Molecular Cloning of Multiple Chitinase Genes in Swimming Crab Portunus trituberculatus

Chitin, a major molecular constituent of the exoskeleton and the peritrophic membrane of insects and crustaceans, is a straight-chain homopolymer of beta-1,4-linked N-acetyl-D-glucosamine units. Chitinases are enzymes widely distributed in animals, plants, fungi, bacteria, and viruses, and catalyze the random hydrolysis of beta-1,4-glycosidic bonds within the chitin polymer. In crustaceans, chitinases play important physiological roles such as digestion of food, ecdysis, and protection against viral pathogens. Although several studies have investigated the chitinase genes from shrimps, there have been no reports on chitinase genes from crab chitinase. In the present study, chitinase genes from Portunus trituberculatus (swimming crab) were successfully cloned and sequenced. The hepatopancreas RNA from P. trituberculatus was isolated, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the complete or partial nucleic acid sequences of three chitinase-encoding cDNA fragments. The comparative analysis of deduced amino acid sequences of PotChi-1 and PotChi-2 revealed that they share a high homology of 82%. The complete nucleotide sequence of PotChi-1 was composed of 1,851 bp, with an open reading frame (ORF) of 1,470 bp, encoding 490 amino acids. The partial nucleotide sequence of PotChi-2 was composed of 1,115 bp. Comparative analysis of deduced amino acid sequences of PotChi-1 and PotChi-2 with the amino acid sequence of Scylla serrata chitinase revealed a significant homology of 86% and 97%, respectively. Expression of PotChi-1 and PotChi-2 in various tissues of P. trituberculatus was evaluated using RT-PCR. The results indicated that PotChi-1 is widely distributed, while it was strongly expressed in the hepatopancreas. PotChi-2 was also expressed in the hepatopancreas, although at relatively lower levels compared with PotChi-1 expression. Phylogenetic analysis based on the amino acid sequences of chitinases from crustaceans and insects indicated that the PotChi-1 and PotChi-2 chitinases were closely related to their crustacean counterparts. These results suggested that PotChi-1 and PotChi-2 chitinases might be involved in the digestion of food in the hepatopancreas. The wide distribution of PotChi-1 throughout the body also suggests a possible role for this chitinase in host defense mechanism.