Cloning and preliminary study on the function of Bcl-2-like gene in Pinctada fucata martensii

As an important apoptosis-regulating gene, Bcl-2 gene plays an important role in endogenous apoptotic pathways. In this study, Pinctada fucata martensii Bcl-2 gene (Pm Bcl-2-like) was cloned by rapid-amplification of cDNA ends (RACE) technology and its characteristics was analyzed by bioinformatics methods. The expression levels of PmBcl-2-like in different tissues, development stages and by immune stimulation were analyzed by quantitative real-time PCR (qPCR). The results showed that the full length of PmBcl-2-like cDNA was 2180 bp, the length of open reading frame was 1650 bp, and it encoded a total of 549 amino acids and the molecular weight was 21.62 ku. Domain prediction showed that PmBcl-2-like contained BH1-4 domains which are typical domains of Bcl-2 family. Multiple sequence alignment and phylogenetic tree analysis showed that PmBcl-2-like was highly conservative and had high similarity with Bcl-2 of other species in phylogenetic tree. Quantitative real-time PCR results showed PmBcl-2-like was expressed in all the tested eight tissues of P. fucata martensii, with the highest expression in the gill, followed by the gonad, and with the lowest expression in the mantle central. At different developmental stages, the expression of PmBcl-2-like was higher in the embryonic stage and the highest in the fertilized egg stage. After being stimulated by LPS, the expression of PmBcl-2-like reached the maximum at 24 h, and then decreased to the minimum at 72 h, with the maximum being about 2.5 times of the minimum. After PGN stimulation, the expression of PmBcl-2-like reached the highest at 6 h and the lowest at 48 h, and the highest was about 7.28 times of the lowest. After Poly: IC stimulation, the relative expression reached its maximum at 3 h, and decreased to its minimum at 12 h, and the maximum was 6.49 times of the minimum. This study suggests that PmBcl-2 -like may play an important role in the development and immune defense response of P. fucata martensii.