猪DKK1基因启动子区的克隆及其活性分析

发布时间：2018-03-24 19:06

本文选题：DKK基因　切入点：启动子　出处：《畜牧兽医学报》2017年06期

【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(P0.01)。-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。
[Abstract]:In order to explore the transcriptional regulation mechanism of DKK1 gene, this study analyzed the sequence characteristics of the promoter region of the gene by using the promoter on-line prediction software. According to the 5'flanking region sequence of porcine DKK1 gene published in Ensembl database, the sequence of 5'flanking region of porcine DKK1 gene was analyzed. Specific PCR primers were designed for amplification and sequencing, and then pGL3-DKK1 double luciferase expression vectors with different deletion fragments in promoter region were constructed and transfected into 293T cells and Hela cells, respectively. The double luciferase reporter gene was detected. The results showed that there was a TATA-box in the promoter of DKK1 gene, and many transcription factors and one promoter of DKK1 gene of CpG island had preference to 239T cells, among which the activity of p16679 / 292bp promoter was the highest. Moreover, it was significantly higher than that of other deletion fragments, P0.01G. -953 679bp as the core promoter region, and there might be negative regulatory elements in the region of -586r-953bp. There may be positive regulatory elements in the region of -953 ~ (-1) 679bp. The bioinformatics analysis of the DKK1 gene and the detection of the activity of the double reporter gene with different length promoter fragments were carried out in this study. It was confirmed that the 5'flanking region of the DKK1 gene had promoter transcriptional activity, and the promoter region of the gene was preliminarily identified, and the core region and the main regulatory region of the promoter were found. It will lay a foundation for further study on the transcriptional regulation mechanism of DKK1 gene.
【作者单位】：河北农业大学动物科技学院;国家北方山区农业工程技术研究中心;河北省山区农业工程技术研究中心;河北工程大学生命科学与食品工程学院;唐山市畜牧工作站;安平县畜牧兽医站;邢台市畜牧站;
【基金】：河北省现代农业产业技术体系专项(HBCT2013070202)
【分类号】：Q78;S828

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