Acta Agriculturae Boreali-Sinica ›› 2022, Vol. 37 ›› Issue (2): 223-230. doi: 10.7668/hbnxb.20192556

Special Issue: Animal husbandry Biotechnology Hot Article

• Animal Husbandry·Fisheries·Veterinarian • Previous Articles     Next Articles

Identification and Transcriptional Activity Analysis of ATGL Gene Promoter in Dairy Goat

LI Jun1,2, YAN Zhihao3, JIA Wanli1, XU Mengmeng1, ZHANG Jingfeng1, XU Qiuliang1, HAN Haoyuan1, QUAN Kai1   

  1. 1.College of Animal Science and Technology,Henan University of Animal Husbandry and Economy, Zhengzhou 450046,China
    2.International Joint Laboratory of Ruminant Nutrition and Feed Resources Development in Henan Province,Zhengzhou 450046,China
    3.Xixia County Agricultural and Rural Bureau,Xixia 474550,China
  • Received:2021-11-03 Published:2022-04-28

奶山羊ATGL基因启动子鉴定与转录活性分析

李君1,2, 闫志浩3, 贾万里1, 许蒙蒙1, 张景锋1, 徐秋良1, 韩浩园1, 权凯1   

  1. 1.河南牧业经济学院 动物科技学院,河南 郑州 450046
    2.河南省反刍动物营养与饲料资源开发国际联合实验室,河南 郑州 450046
    3.西峡县农业农村局,河南 西峡 474550
  • 作者简介:

    作者简介:李 君(1985—),女,河南虞城人,副教授,博士,主要从事反刍动物泌乳调控研究。

  • 基金资助:
    河南牧业经济学院博士科研启动资金项目(2020HNUAHEDF024)

Abstract:

Adipose triglyceride lipase(ATGL)is the main rate limiting enzyme in the process of fat hydrolysis.In order to analyze the structure and transcriptional regulation mechanism of ATGL promoter in dairy goat,the 5' flanking sequence of ATGL was amplified by PCR and analyzed by bioinformatics.Luciferase reporter gene vectors of five deletion fragments with different lengths of promoter were constructed and transfected into mammary epithelial cells of dairy goat.The cells were treated with rosiglitazone and T0901317 respectively to detect the effects of PPARG and SREBP1 on ATGL promoter activity.The results showed that the 5' flanking sequence of ATGL gene of dairy goat was 2 721 bp,including 2 024 bp upstream from the transcription start site.The ATGL promoter contained eukaryotic promoter elements TATA-box and CpG island.It was also found that there were transcription factors-binding sites of FOXO,SREBF1,PPARG,C/EBPα and E2F1 by the online software prediction.The core region of ATGL promoter was located at -256—+1,and there were negative regulatory elements at -527—-256.When cells were treated with rosiglitazone and T0901317,it was found that both of them could significantly up-regulate ATGL promoter activity.The response regions of rosiglitazone and T0901317 were -527—-256 and -882—-527,respectively,indicating that PPARG and SREBP1 could regulate ATGL promoter activity.

Key words: Dairy goat, Adipose triglyceride lipase, Promoter, Core region, Transcriptional activity

摘要:

脂肪甘油三酯水解酶(ATGL)是脂肪水解过程中的主要限速酶,是调控细胞中甘油三酯和脂肪酸之间平衡的关键因子。为了分析奶山羊ATGL基因启动子的结构及转录调控机制,以奶山羊全血DNA为模板,通过PCR技术扩增出ATGL基因5'侧翼序列并进行生物信息学分析;构建了5个不同长度的启动子缺失片段报告基因载体,通过转染奶山羊乳腺上皮细胞并检测其活性,确定其核心区域;分别用PPARG激动剂罗格列酮和SREBP1激动剂T0901317处理细胞,检测其对ATGL启动子活性的影响。结果表明,克隆得到奶山羊ATGL基因5'侧翼序列2 721 bp,包含转录起始位点上游2 024 bp。经在线软件分析发现,ATGL启动子含有真核生物启动子元件TATA-box和CpG岛;对转录因子结合位点预测发现,其上含有FOXO、SREBF1、PPARG、C/EBPα和E2F1等结合位点。启动子活性分析结果表明,ATGL启动子核心区域位于转录起始位点上游-256—+1位,且在-527—-256 位存在负调控元件。用罗格列酮和T0901317处理细胞后发现,二者均能显著上调ATGL启动子活性,且罗格列酮作用的区域为-527—-256 位,T0901317在-882—-527 位发挥作用,表明PPARG和SREBP1调控ATGL启动子活性。

关键词: 奶山羊, ATGL, 启动子, 核心区域, 转录活性