Identification and functional characterization of two Bcl-2 family proteins in swimming crab, Portunus trituberculatus

Bcl-2 family proteins are regulatory factors of the mitochondrial apoptosis pathway that are vital to immune defenses and many other biological processes. However, the effects of Bcl-2 family proteins on apoptosis and immunity in crustaceans is poorly studied. In the study, two novel genes including Bax gene (PtBax) and Bcl-2 (PtBcl-2) from Portunus trituberculatus were identified and characterized. The deduced amino acid sequences showed that PtBax and PtBcl-2 possess domains of typical Bcl-2-related proteins. Both genes were widely expressed in all tested tissues, with highest expression levels in the hepatopancreas. Fluorescence in situ hybridization revealed that PtBax and PtBcl-2 were mainly expressed in the cytoplasm, where they play an important role in pathogen infection. Both PtBax and PtBcl-2 were significantly upregulated in the hepatopancreas at both mRNA and protein levels following challenge with Vibrio parahaemolyticus or white spot syndrome virus (WSSV). Moreover, double-stranded RNA-mediated silencing of both genes altered apoptosis levels in the hepatopancreas and WSSV copy number in muscles. Interactions between PtBax and PtBcl-2 were analyzed at transcriptional and protein levels. Yeast two-hybrid system analysis showed that PtBcl-2 could interact with PtBax directly. These results suggest that PtBax and PtBcl-2 may participate in pathogen infection by regulating apoptosis.