褐菖鲉肝HSC70基因的环介导等温扩增技术的建立及在石油污染剂量效应研究中的应用  

Development of a Loop-mediated Isothermal Amplification Method for Analysis of Heat Shock Cognate Protein HSC70) Gene in Liver of Sebastiscus marmoratus and its Application in Studying the Dose-response Effect of Oil Pollution

作　　者：莫正平[1] 陈荣[1] 黄火清[1] 左正宏[2] 

机构地区：[1]厦门大学环境与生态学院,近海海洋环境科学国家重点实验室,厦门361102 [2]厦门大学生命科学学院,厦门361102

出　　处：《生态毒理学报》2014年第3期524-530,共7页Asian Journal of Ecotoxicology

基　　金：海洋公益性项目"海洋污染生物效应快速监测与评价技术应用示范"子任务-海洋污染效应生物标志物技术集成与研发II(201005016)

摘　　要：从褐菖鲉肝脏中克隆了热休克蛋白HSC70基因,利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立HSC70基因的定量检测方法。为检测该方法的可行性,将褐菖鲉分别暴露于石油水溶性成分(water-soluble fraction,WSF)20、60、180μg·L-11 d后,利用real-time PCR及LAMP技术同时测定褐菖鲉肝HSC70 mRNA表达量,两种方法测定结果基本一致,证实LAMP技术可用于褐菖鲉肝HSC70基因的定量。为更细致了解石油WSF影响褐菖鲉肝脏HSC70基因表达的剂量-效应关系,将褐菖鲉分别暴露于25、50、75、100、125、150、175μg·L-1WSF中,5 d后采样,用LAMP技术定量检测HSC70 mRNA。结果表明,HSC70 mRNA表达量在50μg·L-1浓度组即被显著诱导,在75μg·L-1浓度下达到最大值,这说明褐菖鲉肝HSC70基因对石油污染较敏感,有潜力作为海洋石油污染的生物标志物。Heat shock cognate protein（HSC70） gene was cloned from the liver of Sebastiscus marmoratus. A quantitative loop-mediated isothermal amplification（LAMP） method was established to analyze the mRNA level of HSC70 in S. marmoratus. S. marmoratus had been exposed to different concentrations（20, 60, 180 μg·L-1） of the water-soluble fraction（WSF） of crude oil for 1 day. Then, the contents of HSC70 mRNA in the liver of S. marmoratus weremeasured using real-time PCR and LAMP. The change patterns of HSC70 mRNA levels measured by LAMP were consistent with those measured by real-time PCR, indicating that the LAMP method was feasible for quantify HSC70 mRNA. To clearly understand the dose-effect relationship between WSF and HSC70, S. marmoratus had been exposed to WSF of seven different concentrations（25, 50, 75, 100, 125, 150, 175 μg·L-1） for 5 days, and the mRNA levels of HSC70 in liver were examined by LAMP. The results showed that the HSC70 mRNA levels were significantly increased in 50 μg·L-1group and reached the peak in 75 μg·L-1group, indicating that HSC70 in the liver of S. marmoratus was sensitive to WSF, and could be used as a potential biomarker to oil pollution.

关 键 词：环介导等温扩增 褐菖鲉 HSC70 石油水溶性成分 实时荧光定量PCR 

分 类 号：X171[环境科学与工程—环境科学] X55