Cloning and Specific Expression Analysis of Rat NKx6.1 Promoter

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.017

Abstract

Abstract: This work aims to construct the reporter vector of rat NKx6.1 promoter and verify the activity of transcription factor T3R in the regulation of NKx6.1 promoter. We cloned a 2.4 kb 5' upstream promoter segment of NKx6.1 from the brain tissue of rat by PCR and predicted the binding sites of potential transcription factor T3R in the segment via bioinformatics method. Three promoter-deficient segments with different lengths were obtained by promoter deletion analysis and then cloned into the expression plasmids of luciferase reporter gene（pGL3-Basic）,and corresponding reporter vectors were constructed. The reporter vectors and T3R were co-transfected into rat astroytes,then the activities of the gene’s luciferase were determined. Above results demonstrated that we successfully constructed the reporter vector of NKx6.1 promoter,and the results of dual luciferase assay showed that T3R regulated significantly NKx6.1 promoter,and the region of -1 887 bp-1 507 bp presented the highest activities,i.e.,contained the key cis-regulatory element. In conclusion,we cloned and screened the core promoter region and revealed the transcriptional regulation mechanism of thyroid hormones on NKx6.1 in brain tissue of rat.

Key words: homeobox gene Nkx6.1, thyroid hormones, dual-luciferase reporter assay system, promoter activity

WANG Da-wei ,WANG Bei-lei, YAO Yuan, CHEN Hao ,ZHANG Xin, GUO Gang, ZHANG Rui. Cloning and Specific Expression Analysis of Rat NKx6.1 Promoter[J]. Biotechnology Bulletin, 2016, 32(8): 113-116.

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