Expression Characteristics of Caspase-3 Gene in the Hindgut of Urechis Unicinctus Exposed to Sulfide

GAO Beibei; LI Yue; LI Xueyu; LIU Xiaolong; MA Xiaoyu; ZHANG Zhifeng

Abstract：Caspase-3 is a key protease to the cell apoptosis.In this study,a full length cDNA of Caspase-3 gene from Urechis unicinctus was isolated by rapid amplification of cDNA end technique.The full length cDNA was 1,616 bp with a 759 bp open reading frame encoding a protein of 252 amino acid residues.The deduced amino acid sequence contained a conserved motif (QACRG) of caspase family and two cleavage sites (D96-L97、D135-S136) of Caspase-3.A prokaryotic expression vector pET 28a-Caspase-3 was constructed and transformed into E.coli BL 21(DE 3) to obtain the Caspase-3 protein,and then the recombinant protein was purified and used to preparing its polyclonal antibody.Content of the Caspase-3 protein was detected by Western blotting in the hindgut of U.unicinctus after sulfide stressing.The result showed that no significant difference of Caspase-3 content was found in the hindgut of U.unicinctus when exposed at 50 μmol/L sulfide;however,a significant increase of the Caspase-3 amount was presented in the hindgut of U.unicinctus exposed to 150 μmol/L sulfide for 24 h,1.8-folds higher than that of the control at 72 h.Our finding suggested that the survival of hindgut cells will be not affected obviously at low concentrations of sulfide,while high concentration of sulfide may cause death of the hindgut cells when U.unicinctus is exposed for a long time.