猪DKK1基因启动子区的克隆及其活性分析

doi:10.11843/j.issn.0366-6964.2017.06.020

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (6): 1150-1157.doi: 10.11843/j.issn.0366-6964.2017.06.020

猪DKK1基因启动子区的克隆及其活性分析

胡慧艳¹, 贾青^1,2,3*, 侯胜奎¹, 刘津¹, 赵思思¹, 张伟峰⁴, 张军⁵, 张建亭⁶, 边慧敏⁷

1. 河北农业大学动物科技学院, 保定 071000;
2. 国家北方山区农业工程技术研究中心, 保定 071000;
3. 河北省山区农业工程技术研究中心, 保定071000;
4. 河北工程大学生命科学与食品工程学院, 邯郸 056000;
5. 唐山市畜牧工作站, 唐山 063000;
6. 安平县畜牧兽医站, 安平 053000;
7. 邢台市畜牧站, 邢台 054000

收稿日期:2017-01-06 出版日期:2017-06-23 发布日期:2017-06-23
通讯作者: 贾青,博士,教授,博士生导师,主要从事动物遗传育种研究,E-mail:jiaqing@hebau.edu.cn
作者简介:胡慧艳(1989-),女,河北灵寿人,博士生,主要从事动物遗传育种研究,E-mail:huhuiyan315@163.com
基金资助:
河北省现代农业产业技术体系专项（HBCT2013070202）

Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene

HU Hui-yan¹, JIA Qing^1,2,3*, HOU Sheng-kui¹, LIU Jin¹, ZHAO Si-si¹, ZHANG Wei-feng⁴, ZHANG Jun⁵, ZHANG Jian-ting⁶, BIAN Hui-min⁷

1. College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China;
2. National Engineering Research Center for Agriculture in Northern Mountainous Areas, Baoding 071000, China;
3. Engineering Research Center for Agriculture in Hebei Mountainous Areas, Baoding 071000, China;
4. School of Life Science and Food Engineering, Hebei University of Engineering, Handan 056000, China;
5. Tangshan Animal Husbandry Station, Tangshan 063000, China;
6. Anping Animal Husbandry and Veterinary Station, Anping 053000, China;
7. Xingtai Animal Husbandry Station, Xingtai 054000, China

Received:2017-01-06 Online:2017-06-23 Published:2017-06-23

摘要/Abstract

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/+292 bp启动子片段活性最高，且显著高于其他缺失片段（P<0.01）。-953～-1 679 bp为核心启动子区域，-586～-953 bp区域可能存在负调控元件，在-953～-1 679 bp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:

To further investigate the transcriptional regulatory mechanism of DKK1(Dickkopf1) gene, the sequence features were analyzed by promoter online prediction tools, which were based on the 5'-flanking sequence of swine DKK1 gene published by Ensembl database. Specific primers were designed by Primer Premier 5.0 software to amplify DKK1 gene. To analyze its transcriptional 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 region 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 5'-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 preliminarily 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 mechanism of the DKK1.

中图分类号:

S828
Q785

胡慧艳, 贾青, 侯胜奎, 刘津, 赵思思, 张伟峰, 张军, 张建亭, 边慧敏. 猪DKK1基因启动子区的克隆及其活性分析[J]. 畜牧兽医学报, 2017, 48(6): 1150-1157.

HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHAO Si-si, ZHANG Wei-feng, ZHANG Jun, ZHANG Jian-ting, BIAN Hui-min. Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1150-1157.

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猪DKK1基因启动子区的克隆及其活性分析

Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene

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