Characterization and functional analysis of a chitinase gene: Evidence of Ch-chit participates in the regulation of biomineralization in Crassostrea hongkongensis

Abstract

Chitinase is a key enzyme in many organisms that functions as a defense against chitin-coated pathogens or as a digestive enzyme to hydrolyze dietary chitin. Chitin is also essential for the formation of organic frameworks, involves in various biological processes, including shell formation. However, the function of chitinase of Crassostrea hongkongensis has not yet been determined. In this study, we obtained a full Ch-chit sequence from C. hongkongensis. The full length of Ch-chit cDNA encoded a predicted 470-amino acid protein, contained a signal peptide, a Glyco 18 domain, a low complexity domain, and a transmembrane domain. Phylogenetic analysis showed that Ch-chit clustered with Chit3-lp of other bivalves. In different stages of larvae development, the expression of Ch-chit in the trochophore larvae was the highest and was significantly higher than that in other stages (P < 0.05). In addition, Ch-chit gene was expressed in a variety of tissues, and its expression in mantle and digestive gland was significantly higher than that in other tissues (P < 0.05). In situ hybridization demonstrated that Ch-chit gene was mainly distributed at the edge of mantle, especially in the inner fold. Shell damage experiment revealed that the expression of Ch-chit was significantly up regulated at 6 h and peak at 12 h after shell damage (P < 0.05). When the expression of Ch-chit was interfered by RNA interference (RNAi), abnormal growth occurred on the inner surface of the prismatic layer. The above results imply that Ch-chit is involved in the shell formation, and also provide an important clue for the study of the molecular mechanism of biomineralization in C. hongkongensis.

Keywords

• Chitinase
• Shell formation
• Biomineralization
• Crassostrea hongkongensis