Molecular Cloning of a Chitinase Gene from the Ovotestis of Kuroda’s Sea Hare <i>Aplysia kurodai</i>

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.

Feeding on tea GH19 chitinase enhances tea defense responses induced by regurgitant derived from Ectropis grisescens

“…Endochitinases (EC 3.2.1.14) randomly hydrolyze internal β‐1,4‐linkages of chitin, releasing a mixture of soluble oligomers; whereas exochitinases (EC 3.2.1.29) show catalytic action starting from the non‐reducing end of chitin and release GlcNAcs (Dean et al ). Chitinase can also degrade the cell wall of pathogenic fungi and inhibit hypha growth, spore germination and germ tube elongation of pathogenic fungi (Matsunaga et al , Liu et al ). According to the homology of their amino acid sequence, chitinases can be divided into glycoside hydrolase family 18 (GH18) and family 19 (GH19) (Henrissat ).…”

Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Liver of Golden Cuttlefish <i>Sepia esculenta</i>

“…Chitinases in the fish stomach are classified into two groups based on differences in their primary structure and the patterns of degradation of (GlcNAc) n : acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2) [16] [17] [18] [19]. Conversely, studies reporting the cDNA cloning and expression of chitinases and chitinase-like proteins from bivalves and gastropods, which are mollusks, have noted that these play roles in shell formation [20] [21] [22], immunity [23] [24] [25] [26], and digestion [27]. Chitinase isozymes have been purified and studied from the liver of Decembrachiata (squid and cuttlefish), and are involved in digestion [28] [29] [30] [31].…”