Cloning And Functional Study Of Key Genes In Intrinsic Apoptosis Pathway Of Pinctada Fucata Martensii

Pinctada fucata martensii,is one of the major seawater pearl-cultivating oyster in China.As a member of invertebrates,its innate immune system assumes the responsibility of defending the body against potential pathogens due to the lack of adaptive immune system.Therefore,it has become a key research topic to investigate the mechanism of immune action in the organism.Apoptosis is the active and orderly cell death that occurs under physiological or pathological conditions of the body through gene regulation in order to maintain the homeostasis of its internal environment.Researchers have studied the immune response mechanism of P.f.martensii after different immune stimuli such as allograft and xenograft transplantation,high temperature treatment and Vibrio infection,and the results show that apoptosis plays an important role in all these processes,but some genes or proteins that promote apoptosis play an important role in these processes,and some genes or proteins that inhibit apoptosis play an important role.What is the role of apoptosis in P.f.martensii and how does it work? Based on the above questions,this study was conducted to screen the intrinsic apoptosis pathway key genes in the P.f.martensii based on the genome,transcriptome and proteome databases of the different time points before and after nucleus-inserting operation.The functional studies of the above genes were carried out by real-time fluorescence quantitative PCR,yeast two-hybrid and subcellular localization,and the experimental results were as follows:(1)Cloning of key genes of intrinsic apoptosis pathway in P.f.martensii: Four key genes of intrinsic apoptosis pathway in P.f.martensii,including two Bcl-2 family genes and two Caspase family genes,were screened by combining genomic,transcriptomic and proteomic data.The total length of PmBak c DNA was 1213 bp,encoding 233 amino acids;the total length of PmBcl-2 c DNA was 1052 bp,encoding 250 amino acids;the total length of PmCaspase-9/2 c DNA was 1680 bp and encodes 427 amino acids;PmCaspase-3 c DNA is 2233 bp and encodes 695 amino acids.The Bcl-2 family and Caspase family showed high interspecies homology through multiple sequence alignment and evolutionary tree construction.(2)Analysis of the relative expression levels of key genes in different developmental stages and different tissues: fluorescence quantitative PCR was used to detect the relative expression levels of four genes at different developmental stages,and it was found that the expression levels of PmBak,PmBcl-2 and PmCaspase-9/2 were higher in embryonic stage,and the highest expression levels were found in the fertilized egg.The expression of Pmcaspase-3 was higher in larval stage and the highest in ocular stage.The relative expression levels of these genes in different tissues were detected,and the highest expression levels of Pmbcl-2 and PmCaspase-9/2 were found in gill tissues.The expression levels of PmBak and PmCaspase-3 were the highest in hepatopancreas.(3)Analysis of the expression levels of key genes in gill tissue and hepatopancreas after different immune stimuli: RT-PCR was used to detect the relative expression levels of key genes after different stimuli,and it was found that the two families of genes basically presented similar expression patterns after different stimuli.After LPS,PGN and Poly I:C stimulation,the expression of PmBak,PmBcl-2,PmCaspase-9/2 and Pmcaspase-3 were upregulated at 48 h,24 h and 72 h,respectively.(4)Study on the function of key genes: after RNA interference,PmBak and PmBcl-2 showed different expression patterns at the early stage,but the relative expression levels of the two genes were significantly down-regulated at 48 h,and the downstream genes PmCaspase-9/2 and PmCaspase-3 also showed down-regulated expression.Further yeast two-hybrid experiments showed that PmBak and PmBcl-2 could interact.Through apoptosis induction and subcellular localization,it was found that PmBak protein was translocated to mitochondria during apoptosis,and Caspase-9 and Caspase-3 protease activity showed a consistent expression pattern after receiving the same immune stimulation.In this study,we studied the cloning and function of the key genes of intrinsic apoptosis pathway in P.f.martensii,and preliminary understood the function mode of intrinsic apoptosis pathway after immune stress in P.f.martensii,providing a new idea for understanding the immune response mechanism of invertebrates.