稀有鮈鲫HSP70基因启动子的克隆及功能分析

doi:10.13523/j.cb.20191002

中国生物工程杂志

2019, Vol. 39

Issue (10): 9-16 DOI: 10.13523/j.cb.20191002

研究报告

稀有鮈鲫HSP70基因启动子的克隆及功能分析

黄宇,黄书婷,张夕梅,刘堰(

)

西南大学生命科学学院淡水鱼类资源与生殖发育教育部重点实验室三峡库区生态环境教育部重点实验室重庆 400715

Cloning and Functional Analysis of the Promoter of HSP70 Gene in Gobiocypris rarus

HUANG Yu,HUANG Shu-ting,ZHANG Xi-mei,LIU Yan(

)

Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education),Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education),School of Life Science, Southwest University, Chongqing 400715, China

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摘要：

热休克蛋白70(HSP70)作为一种分子伴侣,在环境毒理学中受到广泛研究。前期研究表明稀有鮈鲫HSP70基因(GrHSP70)表达量与五氯酚(pentachlorophenol, PCP)处理的浓度和时间在肝脏中呈现剂量/时间-依赖效应。为探究启动子在热休克蛋白70表达调控中的作用,根据已知的GrHSP70 cDNA序列,采用染色体步移技术克隆了GrHSP70的5'侧翼区的核苷酸序列。生物信息学分析从预测的转录起始位点(C)起的5'侧翼区域共1 487bp,潜在的转录因子结合位点包括雌激素响应元件(ERE)、Sp1结合位点(Sp1)、糖皮质激素响应元件(GRE)、TATA结合蛋白(TBP)、CCAAT/增强子蛋白结合位点(C/EBP)、Oct-1结合位点(Oct-1)、GATA转录因子结合位点(GATA-1)等。实验构建了含有启动子缺失片段的萤火虫萤光素酶(firefly luciferase)和海肾萤光素酶(renilla luciferase)报告基因表达载体,瞬时转染HeLa细胞后,利用双荧光活性检测确定获得的GrHSP70启动子具有启动活性,其核心启动位点位于转录起始点上游-1 487~-1 093bp。同时,用不同浓度PCP暴露成功转染了重组质粒(pGL-HSP70 promoter-Luc+)的HeLa细胞,培养24h后检测双荧光活性,与对照相比,随PCP浓度的增加,荧光活性均显著增加。说明在稀有鮈鲫肝脏中PCP会通过激活GrHSP70启动子来诱导GrHSP70表达,但PCP在稀有鮈鲫体内通过何种机制来调节HSP70的合成,仍然需要进一步研究。

关键词： 稀有鮈鲫; 热休克蛋白70; 启动子; 瞬时转染; 功能分析

Abstract:

Heat shock protein 70 (HSP70), as a molecular chaperone and extensively studied protein in environmental toxicology. Previous studies had shown that GrHSP70 (the HSP70 gene of Gobiocypris rarus) was reported significantly up-regulated in liver by a concentration/time-dependent manner after pentachlorophenol (PCP) exposure. To explore the role of the promoter in regulating the expression of heat shock protein 70, Based on the HSP70 gene cDNA sequence information of Gobiocypris rarus obtained, the nucleotide sequence of the 5'flanking region of GrHSP70 was cloned using the chromosome walking technology. Biological information analysis showed that the length of 5' flanking region is 1 487bp before the predicted transcriptional start site (C). There were a series of putative transcription factor binding sites include ERE, Sp1, GRE, TBP, C/EBP, Oct-1, GATA-1, etc. Firefly luciferase (Fluc) and renilla luciferase (Rluc) reporter gene vectors with different deletions of GrHSP70 gene were constructed, after transient transfection into HeLa cells, the activity of dual-luciferase reporter gene was detected. It was confirmed that the obtained HSP70 gene promoter was active, and the core region of the GrHSP70 promoter was located between -1 487bp to -1 093bp from the transcriptional start site.Meanwhile, HeLa cells were successfully transfected the recombinant plasmid (pGL-HSP70 promoter-Luc ⁺) and exposure to different concentrations of PCP, after 24 hours cultivation, the double fluorescence activity was examined. Compared with the control, the fluorescence activity increased significantly with the increase of PCP concentration. Indicate that PCP may induce GrHSP70 expression by activating the GrHSP70 promoter. However, the mechanism by which PCP regulates the synthesis of HSP70 in Gobiocypris rarus still needs further research.

Key words: Gobiocypris rarus HSP70 Promoter Transient transfection Functional analysis

收稿日期: 2019-03-16 出版日期: 2019-11-12

ZTFLH:

Q812

通讯作者: 刘堰 E-mail: liuyan@swu.edu.cn

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引用本文:

黄宇,黄书婷,张夕梅,刘堰. 稀有鮈鲫HSP70基因启动子的克隆及功能分析[J]. 中国生物工程杂志, 2019, 39(10): 9-16.

HUANG Yu,HUANG Shu-ting,ZHANG Xi-mei,LIU Yan. Cloning and Functional Analysis of the Promoter of HSP70 Gene in Gobiocypris rarus. China Biotechnology, 2019, 39(10): 9-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191002 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/9

表1 克隆稀有GrHSP70基因5'侧翼区域特异性引物

表2 GrHSP70启动子引物序列

表3 启动子缺失片段引物

图2 GrHSP70基因启动子的活性测定

图3 GrHSP70基因启动子缺失片段电泳图

图4 GrHSP70基因启动子缺失片段活性测定

图5 PCP对稀有GrHSP70基因启动子活性的影响

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