黄鳝FOXL2启动子克隆和报告基因载体活性分析

Cloning of Monopterus albus FOXL2 Gene Promoter and Activity Analysis of Reporter Gene Vector

本研究通过生物信息学软件预测黄鳝FOXL2(Forkhead box L2)基因5′上游3000 bp内的序列,通过聚合酶链反应从黄鳝的基因组DNA PCR扩增出启动子序列,并构建到PGL3-basic载体中,经KpnⅠ/HindⅢ双酶切和测序验证正确性,阳性克隆命名为pGL3-basic-FOXL2。将pGL3-basic-FOXL2和pRL-TK共转染HEK293细胞,采用双荧光素酶检测启动子活性。并采用gene-regulation.com网站的Alibaba2.1及The JASPAR database在线软件预测转录因子结合位点。结果表明成功克隆了FOXL2的启动子,荧光素酶检测结果表明启动子具有活性。同时预测了启动子上具有以下转录因子如GATA-1、Oct-1、AP-1、Sp1、C/EBPalpha、Ftz、Hb、GR、TBP、U SF、SOX9、E4、ER、RXR-beta、Pit-1a、HOX A4、HNF-1等的结合位点。为研究FOXL2参与细胞通路之间的调控研究提供了有力的工具。

In this study,bioinformatics software was used to predict the sequence of 5’upstream 3000 bp of the FOXL2 gene(Forkhead box L2)on Monopterus albus.The promoter sequence was amplified by Polymerase Chain Reaction from the genomic DNA of Monopterus albus and constructed into PGL3-basic vector.The correctness was verified by KpnⅠ/HindⅢdouble digestion and sequencing.The positive clone was named pGL3-basic-FOXL2.HEK293 cells were co-transfected with pGL3-basic-FOXL2 and pRL-TK and double luciferase was used to detect promoter activity.The transcription factor binding sites were predicted by Alibaba2.1 at gene-regulation.com and The JASPAR database online software.The results showed that the promoter of FOXL2 was successfully cloned and the result of luciferase assay showed that the promoter was active.At the same time,it was predicted that the promoter has the following transcription factor binding sites such as GAT A-1,Oct-1,AP-1,Spl,C/EBPalpha,Ftz,Hb,GR,TBP,USF,SOX9,E4,ER,RXR-beta,Pit-la,HOXA4,HNF-1.That provided a powerful tool for studying the regulation of FOXL2 involved in the regulation of cell pathways.