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| 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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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.
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Received: 16 March 2019
Published: 12 November 2019
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Corresponding Authors:
Yan LIU
E-mail: liuyan@swu.edu.cn
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