CLONING OF CDNA OF BCL-2 GENE OF MARSUPENAEUS JAPONICUS AND ITS ROLE IN COLD RESISTANCE

As an important apoptosis regulatory gene, Bcl-2 gene plays an important role in the endogenous apoptosis pathway. In this study, the full-length cDNA sequence of MjBcl-2 gene was cloned by rapid amplification of cDNA ends (RACE) and analyzed by bioinformatics; Real time quantitative PCR (qPCR) was used to analyze the expression level of MjBcl-2 in different tissues of Marsupenaeus japonicus after different temperature stresses and RNA interferences; TUNEL technique was used to detect the apoptosis after the interference of MjBcl-2. The results showed that the full length of MjBcl-2 cDNA was 2432 bp, the open reading frame was 726 bp, encoding 241 amino acids with a molecular weight of 26.80 kD; Domain prediction analysis showed that MjBcl-2 contained typical conserved domains of Bcl-2 family. The results of multiple sequence alignment and phylogenetic tree construction showed that MjBcl-2 was highly conserved and similar to other species. Quantitative PCR results showed that MjBcl-2 was expressed in all tissues of M. japonicus, with the highest expression in muscle, the second in gill and the lowest in heart. Under the stress of low temperature (10℃ and 16℃), the expression of Bcl-2 gene in gill and hepatopancreas of M. japonicus increased gradually, and reached the highest point at 72h; After interference with Bcl-2 gene, the expression of Bcl-2 gene was down-regulated and Caspase-3 gene was up-regulated at all temperatures (P<0.05); There were only a few apoptotic cells in the 28℃ treatment group. TUNEL results showed that the number of apoptotic cells increased with the decrease of temperature in RNAi group and NC group (control group). There were a small number of apoptotic cells in 28℃ treatment group, but there was no significant change in the number of apoptotic cells in RNAi group and NC group. The number of apoptotic cells in 10℃ hypothermia treatment group was significantly higher than that in NC group; After 12h, the apoptosis rates of gill tissue in 28℃ NC group, 28℃ RNAi group, 10℃ NC group and 10℃ RNAi group were 1.73%, 2.35%, 21.59% and 33.70% respectively. The results showed that MjBcl-2 may play an important role in the immune defense response of M. japonicus to low temperature stress.