转录因子PAX4调控湖羊FSHR基因转录活性

doi:10.11843/j.issn.0366-6964.2018.05.006

摘要/Abstract

摘要：

本研究旨在了解湖羊FSHR基因5'-UTR序列特征和转录因子调控，为揭示湖羊FSHR基因转录调控机制提供依据。采集3只成年湖羊母羊的心、肝、脾、肺、肾、胃、肌肉、大肠、小肠、子宫和卵巢11种组织，采用PCR扩增和克隆测序技术获得湖羊FSHR基因5'-UTR序列，生物信息学方法分析其序列特征，RT-PCR技术分析转录因子PAX4在湖羊11种组织中的表达情况；合成湖羊PAX4基因过表达载体pcDNA3.1-PAX4，并将其转染到猪卵巢颗粒细胞（GCs），用流式细胞仪测定了GCs的细胞凋亡率。双荧光素酶报告基因系统检测PAX4对湖羊FSHR基因转录活性的影响。结果表明，湖羊FSHR基因5'-UTR全长为161 bp，含有典型的E-box、CAAT-box和GC-box等转录调控元件以及GATA-2、Sp1和PAX4等转录因子结合位点。转录因子PAX4在湖羊各组织中均有表达，其中在卵巢组织中等表达。湖羊PAX4过表达载体可在卵巢颗粒细胞高效表达（P<0.01）。过表达湖羊PAX4基因后，卵巢颗粒细胞凋亡率显著上升（P<0.05），卵巢颗粒细胞和COS-7细胞中FSHR基因5'-UTR双荧光素酶报告载体的荧光素酶活性显著（P<0.05）或极显著（P<0.01）下调。综上表明，转录因子PAX4抑制湖羊FSHR基因转录活性，从而促进卵巢颗粒细胞凋亡。

Abstract:

The present study aimed to understand the sequence characterization and transcriptional regulation of the 5'-UTR of Hu sheep FSHR gene, thus provided the basis for further exploring of transcription regulatory mechanism of FSHR gene. Adult female Hu sheep (n=3) were slaughtered for 11 tissues sampling of heart, liver, spleen, lung, kidney, stomach, muscle, rectum, intestinal, uterus and ovary. PCR amplification and clone sequencing were performed to determine the 5'-UTR sequence, and then the sequence characterization of Hu sheep FSHR gene was analyzed by bioinformatic method. Tissue expression patterns of transcription factor PAX4 of Hu sheep were detected by RT-PCR. Overexpression vector (pcDNA3.1-PAX4) of Hu sheep PAX4 gene was synthesized, which was then transfected into pig ovarian granulosa cells (GCs), and the apoptosis rate of GCs was measured by flow cytometry. The dul-luciferase reporter assay system was used to evaluate the effect of PAX4 on transcriptional activity of Hu sheep FSHR gene. The results showed that the full length of the 5'-UTR of Hu sheep FSHR gene was 161 bp, which contained several typical transcriptional regulatory elements such as E-box, CAAT-box and GC-box. A few of binding sites for transcription factor were also found, such as GATA-2, Sp1 and PAX4. RT-PCR showed that transcription factor PAX4 was expressed in 11 tissues of Hu sheep, and moderately expressed in ovarian tissue. After transfecting with pcDNA3.1-PAX4, the mRNA levels of PAX4 was increased (P<0.01) in GCs. Overexpression of PAX4 significantly increased the apoptosis rate of GCs (P<0.05), and decreased the luciferase activity of luciferase reporter vectors containing FSHR 5'-UTR in both GCs (P<0.05) and COS-7 cells (P<0.01). Together, the findings demonstrated that transcription factor PAX4 could enhance GC apoptosis by inhibiting transcriptional activity of FSHR gene in Hu sheep.

中图分类号:

S826.2

王德迪, 李隐侠, 姚一龙, 潘增祥, 决肯, 依明, 曹少先, 李齐发. 转录因子PAX4调控湖羊FSHR基因转录活性[J]. 畜牧兽医学报, 2018, 49(5): 919-926.

WANG De-di, LI Yin-xia, YAO Yi-long, PAN Zeng-xiang, JUE Ken, YI Ming, CAO Shao-xian, LI Qi-fa. Transcription Factor PAX4 Regulates the Transcriptional Activity of FSHR Gene in Hu Sheep[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(5): 919-926.

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