研究以唐鱼(Tanichthys albonubes)鳃部组织为材料,利用同源克隆和RACE方法,首次克隆得到唐鱼Caspase-3和Caspase-9 cDNA全长序列,并对其生物信息学进行了分析和鉴定.实验结果表明唐鱼Caspase-3 cDNA全长2153 bp,开放读码框(Open Reading Frame,ORF)为840 bp,编码一段长为279个氨基酸的多肽序列;Caspase-9 cDNA全长为1758 bp,ORF为1323 bp,编码440个氨基酸.生物信息学分析表明Caspase-3和Caspase-9与其他鱼类在蛋白结构上较为保守,在重要的功能域呈现高度的保守性.应用实时荧光定量PCR技术,分析了氯氰菊酯对唐鱼鳃部组织表达Caspase-3和Caspase-9的影响,在氯氰菊酯的胁迫下,Caspase-3和Caspase-9的表达呈现抑制状态,且呈现明显的浓度和时间依赖效应.%The white cloud mountain minnow (Tanichthys albonubes) is an important aquarium fish, and also a potential experimental model organism. In order to get the knowledge of apoptosis under environmental stress in this species, we have firstly cloned and sequenced the full-length cDNAs of Caspase-3 and Caspase-9 from the gill tissue of T. albonubes using homology cloning approach combined with RACE method. In this study, the bioinformatics analysis of Caspase-3 and Capase-9 full-length cDNA sequences were presented. The results indicated that the full-length cDNA of Caspase-3 was composed of 2153 base pairs (bp), which included a 840 bp open reading frame (ORF) encoding a 279-amino acid peptide, whereas the full-length cDNA of Caspase-9 was composed of 1758 bp, which included a 1323 bp ORF encoding a 440-amino acid peptide. The identity comparison and the phylogenetic analysis showed that the predicted Caspase-3 and Caspase-9 proteins of T. albonubes were structurally similar to those of other fish, with high conservation in important functional domains. Real-time PCR analysis showed that the expression levels of Caspase-3 and Caspase-9, under cypermethrin-induced stress conditions, were suppressed compared to control group, and that Caspase-3 and Caspase-9 mRNA levels were significantly affected in a dose- and time-dependent manner by cypermethrin. These results suggest that cypermethrin plays a role in the suppression of apoptosis of T. albonubes, and that the anti-apoptotic effect of cypermethrin may be beneficial to this fish so that it can survive in harsh environments.
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