Molecular Cloning and Expression Analysis of Endogenous Cellulase EsGH9-1 from Eriocheir sinensis and Its Response to Different Diets

Abstract

Cellulases are a class of modular enzymes with a catalytic glycoside hydrolase (GH) module that hydrolyzes the β-1, 4-glucosidic bond of the cellulose chain. Cellulases had been broadly divided into three types: endo-β-1, 4-glucanases, exo-β-1, 4-glucanases and β-glucosidases, which of synergistic effect lead to the complete degradation of cellulose to glucose. The endo-β-1, 4-glucanase is one of the key cellulases in cellulose digestion, which are likely to be the product of a glycosyl hydrolase family 9 (GHF9) gene. In this study, an endogenous cellulase gene, EsGH9-1, was identified from the transcriptome database of the Chinese mitten crab, Eriocheir sinensis, and the sequence of EsGH9-1 was cloned by PCR. The expression levels of EsGH9-1 in different tissues and different developmental stages were studied. In addition, EsGH9-1 mRNA expression and corresponding enzyme activity under different diet conditions were analysed. The result showed that EsGH9-1 genomic DNA (11 679 bp) consists of 15 exons interrupted by 14 introns; EsGH9-1 cDNA (2044 bp) contains an open reading frame of 1740 bp, corresponding to a polypeptide of 580 amino acid residues, with a typical catalytic domain of the glycosyl hydrolase family 9 (GHF9). EsGH9-1 was expressed in various tissues of E. sinensis, particularly high in the hepatopancreas. A significant increase in expression of EsGH9-1 was also observed in megalopa and early-stage larvae. Under different dietary conditions, the relative expression of EsGH9-1 in the plant diet group was significantly up-regulated, which was consistent with the trend of β-1, 4-endoglucanase activity. The finding of the endogenous cellulase gene (EsGH9-1) in E. sinensis implies that EsGH9-1 may be involved in the decomposition of plant diet. Furthermore, this work is a fundamental step toward understanding the role of endogenous cellulase in the digestion mechanism of aquatic crustaceans.

Keywords

• eriocheir sinensis
• cellulase
• β-1, 4-endoglucanase
• gene cloning
• expression analysis
• different diet