Cloning and characterization of a novel hydrolase gene from Hong Kong oyster Crassostrea hongkongensis

Abstract

Hydrolase genes play an important role in the immune defense system in mollusks. We found a new hydrolase gene in Crassostrea hongkongensis. To explore its function, we cloned and characterized the hydrolase gene (Chhd) from C. hongkongensis. The full-length cDNA of Chhd is 936 bp, which is composed of a 531 bp open reading frame (ORF), a 92 bp 5′ -untranslated region (UTR) and a 313 bp 3′ UTR, the ORF encodes a polypeptide of 176 amino acids which contains a hydrolase_2 domain. qRT-PCR results showed that Chhd was ubiquitously expressed in tissues of C. hongkongensis, and the expression was highest in the mantle. After bacterial challenge, the expression of Chhd in hemocytes was significantly up-regulated. Recombinant pEGFP-N1-Chhd was distributed in the cytoplasm of HEK293T cells. The results of prokaryotic expression showed that pET28a-Chhd protein could express in large quantities. The expression of Chhd in total protein of C. hongkongensis was detected by western blot assay. In addition, Chhd inhibited the activities of NF-κB/p21-Luc in HEK293T cells, and RNA interference of Chhd significantly reduced the apoptosis rate of C. hongkongensis hemocytes. Taken together, our finding indicated that Chhd was involved in immunity and apoptosis regulation of C. hongkongensis, and would provide basic data for understanding the immune mechanism of shellfish.

Keywords

• Crassostrea hongkongensis
• Hydrolase gene
• Innate immunity
• RNAi
• Apoptosis