唐鱼Caspase-3和Caspase-9 cDNA全长克隆及胁迫表达分析
CLONING AND EXPRESSION OF WHITE CLOUD MOUNTAIN MINNOW(TANICHTHYS ALBONUBES) CASPASE-3 AND CASPASE-9 cDNAs
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摘要: 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。Abstract: 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.albo-nubes 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 cyperme-thrin.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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Keywords:
- Tanichthys altbonubes /
- Caspase-3 /
- Caspase-9 /
- Cloning /
- Cypermethrin /
- Expression
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