猪StAR基因启动子区克隆及其转录活性研究

doi:10.11843/j.issn.0366-6964.2018.07.023

摘要/Abstract

摘要：

旨在通过分析猪StAR基因启动子活性区域，探究猪StAR基因的转录调控机制，从育种学角度为提高猪繁殖力提供新思路。本研究根据Ensembl数据库已公布的猪StAR基因的5'侧翼区序列，利用在线预测软件对该基因启动子区序列信息进行分析，以大白猪基因组DNA为模板，利用特异性引物，进行PCR扩增、测序，进而构建启动子区不同缺失片段的pGL3-StAR双荧光素酶表达载体，转染293T细胞并进行活性检测。结果显示，StAR基因5'侧翼区不含有典型的TATA-box和CpG岛；成功克隆了10个含有不同长度的启动子片段，并构建了各片段与表达载体的重组质粒；转染293T细胞后经双荧光素酶活性检测发现，大白猪StAR基因5'侧翼区存在着核心启动子，其中-196~+127 bp这一区域活性值最高，且显著高于其他缺失片段（P<0.01），表明在+127~-196 bp的区域内存在重要的正调控因素，外显子1对启动子活性起重要的调控作用。-41~-196 bp为核心启动子区域，该区域存在着关键的正调控元件，包含GATA2、GATA4、SP1、ZNF263、Hoxa9、KLF16和ZNF740转录因子结合位点。本试验通过对StAR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StAR基因的5'侧翼区序列具有启动子转录活性。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StAR基因转录调控机制提供理论依据。

Abstract:

The research was designed to analyze the activity region in the promoter of StAR gene, and to explore the mechanism of expression regulation of StAR gene, thus provide new ideas for improving the fertility of the pigs from the perspective of breeding. The sequence of the promoter activity region was analyzed by online tools based on the 5'-flanking sequence of swine StAR gene published by Ensembl database. The specific primer was designed and the PCR was used to amplify the gene promoter sequence based on the reference genomic sequence of the pigs, pGL3-StAR promoter luciferase reporter gene vectors were constructed and transfected into 293T cells, the relative luciferase activity was measured by dual luciferase assay system. The results showed that the 5'-flanking sequence of swine StAR gene didn't contain the typical TATA-box and CpG island. Ten promoter fragments with different lengths were obtained and luciferase reporter gene vectors were constructed, and then to analyze their transcriptional activity through transfected into 293T cells, respectively. The core promoter region of StAR gene was located in the 5'-flanking sequence of swine StAR gene, among them, -196-+127 bp region had the highest activity value, which was significantly higher than other deletion fragments(P<0.01),suggested that the region of +127——196 bp existed an important positive regulatory element. The exon 1 played a critical role in regulating the activity of the promoter. -41——196 bp as the core promoter region, contained a primary positive regulatory element, in which there were a number of transcription factor binding sites, including GATA2, GATA4, SP1, ZNF263, Hoxa9, KLF16 and ZNF740. Bioinformatics analysis of StAR gene and detection of dual reporter gene activity with different length promoter fragments confirmed that 5'-flanking sequence of swine StAR had promoter transcriptional activity. The promoter region of the gene had been preliminarily identified, the promoter region and the main regulatory region were found, which provided a theoretical basis for further study of StAR gene transcriptional regulation mechanism.

中图分类号:

S828.2
Q785

胡慧艳, 贾青, 侯胜奎, 刘津, 张婧, 张伟峰, 锡建中. 猪StAR基因启动子区克隆及其转录活性研究[J]. 畜牧兽医学报, 2018, 49(7): 1524-1532.

HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHANG Jing, ZHANG Wei-feng, XI Jian-zhong. Cloning and Transcriptional Activity of Swine StAR Gene Promoter[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1524-1532.

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