牦牛Smad 4基因3′UTR区双荧光素酶载体构建及与bta-miR-146a的靶向验证

Construction of Yak Smad 4 Gene 3′UTR Dual-luciferase Reporter Vector and Its Targeting Validation to bta-miR-146a

旨在鉴定牦牛Smad4基因与bta-miR-146a的靶向关系,为探究它们在牦牛卵巢卵泡发育过程中的可能分子调控机制提供理论基础。利用miRBase数据库和MEGA 6.0软件分析miR-146a在不同物种中的保守性,同时利用TargetScan软件预测其潜在靶基因,通过构建psiCHECK2双荧光素酶报告基因载体进行靶向验证。生物信息学分析结果显示,miR-146a成熟序列在脊椎动物中具有高度的保守性,并预测到178个与卵巢卵泡发育相关的潜在靶基因,从中选取Smad4基因分析发现其3′非编码区域(3′UTR)与bta-miR-146a存在结合位点;进一步成功构建牦牛Smad4基因3′UTR野生型及突变型psiCHECK2双荧光素酶报告质粒,荧光素酶活性检测结果显示,bta-miR-146amimic对牦牛Smad4基因3′UTR野生型报告质粒荧光活性有明显的下调作用,分别与突变型及NC对照组差异显著(P<0.05)。本研究初步证实,牦牛Smad4基因是bta-miR-146a的靶基因,从而提示btamiR-146a可能通过调控Smad4基因在牦牛卵巢卵泡发育过程中的表达发挥作用。

The aim of this study was to identify the targeting relationship between Smad 4 gene and bta-miR-146 a,which would provide a theoretical basis for their regulating mechanism in follicular development of yak ovary.The conservation of miR-146 ain different species was analyzed by miRBase database and MEGA 6.0 software.At the same time,TargetScan software was used to predict the potential target genes of miR-146 a,and the targeting validation was performed by construction of psiCHECK2 dual-luciferase reporter vector.The results of bioinformatics anal-ysis showed that the miR-146 amature sequence was highly conserved in vertebrates,and a total of 178 potential target genes related to ovarian follicular development were predicted,then,among these genes,Smad 4 gene was selected to be further analyzed,showing that its 3＇UTR had the binding site to bta-miR-146 a.The wild and mutant type psiCHECK2 dual-luciferase reporter vectors of 3′UTR of yak Smad 4 gene were successfully constructed,and the dual-luciferase assay result showed that the luciferase activity of wild vector of 3＇UTR of yak Smad 4 gene was distinctly down-regulated by the bta-miR-146 amimics,and was significantly lower than that of mutant vector and NC group（P〈0.05）,respectively.In conclusion,this research preliminarily confirmed that yak Smad 4 gene was the target gene of bta-miR-146 a,suggesting that btamiR-146 amight play roles in the development of yak follicles by regulating Smad 4 gene.