM THE CHINESE MITTEN CRAB, ERIOCHEIR SINENSIS H. MILNE EDWARDS, 1853

摘要

Cryptocyanin is a copper-free non-respiratory protein and plays an important role in the physiological process of decapod crustaceans. In the present study, the full length cDNA encoding cryptocyanin (EsCc) was cloned from the haemocytes of the Chinese mitten crab Eriocheir sinensis, which was 2630 bp, consisting of a 5'-untranslated region of 237 bp, a 3'-untranslated region of 359 bp, and an open reading frame of 2034 bp. The deduced protein possessed 678 amino-acid residues, and the predicted molecular mass was 79.09 kDa. The phylogenetic analysis revealed that it belonged to the arthropod haemocyanin superfamily and shared 78% homology to Dungeness crab Metacarcinus magister cryptocyanin subunit 2. The expression pattern of EsCc in five tissues was investigated in premoult, intermoult and postmoult stages to understand its role during the moulting cycle. In hepatopancreas, the significant up-regulation of EsCc expression was observed in both premoult and intermoult stages, while the significant change of EsCc expressions in muscle and haemocytes was only found in premoult stages. To gain insight into the antimicrobial activity of EsCc in inflammatory processes, its expression profiles in the haemolymph of Chinese mitten crab challenged with Vibrio anguillarum and Pichia pastoris were examined. The challenge of V. anguillarum induced higher expression of EsCc as early as 6 h post-infection. Instead, a significant up-regulation of EsCc transcript was observed at 12 h post P. pastoris challenge. The above results implied that crustacean cryptocyanin was involved in the new exoskeleton formation. Also, it might be induced to resist invasions of pathogens.