The Study Of Apoptosis-rclatcd Genes In Zhikong Scallop Chlamys Farreri

Zhikong scallop (Chlamys farreri) is an important marine aquaculture species in China.Since1990s, Zhikong scallop has been suffering from some desease resulting in serousconsequence such as summer mass mortality, which caused a great economic loss. Thecultivation of high-resistance scallop breeds is the primary strategy to resolve the problem.In the paper, studies on genes involved in apoptosis in Zhikong scallop were performed,which can provide the basic experimental data improving our understanding of the apoptosissystem of scallop and contribute to the scallop breeding..In the present study, five full length cDNA sequences including CfIAP-1, CfIAP-2,CfBcl-2, CfBax and CfBI-1were first obtained by Rapid Amplification of cDNA Ends(RACE) technique. The tissue distribution and the temporal expression of the mRNA levelsof these genes with the treatment of air exposure, liopopolysaccharides (LPS) and AcuteViral Necrobiotic Disease Virus (AVNV) were measured by Real-time RT-PCR. Yeasttwo-hybrid technology and RNA interference (RNAi) technology were used to study theinteraction between CfIAP-1, CfIAP-2and CfCaspase3.The full length cDNA of CfIAP-1consisted of1552nucleotides containg an openreading frame (ORF) of756bp encoding a251amino-acid polypeptide (MW=28.6kDa,PI=6.00). The predicted amino acid sequence comprised of two BIR domains. Phylogeneticanalysis revealed that CfIAP-1showed high homology with that of Litopenaeus vannamei.The full length cDNA of CfIAP-2consisted of1243nucleotides containg an ORF)of1071bpencoding a356amino-acid polypeptide (MW=40.16kDa, PI=6.23). The predicted aminoacid sequence comprised of one BIR domain and a RING domain. Phylogenetic analysisrevealed that CfIAP-2showed high homology with Livin of Homo sapiens. The CfIAP-1and CfIAP-2mRNA expression existed in all tested tissues of C. farreri, including adductormuscle, digestive gland, gill, mantle, gonad and haemolymph. CfIAP-1mRNA level was highest in adductor muscle and CfIAP-2mRNA level was highest in gill. Subcellularlocalization results showed that CfIAP-1protein was mainly localized in the cytoplasm andCfIAP-2protein was expressed in the cytoplasm and nucleus. The levels of CfIAP-1andCfIAP-2mRNA clearly increased after air exposure stress, LPS and AVNV infection,implying that CfIAP-1and CfIAP-2may be involved in the response and the apoptosisprocess induced by these stimuli. RNAi technology was applied to detect the activity ofCfCaspase3after the expression of CfIAP-2mRNA was inhibited. The results showed thatthe expression of CfIAP-2was inhibited using siRNA injection and the activity ofCfCaspase3increased significantly.The full length cDNA of CfBcl-2was of944bp and contained an ORF of678bpencoding a225amino-acid polypeptide (MW=24.99kDa, PI=5.71). The predicted aminoacid sequence comprised of a transmembrane domain in C-terminus and four BH domainsincluding BH1, BH2, BH3and BH4. The full length cDNA of CfBax contained an ORF of348bp that encoded a115amino-acid polypeptide (MW=13.39kDa kDa, PI=6.51). Thepredicted amino acid sequence comprised of a transmembrane domain in C-terminus andthree BH domains including BH1, BH2and BH3. The homology and phylogenetic analysisrevealed that CfBcl-2showed high homology with that of Crassostrea gigas and the aminoacid sequence of CfBax showed high homology with that of C. gigas and Mytilusgalloprovincialis. CfBcl-2and CfBax expression existed in all tested tissues of C. farreri.CfBcl-2expression was highest in adductor muscle and CfBax expression was highest in gill.The levels of CfBcl-2and CfBax mRNA clearly increased after air exposure stress, LPS andAVNV infection, implying that CfBcl-2and CfBax may be involved in the apoptosis processinduced by air exposure stimulation, LPS and AVNV infection. CfBcl-2could interact withCfBax through the yeast two-hybrid experiments. The result further indicated that Bcl-2canprobably form homo-or hetero-dimers with Bax to achieve regulatory role in apoptosis.The full length cDNA of CfBI-1was of957bp and contained an ORF of714bp encodinga237amino-acid polypeptide (MW=27kDa). The homology and phylogenetic analysisrevealed that the amino acid sequence of CfBI-1showed high homology with that of otherspecies, indicating that CfBI-1was highly conservative. We used quantitative real-time PCRto estimate mRNA expression level of CfBI-1in various tissues and its induced expression following air exposure stimulation, LPS infection and scallop AVNVinfection, respectively.The results showed that CfBI-1expression existed in all tested tissues of C. farreri,including adductor muscle, digestive gland, gill, mantle, gonad and haemolymph. CfBI-1expression was highest in adductor muscle and lowest in haemolymph. The levels of CfBI-1mRNA clearly increased after air exposure stress and AVNV infection, implying that CfBI-1may be involved in the apoptosis process induced by air exposure stimulation and AVNVinfection.In conclusion, the CfIAP-1, CfIAP-2, CfBcl-2, CfBax and CfBI-1may play an importantrole in apoptosis of C. farreri, which will provide new data for the study of apoptosismechanism of scallop and richen the knowledge of apoptosis system of molluscs.