黄鳝Dmrt1基因5′端侧翼序列克隆和报告基因载体活性分析

Cloning and activity analysis of Monopterus albus Dmrt1 gene 5′flanking region

为深入研究黄鳝Dmrt1基因5′端侧翼序列在性别决定与分化中细胞通路之间的转录调控作用。本研究以黄鳝基因组DNA为模板,PCR扩增出Dmrt1基因5′端侧翼序列,并对该序列进行了生物信息学分析,然后将克隆获得的Dmrt1基因5′端侧翼序列构建到PGL3-enhancer载体中,命名为pGL3-enhancer-Dmrt1,最后将pGL3-enhancer-Dmrt1和内参质粒pRL-TK共转染至HEK293细胞,采用双荧光素酶检测系统对Dmrt1基因5′端侧翼的启动活性进行了检测。实验结果显示:克隆获得的Dmrt1基因5′端侧翼序列为1519 bp;利用TESS-Transcription Element Search System转录因子结合位点在线预测数据库对黄鳝Dmrt1基因5′端侧翼序列进行预测分析结果表明,该序列存在AP-1、Oct-1、C/EBPa和GATAx等真核生物通用的转录因子结合位点以及与性别相关基因的蛋白结合位点,如SRY、Sox3和Sox5等;双荧光素酶检测结果显示,该5′端侧翼序列具有启动活性。

The 5′flanking region of Dmrt1 gene was amplified by genomic DNA PCR of Monopterus albus and bioinformatics analysis was performed in this study.Then,the cloned 5′flanking region of Dmrt1 gene was constructed into pGL3-enhancer vector,named pGL3-enhancer-Dmrt1.Finally,pGL3-enhancer-Dmrt1 and PRL-TK were co-transfected into HEK293 cells,and the activation activity of the Dmrt1 gene 5′flanking region was analyzed using a dual luciferase assay system.The experimental results showed that the sequence of Dmrt1 gene 5′flanking region obtained by cloning was 1519 bp.Using TESS-Transcription Element Search System online database to predict the binding sites of potential transcription factors,it showed that in the sequence of Dmrt1 gene 5′flanking region,there were transcription factor binding sites commonly used in eukaryotes such as AP-1,OCT-1,C/EBPA and GATAX,as well as protein binding sites associated with sex-related genes such as SRY,Sox3,Sox5 and so on.The double luciferase assay indicated that the 5′flanking region had promoter activity.This study lays the foundation for further investigations on the transcriptional regulation mechanism between the cellular pathways of sex determination and differentiation.