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| 牛FoxO1基因启动子转录调控分析 |
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赵毅昂1,魏大为1,2*,张久盘3,宋雅萍1,姜超1,王兴平1,2,罗仍卓么1,2,马云1,2
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| (1.宁夏大学 农学院, 银川 750021;2.宁夏大学 宁夏反刍动物分子细胞育种重点实验室, 银川 750021;3.宁夏农林科学院 动物科学研究所, 银川 750021) |
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| 摘要: |
| 为鉴定牛叉头框转录因子1(Forkhead box，FoxO1)的核心启动子区及其关键转录因子，利用PCR扩增牛FoxO1基因的启动子序列，同时设计7个逐段缺失片段引物扩增其启动子逐段缺失片段，进一步将其构建双荧光素酶报告载体并分别转染小鼠C2C12和3T3-L1细胞系，通过检测不同缺失片段荧光素酶报告载体的活性，以确定牛FoxO1启动子的核心区域；使用Genomatix和JASPAR在线软件预测牛FoxO1基因启动子核心区域的关键转录因子，采用定点突变试验在小鼠C2C12细胞系中初步鉴定预测的关键转录因子对FoxO1的转录调控作用。结果表明:1)成功扩增了牛FoxO1的启动子区1 920 bp序列，获得了FoxO1基因启动子序列的7个逐段缺失片段序列；2)经双荧光素酶报告试验进一步证实，牛FoxO1基因核心启动子位于-285/-27区域；3)预测并通过定点突变试验鉴定出MEF2A、KLF4、HOXA5和KLF5转录因子对FoxO1基因的转录活性具有关键调控作用。综上，本研究初步鉴定出牛FoxO1基因启动子核心区(-285/-27)内转录因子MEF2A、KLF4、HOXA5和KLF5对FoxO1基因有重要的转录调控作用，为FoxO1基因对牛肌肉脂肪发育过程中的转录调控机制奠定了一定理论基础。 |
| 关键词: 牛 FoxO1基因 启动子活性 转录调控 转录因子 |
| DOI：10.11841/j.issn.1007-4333.2022.09.05 |
| 投稿时间：2021-11-11 |
| 基金项目:国家自然科学基金项目(32060744)；宁夏自然科学基金项目(2021AAC05007)；宁夏重点研发(引才专项,2020BEB04011)；宁夏重点研发计划(2021BEF02029)；宁夏大学科研启动项目(030900001926) |
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| Transcription regulation analysis of bovine FoxO1 gene |
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ZHAO Yiang1,WEI Dawei1,2*,ZHANG Jiupan3,SONG Yaping1,JIANG Chao1,WANG Xingping1,2,LUORENG Zhuoma1,2,MA Yun1,2
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| (1.School of Agriculture Ningxia University, Yinchuan 750021, China;2.Key Laboratory of Ruminant Molecular Cell Breeding, Ningxia University, Yinchuan 750021, China;3.Institute of Animal Sciences, Ningxia Academy of agricultural and Forestry Sciences, Yinchuan 750021, China) |
| Abstract: |
| In order to identify the core promotor region of Forkhead box transcription factor 1(FoxO1)and its key transcription factors, the promoter sequence of bovine FoxO1 gene was amplified by PCR. At the same time, seven segment by segment deletion fragment primers were designed to amplify the segment. Furthermore, a dual luciferase reporter vector was constructed to transfect C2C12 and 3T3-L1 cells, respectively. The core region of the bovine FoxO1 promoter was determined by detecting the activity of luciferase reporter vectors of different deletion fragments. Additionally, Genomatix and JASPAR online software were used to predict key transcription factors in the core region of the bovine FoxO1 promoter. The transcriptional regulation of FoxO1 by the predicted key transcription factors was preliminarily identified in mouse C2C12 cell line by site directed mutation test. The results showed that: 1)The 1 920 bp sequence of bovine FoxO1 promoter and seven truncated promoter sequences were successfully amplified. 2)Dual-Luciferase assay further confirmed that the core promoter was located in the -285/-27 region. 3)MEF2A, KLF4, HOXA5 and KLF5 transcription factors were predicted and identified by site-directed mutation as key regulatory factors of FoxO1 gene transcriptional activity. In summary, the study preliminarily identified that the transcription factors MEF2A, KLF4, HOXA5 and KLF5 in the core region -285/-27 of bovine FoxO1 gene promoter have important transcriptional regulation effects on FoxO1 gene, which laid a theoretical foundation for the transcriptional regulation mechanism of FoxO1 gene in the process of muscle and fat production in bovine. |
| Key words: bovine FoxO1 gene promoter activity transcriptional regulation transcription factors |
