摘要
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。
To further investigate the transcriptional regulatory mechanism of DKK1 （Dickkopfl） gene, the sequence features were analyzed by promoter online prediction tools, which were based on the 5r-flanking sequence of swine DKK1 gene published by Ensembl database. Specific prim- ers were designed by Primer Premier 5.0 software to amplify DKK1 gene. To analyze its tran- scriptional activity, pGL3-DKK1 promoter luciferase reporter gene vectors were constructed and transfected into 293T cells and Hela cells, respectively. The results showed that the promoter re- gion of DKK1 gene contained a TATA-box, a variety of transcription factors and a CpG island. Meanwhile, the promoter of DKK1 gene had a preference for 239T cells, and the sequence of --1 679/＋292 bp had the highest promoter activity, which was obviously higher than that of other fragments （P〈0. 01）. Further analysis revealed that there were core promoter region （-953/- -1 679）, negative （-586/- -953） and positive （-953/- -1 679） regulatory regions, respectively. The 5r-flanking sequence of swine DKK1 was analyzed by bioinformatics combined with the reporter gene activity detection of promoter fragments with different length, this result demonstrated that it had transcriptional activity of promoter, and its promoter region was prelim- inarily determined, and the core promoter region and the main regulatory region were successfully identified, which laid a foundation for further studying on the transcriptional regulatory mecha- nism of the DKK1.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第6期1150-1157,共8页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
河北省现代农业产业技术体系专项(HBCT2013070202)
