稀有鲫热激蛋白70的分子克隆及表达特征分析

MOLECULAR CLONING AND EXPRESSION CHARACTERIZATION OF HEAT SHOCK PROTEIN 70 FROM GOBIOCYPRIS RARUS

  • 摘要: 为研究稀有鲫(Gobiocypris rarusHSP70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。

     

    Abstract: Heat shock protein (HSP) 70, as a molecular chaperone and extensively studied protein in environmental 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, Gobiocypris 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 Gobiocypris 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.

     

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