大鼠NKx6.1启动子克隆及特异性表达分析

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

生物技术通报 ›› 2016, Vol. 32 ›› Issue (8): 113-116.doi: 10.13560/j.cnki.biotech.bull.1985.2016.08.017

大鼠NKx6.1启动子克隆及特异性表达分析

王大玮,汪蓓蕾,姚远,陈皓,张欣,郭刚,张瑞

天津医科大学代谢病医院内分泌研究所卫生部激素与发育重点实验室,天津 300070

修回日期:2015-10-30 出版日期:2016-08-25 发布日期:2016-08-25
作者简介:王大玮,男,硕士研究生,研究方向：生物化学与分子生物学;E-mail：grabbydowa@sina.com

Cloning and Specific Expression Analysis of Rat NKx6.1 Promoter

WANG Da-wei ,WANG Bei-lei, YAO Yuan, CHEN Hao ,ZHANG Xin, GUO Gang, ZHANG Rui

Key Laboratory of Hormones and Development（Ministry of Health）,Institute of Endocrinology,Metabolic Disease Hospital of Tianjin Medical University,Tianjin 300070

Revised:2015-10-30 Published:2016-08-25 Online:2016-08-25

摘要/Abstract

摘要： 旨在构建大鼠NKx6.1启动子的报告载体,验证转录因子T3R对NKx6.1启动子的调控活性。用PCR扩增大鼠脑组织NKx6.1的 5'上游启动子片段2.4 kb,应用生物信息学方法预测该片段上潜在的转录因子T3R结合位点,根据不同的结合位点作系列截短,获得3段长度不等的启动子缺失片段,分别克隆到荧光素酶报告基因表达质粒（pGL3-Basic）上,构建相应的报告载体。将报告载体和T3R共转染大鼠星形胶质细胞（rat astrocytes,RA）,并检测报告基因荧光素酶的活性。结果显示,成功构建大鼠NKx6.1启动子报告载体,双荧光素酶报告基因活性检测表明T3R对NKx6.1启动子有明显调控作用,其中-1 887 bp-1 507 bp活性最高,即存在关键顺式调控元件。克隆并筛选出启动子核心区域,揭示了甲状腺激素对大鼠NKx6.1的表达调控机制。

关键词: 同源盒基因NKx6.1, 甲状腺激素, 双荧光素酶报告检测, 启动子活性

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

王大玮,汪蓓蕾,姚远,陈皓,张欣,郭刚,张瑞. 大鼠NKx6.1启动子克隆及特异性表达分析[J]. 生物技术通报, 2016, 32(8): 113-116.

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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大鼠NKx6.1启动子克隆及特异性表达分析

Cloning and Specific Expression Analysis of Rat NKx6.1 Promoter

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