猪速激肽3基因启动子活性及其表达调控的初步研究  

Analysis of the promoter activity and its regulation of TAC3 gene expression in pig

作　　者：李忠慧 袁晓龙[1] 邢燕 林长光 张哲[1] 张豪[1] 李加琪[1] LI Zhonghui;YUAN Xiaolong;XING Yan;LIN Changguang;ZHANG Zhe;ZHANG Hao;LI Jiaq i(College of Animal Science, South China Agricultural University, Guangzhou 510642, China;Fujian Guanghua BEST Eco-agricuhure and Animal Husbandry Development Co., Ltd., Sanming 365106, Chin)

机构地区：[1]华南农业大学动物科学学院,广东广州510642 [2]福建光华百斯特生态农牧发展有限公司,福建三明365106

出　　处：《畜牧与兽医》2018年第6期12-18,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家现代农业产业技术体系(CARS-35);福建省科技重大专项(2012NZ0003-3);国家科技基础性工作专项(2014FY120800)

摘　　要：为了确定猪速激肽3基因（tachykinin3,TAC3）核心启动子区域,探索作用于该区域的转录因子对TAC3的调控作用,以猪耳组织DNA为模板,PCR扩增TAC3基因5＇端不同长度缺失片段,并构建至p GL3-basic载体,转染猪卵巢颗粒细胞,通过双荧光素酶活性分析确定核心启动子区域,染色质免疫共沉淀技术（Ch IP）验证转录因子与基因启动子区的作用,构建转录因子超表达载体和小干扰RNA（siRNA）转染至猪卵巢颗粒细胞,qRT-PCR检测TAC3基因mRNA表达变化。结果显示：成功构建了TAC3基因5＇端不同长度缺失片段,通过双荧光素酶报告系统分析发现,-1 310/-544区域为TAC3基因的核心启动子区,-2 486/-1 633区域可能存在负向调控元件,-1 310/-544区域可能存在正向调控元件。Ch IP检测发现转录因子C/EBPβ和YY1分别结合在TAC3基因的-653/-639和-873/-856区域;超表达C/EBPβ和YY1后,TAC3基因启动子活性显著升高（P〈0.05）、mRNA表达水平显著上调（P〈0.01）;干扰C/EBPβ后,TAC3基因mRNA表达水平显著下调（P〈0.01）;干扰YY1后,TAC3基因启动子活性显著降低（P〈0.05）。结果表明：转录因子C/EBPβ、YY1结合在TAC3基因的核心启动子区域,促进TAC3基因的转录活性。This study was to determine the sequence of the core promoter of tachykinin 3 gene（ TAC3）,and to explore the role of transcription factors in regulation of the TAC3 core promoter region. The deletion fragments of 5＇end of TAC3 were amplified by PCR,taking the porcine ear genomic DNA as a model. The fragments were cloned into the p GL3-basic vector to build gene promoter recombinant plasmids,and then were transfected into porcine ovarian granulosa cells. The core promoter region was determined by double luciferase activity analysis.Chromatin immunoprecipitation assay（ Ch IP） was performed to determine if the transcription factors physically bound to the promoters of TAC3. The transcription factor overexpression vector and the small interfering RNA（ siRNA） transfected porcine ovarian granulosa cells were formed,and qRT-PCR was used to detect changes in expression of TAC3 mRNA. The results showed that the deletion fragments of the 5＇end of TAC3 were successfully constructed. The double-luciferase reporter system showed that the core promoter region of TAC3 was at-1 310/-544. The-2 486/-1 633 region might contain negative regulation elements,and the-1 310/-544 region might contain positive control elements. Ch IP assay showed that C/EBPβ and YYl bound to the-653/-639 and-873/-856 regions of TAC3,respectively. Overexpression of C/EBPβ and YY1 enhanced the activity of the TAC3 gene promoter（ P〈0. 05） and raised the mRNA expression level（ P〈0. 01）. Disturbing the expression of C/EBPβ reduced the expression of mRNA of TAC3（ P〈0. 01）; and disturbing the expression of YY1 also reduced the gene promoter activity（ P〈0. 05）. The findings here suggested that the C/EBPβ and YY1 genes acted on the core promoter region of TAC3,and contributed to the transcriptional activity of the gene.

关 键 词：TAC3基因 5'端缺失片段 C/EBPΒ YY1 猪 

分 类 号：S828[农业科学—畜牧学]