稀有鲫热激蛋白70的分子克隆及表达特征分析

摘要

Heat shock protein (HSP) 70, as a molecular chaperone and extensively studied protein in environ-mental toxicology, plays a key role in membrane translocation, protein degradation, and regulatory process. However, as toxicology and risk assessment of freshwater, characterizations of HSP70 in rare minnow, Gobio-cypris rarus, remain unknown. In this paper, we described the full-length cDNA of HSP70, designated as GrHSP70, by cloning through rapid-amplification of cDNA ends (RACE) technique from the liver of Gobio-cypris rarus for the first time. The GrHSP70 shares high nucleotide sequence homolog, corresponding to amino acid sequence homology (>95%) with other fish species by NCBI blast analysis. Through quantitative real-time PCR (RT-PCR), we obtained the distribution of GrHSP70 in thirteen tissues of the untreated Gobio-cypris rarus juveniles and its expression profile in liver after pentachlorophenol (PCP) exposure. The re-sults indicated that mRNA transcription level of GrHSP70 is tissue-dependent, with the highest expression in the gonad tissue. Moreover, GrHSP70 is significantly up-regulated in liver by a concentration/time-depen-dent manner after PCP exposure. In summary, our results suggest that GrHSP70 may serve as a very sensi-tive biomarker for a toxicology and risk assessment of freshwater pollution in China.%为研究稀有逗鲫(Gobiocypris rarus) HSP70用于淡水毒理学和风险评估方面的可行性，通过cDNA末端快速扩增的方法首次从稀有逗鲫肝脏中成功克隆出HSP70基因的cDNA全长，命名为GrHSP70。NCBI比对分析结果显示， GrHSP70核苷酸序列与其他鱼类的HSP70基因的核苷酸序列相似性极高，相应的氨基酸序列同源性也高(大于95%)。通过实时定量PCR获得未经污染物暴露的稀有逗鲫幼鱼体内13种组织中GrHSP70的分布以及PCP暴露后肝脏中该基因的表达图谱，结果表明GrHSP70在稀有逗鲫体内的表达呈现出组织依赖性，在性腺组织中的表达最高。此外，在不同时间﹑不同浓度的PCP暴露下稀有逗鲫肝脏中GrHSP70的表达模式呈现出显著的时间-剂量依赖效应。总之， GrHSP70可作为一种非常敏感的生物标志物，适用于中国淡水环境污染的毒理学研究及风险评估。