论文部分内容阅读
旨在初步探索DKK1基因转录调控机制,本研究利用启动子在线预测软件分析了该基因启动子区序列特征,根据Ensembl数据库已公布的猪DKK1基因的5′侧翼区序列,设计特异性PCR引物进行扩增、测序,进而构建启动子区不同缺失片段的pGL3-DKK1双荧光素酶表达载体,分别转染293T细胞和Hela细胞,并进行双荧光素酶报告基因检测。结果显示,DKK1基因启动子中含有1个TATA-box、多种转录因子和1个CpG岛;DKK1基因启动子对239T细胞具有偏好性,其中p-1 679/+292bp启动子片段活性最高,且显著高于其他缺失片段(P<0.01)。-953~-1 679bp为核心启动子区域,-586~-953bp区域可能存在负调控元件,在-953~-1 679bp区域可能存在正调控元件。本试验通过对DKK1基因进行生物信息学分析并结合不同长度启动子片段双报告基因活性检测,证实了DKK1基因的5′侧翼区序列具有启动子转录活性,并初步确定了该基因的启动子区域,找到了启动子的核心区域和主要调控区域,为进一步研究DKK1基因转录调控机制奠定基础。
The purpose of this study was to explore the mechanism of DKK1 gene transcriptional regulation. In this study, promoter sequence prediction software was used to analyze the sequence of promoter region of the gene. Based on the 5 ’flanking region of pig DKK1 gene published in Ensembl database, The pGL3-DKK1 dual-luciferase expression vector was constructed and transfected into 293T cells and Hela cells respectively, and the dual luciferase reporter gene was detected. The results showed that the promoter of DKK1 gene contained one TATA-box, multiple transcription factors and one CpG island. The DKK1 gene promoter had a preference for 239T cells. The promoter of p-1 679 / + 292bp was the highest, And significantly higher than other deletion fragments (P <0.01). -953 ~ -1 679bp as the core promoter region, -586 ~ -953bp region may have negative regulatory elements, -953 ~ -1 679bp region may exist positive regulatory elements. In this study, bioinformatics analysis of DKK1 gene combined with dual reporter gene activity test of different length promoter fragments confirmed that the 5 ’flanking region of DKK1 gene has promoter transcriptional activity and initially identified the promoter region of the gene , Found the core promoter region and the main regulatory region, which laid the foundation for further study on the transcriptional regulation of DKK1 gene.