转录因子PAX4调控湖羊FSHR基因转录活性

Transcription Factor PAX4 Regulates the Transcriptional Activity of FSHRGene in Hu Sheep

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161bp,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。

The present study aimed to understand the sequence characterization and transcriptional regulation of the 5′-UTR of Hu sheep FSHRgene,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 sheepPAX4 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 was161 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 in11 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 FSHR5′-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 FSHRgene in Hu sheep.