Molecular characterization and expression analysis of five chitinases associated with molting in the Chinese mitten...

Chitinases are crucial enzymes required for chitin degradation in crustaceans. Five chitinase genes, namely, EsCht1, EsCht2, EsCht3, EsCht4, and EsCht6, were identified in the Chinese mitten crab, Eriocheir sinensis. The open reading frames (ORF) of EsCht1, EsCht2, EsCht3, EsCht4 and EsCht6 ranged from 1182 bp to 1926 bp with encoding proteins between 393 and 641 amino acid residues. Domain analysis of the chitinase proteins showed that most EsChts contained the catalytic domain and the chitin-binding domain (CBD) connected with the serine/threonine (S/T)-rich linker region. Phylogenetic analyses revealed that EsChts with orthologs in crustaceans were divided into six groups. The tissue-dependent, developmental stage-related and molting stage-related differential expression patterns of chitinase genes were determined using end-point polymerase chain reaction and real-time quantitative polymerase chain reaction. During the molting cycle, EsCht2 mRNA expression in the cuticle and EsCht4 and EsCht6 mRNA expression in the hepatopancreas were 108-fold (P<0.05), 19-fold (P<0.05) and 12-fold (P<0.05) higher in the premolt (D(0-1)) than in the intermolt stage, respectively. The results indicated that EsCht1 from group 1 might play a role in the digestion of chitin-containing food; EsCht2 from group 2 likely played a role in the degradation of chitinous cuticle during molting for growth and during the post-embryonic development; EsCh3 from group 3 potentially had a dual role in the digestion of chitin-containing food and defense against chitin-bearing pathogens. EsCht3, EsCht4, and EsCht6 were highly expressed in the reproductive system, indicating their potential roles in reproductive molting. Differential expression patterns of the chitinase genes suggested that they might have distinct biological functions in developmental stage- and physiological growth-related and reproductive molting processes, which are essential for metamorphosis, growth, and reproduction. The findings of this study might form a basis for further studies on the functions of chitinases in E. sinensis and other crustaceans.