Molecular Cloning, Recombinant Expression And Expression Analysis Of Caspase Gene In Pacific Oyster Crassostrea Gigas

In this paper, a caspase gene (CgCASPl) was cloned from Crassostrea gigas, which involves in cell apoptosis. The real-time PCR assay was used to detect the distribution of CgCASP1in different larval stages and adult tissues. Expression of CgCASP1was analyzed with the prolonged of the cell culture time and the H2O2stimulating of the primary cell. The CgCASP1and Relish gene were analyzed both in mRNA and protein levels after PDTC and H2O2induced apoptosis of primary cells. The CgCASP1recombinant protein was obtained in prokaryotic and eukaryotic expression system. The key results are as follows:The full-length cDNA of CgCASPl gene consists of3613bp, including473bp5’UTR,1526bp3’UTR and an ORF encoding a541amino acids protein. The putative CgCASP1protein possesses caspase family’s conservative five-peptide sequence QACRS and characteristic domains, namely P20large subunit and P10small subunit. A unique fragment of CgCASP1was found and confirmed both in cDNA and genomic DNA sequences. The real-time PCR assay showed the CgCASP1mRNA expressed in all larval stages, in adult, it had highest expression in gill, but lower expression in mantle and liver. We also found that with the prolonged of the cell culture time of the primary cell, the expression of caspase was up-regulated correspondingly, which suggested that CgCASP1gene was might related with cell apoptosis. There were no difference in Relish mRNA expression but an up-regulation in CgCASPl mRNA after H2O2stimulation; differently, the PDTC stimulation had no influence on CgCASPl gene but a down-regulation of Relish mRNA. The Relish protein was reduced both in cytoplasm and nucleus after PDTC stimulation, but no changes with H2O2stimulation. On the basis of the full length cDNA of CgCASP1gene, the prokaryotic expression vector CgCASP1-pEASY-E2and the eukaryotic expression vector CgCASP1-pT7CFE1-Chis were constructed. Recombinant protein about70kD was obtained in prokaryotic expression system, furthermore the activity was assayed through eukaryotic expression system.In conclusion, we cloned the apoptosis related gene CgCASP1then analyzed the characteristics of the gene sequence and it’s correlation with apoptosis. It will helpful for understanding of the apoptosis mechanism, as well as ontogenetic, morphogenetic and innate immunity in mollusks.