Caspase-apoptosis Pathway Effects On Stress And Embryonic Development In The Small Abalone Haliotis Divercolor

Caspase apoptosis signal pathway plays an important role in stress and development. Some partial sequences of caspase apoptosis signal pathway were obtained from our EST database of small abalone Haliotis divercolor. In our studies, five Caspase apoptosis signal pathway elements, caspase-3(saCASP-3), caspase-8(saCASP-8), caspase 3-like protein(saCASP-3lp),transcription factor activator protein-1(saAP-1) and defender against apopototic cell death 1(saDAD-1) were cloned by SMART RACE techniques. Real-time quantitative PCR(RT-qPCR)and gene silencing were used to reveal the role of caspase apoptosis signal pathway in larval development, immune stress, heat stress and hypoxia. The results were reported as follows:1) The length cDNA of saCASP-3 is 1192 bp, encodes a protein of 276 amino acids. The analysis of conserved domains revealed that saCASP-3 includes the presence of a P20 domain(P20) and a P10 domain(P10). Additionally, two CASP characteristic motifs were identified from the putative amino acid sequence of saCASP-3:P20 domain histi-din signature(HKDTDCFVCVILTHG) and P20 domain cysteine active site(KPKLFFIQACRG). qPCR was employed to investigate the tissue distribution of saCASP-3 mRNA, and its expression in abalone under bacteria 、 megatherm 、 hypoxia challenge and larvae at different developmental stages. The saCASP-3 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes;The saCASP-3 expression level at 12 h after V. parahaemolyticus challenge was up-regulated significantly(P<0.05) in hemocytes; The saCASP-3 expression level was up-regulated after megatherm challenge immediately(P<0.05) in gill,It was also quickly up-regulated at 24 h(P<0.05) in hemocytes and hepatopancreas; The saCASP-3 expression level at 96 h after hypoxia challenge was up-regulated significantly(P<0.05) in gill, It was also quickly up-regulated at 4 h(P<0.05) in hemocytes; saCASP-3 was constitutively expressed at all examined developmental stages. RNAi was employed to diminish the expression of endogenous saCASP-3 in late trochophore. The statistical analysis showed that the saCASP-3 dsRNAinterference induced a 33.1% increase(P<0.01) in larval aberration compared with the non-treated larvae.2) The length cDNA of saCASP-8 is 1001 bp, encodes a protein of 260 amino acids. The analysis of conserved domains revealed that saCASP-8 includes the presence of a P20 domain(P20) and a P10 domain(P10).Quantitative real-time PCR was employed to investigate the tissue distribution of saCASP-8 mRNA, and its expression in abalone under bacteria 、 megatherm 、anoxia challenge and larvae at different developmental stages. The saCASP-8 mRNA could be detected in all examined tissues, with the highest expression level in hepatopancreas; The saCASP-8 expression level at 3 h after V. parahaemolyticus challenge was up-regulated significantly(P<0.05) in hemocytes;The saCASP-8 expression level was up-regulated after megatherm challenge immediately(P<0.05) in hemocytes; The saCASP-8 expression level at 96 h after anoxia challenge was up-regulated significantly(P<0.05) in hemocytes.3) The full-length cDNA of saCASP-3lp is 1260 bp, and the complete open reading frame of783 bp encodes a protein of 260 amino acids. Amino acid sequence analysis revealed saCASP-3lp shares CARD, DEATH-like domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saCASP-3lp mRNA, and its expression in abalone under bacteria 、 megatherm 、 anoxia challenge and larvae at different developmental stages. The saCASP-3lp mRNA could be detected in all examined tissues, with the highest expression level in mucous gland;The saCASP-3lp expression level at 6 h after V. parahaemolyticus challenge was up-regulated significantly(P<0.05) in hemocytes; The saCASP-3lp expression level at 24 h after megatherm challenge was up-regulated significantly(P<0.05) in hemocytes. The saCASP-3lp expression level at 192 h after anoxia challenge was up-regulated significantly(P<0.05) in hemocytes. RNAi was employed to diminish the expression of endogenous saCASP-3lp in late trochophore. The statistical analysis showed that the saCASP-3lp dsRNA interference induced a 30.5% increase(P<0.01) in larval aberration compared with the non-treated larvae.4) The full-length cDNA of saAP-1 is 1482 bp, and the complete open reading frame of 948 bp encodes a protein of 315 amino acids. Amino acid sequence analysis revealed saAP-1 shares Jun transcription factor domain(JTFD), The basic leucine zipper domain(b ZIP). Quantitative real-time PCR was employed to investigate the tissue distribution of saAP-1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saAP-1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes; The saAP-1 expression level at 24 h after V. parahaemolyticus challenge was up-regulated significantly(P<0.05) in hemocytes; qPCR showed that saAP-1 mRNA transcriptswere rapidly increased in early trochophore stage, and constitutively highly expressed with the larval development(P<0.01).5) The full-length cDNA of saDAD-1 is 553 bp, and the complete open reading frame of393 bp encodes a protein of 130 amino acids. Amino acid sequence analysis revealed saDAD-1shares DAD. Quantitative real-time PCR was employed to investigate the tissue distribution of saDAD-1 mRNA, and its expression in abalone under bacteria、megatherm、anoxia challenge and larvae at different developmental stages. The saDAD-1 mRNA could be detected in all examined tissues, with the highest expression level in digestive tract;The saDAD-1 expression level at 12 h after V. parahaemolyticus challenge was up-regulated significantly(P<0.05) in hemocytes; The saDAD-1 expression level at 4 h after megatherm challenge was up-regulated significantly(P<0.05) in hemocytes; The saDAD-1 expression level at 96 h after anoxia challenge was up-regulated significantly(P<0.05) in hemocytes.