牛CART基因核心启动子鉴定及转录调控分析  

Identification and Transcriptional Regulation Analysis of Core Promoter of Bovine CART Gene

作　　者：王栋梁[1] 任静 郝琴琴 李鹏飞[2] WANG Dongliang;REN Jing;HAO Qinqin;LI Pengfei(Shuozhou Vocational Technology College,Shuozhou 036002,China;College of Life Science,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]朔州职业技术学院,朔州036002 [2]山西农业大学生命科学学院,太谷030801

出　　处：《畜牧兽医学报》2023年第9期3689-3699,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金面上项目(31873002);山西省应用基础研究计划面上项目(20210302123380);山西农业大学横向科技项目(2022HX010,2021HX23,2020HX06,2019HX03)。

摘　　要：旨在筛选牛CART基因核心启动子区并鉴定调控CART表达的转录因子,探究其转录调控机制。本研究采集3头健康母牛下丘脑组织,提取基因组DNA,通过PCR扩增、测序获得牛CART启动子序列,EMBOSS、MethPrimer、New PLACE数据库分析启动子结构特征;构建4个包含不同截短长度的CART启动子报告基因载体,双荧光素酶报告基因活性检测鉴定核心启动子区;应用DNA pull down结合质谱分析(n=3),功能聚类及结合位点预测分析筛选核心启动子区候选转录因子;构建转录因子过表达载体,转染至293T细胞,分析转录因子对牛CART核心启动子区的转录调控功能(n=3)。结果表明,牛CART基因-1200 bp~+22 bp区域存在CpG岛和TATA box、CAAT box等顺式作用元件;构建的4个截短启动子报告基因载体均具有转录起始活性,-292 bp~+22 bp片段转录起始活性最强,为牛CART基因核心启动子区;转录因子RFX5、CREB、RFX1、JUND、TEAD4、TFAP2D、RELA可与牛CART基因核心启动子区特异性结合;进一步研究证实RFX5、RFX1、TEAD4抑制CART转录(P<0.01),CREB与RELA激活CART转录(P<0.001)。本研究成功扩增获得牛CART启动子区域,筛选鉴定-292 bp~+22 bp为牛CART基因的核心启动子区,证实RFX5、RFX1、TEAD4为CART转录抑制因子,CREB、RELA为CART转录激活因子。研究结论为丰富牛下丘脑CART表达调控机制,深入研究CART调控卵泡发育机理提供依据。The present study aimed to screen the core promoter of bovine CART gene and identify the transcription factors regulating CART expression,and explore its transcriptional regulation mechanism.The hypothalamus of 3 healthy cow were collected for genomic DNA extraction.The promoter of bovine CART gene was obtained through PCR amplification and clone sequencing,and its sequence characteristics were analyzed by EMBOSS,MethPrimer,New PLACE database.The 4 CART gene promoter reporter vectors containing different truncated lengths were constructed to identify the core promoter region by dual luciferase reporter gene activity assay.DNA pull down and mass spectrometry,functional cluster and binding site prediction analysis were used to screen the candidate transcription factors in the core promoter region(n=3).The transcription factors overexpression vectors were constructed and transfected to 293T cells to analyze the regulatory function of transcription factors on bovine CART gene transcription(n=3).The results showed that there were CpG island and cis-acting elements such as TATA box and CAAT box in the-1200 bp-+22 bp region of bovine CART gene.All the 4 reporter vectors with truncated promoters had transcription initiation activity and the-292 bp-+22 bp was the highest,this region was the core promoter of bovine CART gene.RFX5,CREB,RFX1,JUND,TEAD4,TFAP2D,RELA could specifically bind to the core promoter region of the bovine CART gene.Furthermore,it was proved that RFX5,RFX1,TEAD4 could inhibit CART gene transcription(P<0.01);CREB and RELA could activate CART gene transcription(P<0.001).In this study,bovine CART promoter region was successfully amplified,and identify the-292 bp-+22 bp was core region of bovine CART gene promoter.And it was proved that the RFX5,RFX1,TEAD4 were inhibiting transcription factors of CART gene;the CREB and RELA were activating transcription factors of CART gene.These findings will enrich the regulatory mechanism of CART gene expression in bovine hypothalamus,and strengthen the understanding of

关 键 词：牛 CART 核心启动子 转录调控 DNA pull down 

分 类 号：S823.2[农业科学—畜牧学]