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| 牦牛bta-miR-1434-5p和Smad1基因组织表达谱及荧光素酶报告载体构建 |
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牛家强1, 索朗斯珠1, 徐业芬1, 王玉恒1, 商鹏1, 强巴央宗1, 郭敏2, 席广银2, 赵良栋1
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| (1.西藏农牧学院 动物科学学院, 西藏 林芝 860000;2.中国农业大学 动物科学技术学院, 北京 100193) |
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| 摘要: |
| 为探索牦牛Smad1基因与bta-miR-1434-5p是否存在靶向关系，探究bta-miR-1434-5p分子调控可能机制，利用RT-PCR技术对bta-miR-1434-5p和Smad1 mRNA在牦牛组织中的表达谱进行检测，并构建pmiR-RB-REPORTTM双荧光素酶报告载体对牦牛Smad1基因3'端非翻译区（3'UTR）与bta-miR-1434-5p的靶向关系进行研究。结果表明：Smad1基因mRNA在牦牛下丘脑、垂体、心、肝、脾、肺、肾、骨骼肌、淋巴结、卵巢、输卵管、子宫组织中均有广泛表达；bta-miR-1434-5p除在牦牛输卵管中没有表达外，在其他组织均与Smad1基因mRNA共表达；获得牦牛Smad1基因3'UTR序列并成功构建野生型及突变型pmiR-RB-REPORTTM双荧光素酶报告质粒；荧光素酶活性检测发现bta-miR-1434-5p mimics对Smad1野生型质粒报告荧光没有明显的下调作用，由此推测bta-miR-1434-5p与牦牛Smad1基因的该段3'UTR之间未发现明显互作。 |
| 关键词: 牦牛 bta-miR-1434-5p Smad1基因 组织表达谱 载体构建 |
| DOI：10.11841/j.issn.1007-4333.2018.05.006 |
| 投稿时间：2017-09-26 |
| 基金项目:国家自然科学基金项目（31460604）；中西部高校综合实力提升计划：西藏农牧学院动物遗传育种与繁育学科建设（502000105）；国家肉牛牦牛产业体系项目（CARS-37） |
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| Tissue expression profiles of yak bta-miR-1434-5p and Smad1 gene and the construction of luciferase reporter vector |
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NIU Jiaqiang1, Suolangsizhu1, XU Yefen1, WANG Yuheng1, SHANG Peng1, Qiangbayangzong1, GUO Ming2, XI Guangying2, ZHAO Liangdong1
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| (1.Department of Animal Science, Xizang Agriculture and Animal Husbandry College, Linzhi 860000, China;2.College of Animal Science and Technology, China Agricultural University, Beijing 100193, China) |
| Abstract: |
| The aim of this study was to determine the target relationship between yak Smad1 gene and bta-miR-1434-5p,establishing foundation to clarify the molecular mechanism of bta-miR-1434-5p regulation.The bta-miR-1434-5p and Smad1 mRNA expression profiles of yak tissues were detected by RT-PCR,and the targeting relationship between the 3'untranslated region (3'UTR) of Smad1 gene and bta-miR-1434-5p was explored through the construction of pmiR-RB-REPORTTM dual-luciferase reporting vector.The results showed that:Smad1 mRNA was widely expressed in many tissues of yak such as hypothalamus,pituitary,heart,liver,spleen,lung,kidney,skeletal muscle,lymph node,ovary,oviduct and uterus;bta-miR-1434-5p was co-expressed with Smad1 mRNA in other tissues of yak except oviduct.The 3'UTR of yak Smad1 gene sequence was obtained and the wild and mutant type of pmiR-RB-REPORTTM dual luciferase reporting vectors were successfully constructed.Luciferase activity was detected after co-transfection of the wild or mutant type vector with bta-miR-1434-5p mimics or non-target control (NC) in 293T cells,respectively,and the down-regulation effect of the luciferase expression level of the bta-miR-1434-5p mimics to wild type reporter vector of Smad1 gene was not significant.It is concluded that bta-miR-1434-5p could not target to this fragment of the 3'UTR of yak Smad1 gene. |
| Key words: yak bta-miR-1434-5p Smad1 gene tissue profile vector construction |
