大鼠 NKx6.1启动子克隆及特异性表达分析

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-1887 bp-1507 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。%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.