Identification And Characterization Of RpFADD And RpMacin From Manila Calms Ruditapes Philippinarum

Manila clam Ruditapes philippinarum is an important marine economic mollusk in China.Recent mass mortalities in manila clams have been attributed to pathogen invasion and environmental deterioration.It is important to explore the immune regulation and immune defense mechanism of R.philippinarum,which can provide a scientific basis for the disease prevention of manila clams.The NF-κB signaling pathway is an important signaling pathway in innate immunity,which regulates many responses to inflammatory and immune responses.In general,NF-κB signaling pathway not only inhibits apoptosis but also promotes apoptosis.Meanwhile apoptosis-related genes are involved in the regulation of NF-κB signaling pathway.In this study,we studied the immune functions of the apoptosis-related factors RpFADD in the R.philippinarum,and briefly investigated the antibacterial activities of the immune effector RpMacin.These results lay the foundation for better understanding the innate responses of R.philippinarum against bacterial challenge.The main results are as followed:1.Molecular cloning and functional analsis of RpFADDIn the study,an FADD(designated as RpFADD)was identified and characterized from R.philippinarum by RACE approaches.Multiple alignments and phylogenetic analysis strongly suggested that RpFADD was a new member of the FADD family.The RpFADD transcripts were constitutively expressed in a wide range of tissues,and dominantly expressed in hemocytes.After challenged with Vibrio anguillarum or Micrococcus luteus,the expression level of RpFADD transcripts was significantly induced and reached the maximum level at 72?h and 48 h,respectively.Knockdown of RpFADD down-regulated the transcript levels of RpIKK,RpTAK1 and RpNF-κB with the exception of RpIκB.Moreover,RpFADD primarily localized in the cell cytoplasm,and its over-expression promoted the apoptosis of HeLa cells.Theseresults revealed that RpFADD perhaps regulated the NF-κB signaling pathways positively,which provided a better understanding of RpFADD in innate immunity.2.Molecular cloning and immune fuctions of RpMacinNested PCR was used to clone the full-length cDNA of RpMacin.Based on multiple sequence alignments and phylogenic analysis,RpMacin is a member of Macin family.The RpMacin transcripts were constitutively expressed in a wide range of tissues,and dominantly expressed in gills and hepatopancreas.RpMacin transcripts were significantly induced in hemocytes at different time intervals after V.anguillarum challenge.Recombinant RpMacin showed obvious antibacterial activity against all examined bacteria.Moreover,membrane integrity analysis revealed that rRpMacin increased the membrane permeability of pathogenic bacteria and then resulted in cell death.These results suggested that RpMacin had an important function in host defense against invasive pathogens,especially through membrane-disruptive activity.In summary,the present study investigated the immune responses of the apoptosis-related factors RpFADD against V.anguillarum or M.luteus challenge,and the antibacterial mechanism of the newly identified immune effector RpMacin.The results have a good scientific significance for the enrichment and development of immunology research in mollusks.