“-GCT(A)GCGAGATC-”序列负向调控Nanog基因启动子-135/-230区活性研究

Activity of Nanog promoter -135/-230 could be reverse-regulated by "-GCT( A) GCGAGATC-" sequence

目的探究Nanog启动子-332/+50区与双荧光素酶报告载体p GL4. 10连接后负向调节Nanog-135/-230区活性的调节序列。方法截短法将Nanog启动子-812-+171区不同片段与p GL4. 10载体连接,脂质体转染法将各载体分别与内参载体p RL-TK共转入细胞,双荧光素酶系统报告系统检测报告基因的表达变化。结果Nanog启动子-812/-332区、+50/+171区及-332/+51(或+56)区均不含Nanog-135/-230区的负向调控元件,改变Naong-332/+50区与报告载体p GL4. 10的连接位点(NheⅠ,BglⅡ)行成序列"-GCT (A)GCGAGATC-"即可消除Nanog启动子-332/+50对报告基因的正向调控作用。结论待检测启动子与载体连接构成"-GCT(A) GCGAGATC-"序列后可负向调控Nanog-135/-230区的活性。

Objective Identify the regulatory sequence of Nanog promoter -332/＋50 that down-regulates Nanog -135/-230＇s activity. Methods Construct a set of plasmids that ligate different Nanog promoter -812-/＋171 truncates with pGL4.10, co-transfect pRL-TK with the plasmid minded to cells using Lipofectamine 2000, testing the promoter activity using the Dual Luciferase Reporter assay system. Results There have no regulating sequence in the Nanog promoter -812/-332, ＋50/＋171, and -332/＋51. A change of ligated sequence between the NheⅠ, BglⅡ restriction sites maintained in pGL4.10 and Nanog promoter -332/＋50 sequence, “-GCT（A）GCGAGATC-”, could counteract the regulatory effect of Nanog promoter -332/＋50 on report gene expression. Conclusion Sequence of “-GCT（A）GCGAGATC-” could reversely regulate Nanog 332/＋50 promoter activity.