Abstract
In this study, we report that we successfully cloned and sequenced a chitinase gene from the ovotestis of Kuroda’s sea hare Aplysia kurodai. By using reverse transcription-polymerase chain reaction (RT-PCR) and a system for the 5’ and 3’ rapid amplification of cDNA ends, we obtained a 1352 bp chitinase gene (AkChi) from the ovotestis of A. kurodai. AkChi contains a 1263 bp open reading frame that encodes 421 amino acids. The domain structure predicted from the deduced amino acid sequence was an N-terminal signal peptide and a catalytic domain of glycoside hydrolase (GH) family 18 chitinase. A comparative analysis of the deduced amino acid sequences of AkChi with those of the acidic mammalian chitinase of the California sea hare Aplysia californica revealed the highest homology at 83%. The purified chitinase from the ovotestis was digested by trypsin, and 119 residues of digested peptides were consistent with the deduced amino acid sequence of AkChi. We used RT-PCR to evaluate the expression of AkChi in various tissues of A. kurodai, and we observed that AkChi was expressed only in the ovotestis. A phylogenetic tree analysis, performed using the amino acid sequences of AkChi and known GH family 18 chitinases, showed that AkChi was separated from the molluscan chitinases with a chitin binding domain. To our knowledge, this is the first study demonstrating the cDNA cloning of an ovotestis chitinase from a sea hare.
Highlights
Chitin, a major molecular constituent of the exoskeleton of insects and crustaceans, is a straight-chain homopolymer of β-1,4-linked N-acetyl-D-glucosamine units [1]-[3]
Six degenerate primers were designed for the reverse transcriptase-polymerase chain reaction (RT-PCR) from conserved sequences of molluscan chitinase, including those from California sea hare (Aplysia californica; GenBank: XM_005112601), triangle sail mussel (Hyriopsis cumingii; GenBank: JN582038), Pacific oyster (Crassostrea gigas; GenBank: AJ971239), Hawaiian bobtail squid (Euprymna scolopes; GenBank: KF015222), and golden cuttlefish (Sepia esculenta; GenBank: AB986212)
In order to classify the chitinase from the ovotestis of A. kurodai among the glycoside hydrolase (GH) family 18 chitinases, we constructed a phylogenetic tree based on the enzyme precursor sequences by the neighbor-joining method, using the ClustalW program
Summary
A major molecular constituent of the exoskeleton of insects and crustaceans, is a straight-chain homopolymer of β-1,4-linked N-acetyl-D-glucosamine units [1]-[3]. Chitinases (EC 3.2.1.14) are enzymes that randomly hydrolyze the β-1,4 glycosidic bonds of chitin [4]. They have been found in various organisms, and they play important physiological roles in functions such as attack, defense, morphological changes, and digestion [5] [6]. The characterization and cDNA cloning of chitinases from several fishes have been reported [7]-[9]. A. kurodai is a kind of herbivorous gastropoda seen in the vicinity of the coast from April to June This creature was allowed to degenerate shells despite the shellfish. We cloned the cDNA encoding chitinase from the ovotestis of A. kurodai and determined the primary structure of the chitinase
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