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
Tissue expression profiles of yak bta-miR-1434-5p and Smad1 gene and the construction of luciferase reporter vector

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。
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.