农业生物技术学报2018，Vol.26Issue(2)：234-245,12.DOI:10.3969/j.issn.1674-7968.2018.02.006
绵羊卵巢oar-mir-150靶向调节类固醇激素合成急性调节蛋白基因的表达
Target Regulation of the Expression of Steroidogenic Acute Regulatory Protein Gene in Sheep (Ovis aries) Ovaries by oar-mir-150
摘要
Abstract
In order to study the regulation of Turpan black sheep (Ovis aries) in breeding and cultivate the high fecundity breeds of sheep,ovis aries microRNA-150(oar-mir-150),which was significantly differently expressed in follicular and luteal phase of Turpan black sheep,was studied as the research object in this study.The target genes of oar-mir-150 were predicted by bioinformatics,and the expression of oar-mir-150 and steroidogenic acute regulatory protein gene(STAR) in follicular and luteal phase of Turpan black sheep ovaries were detected by qRT-PCR.In addition,the base sequence of wild,mutant and deletant-type STAR gene were synthesized and ligated with the PGL3-control vector after annealing for constructing the luciferase reporter gene vector.Then,the oar-mir-150 mimic or negative control mimic with the recombinant plasmid were co-transfected into 293T cells,and the luciferase activity was detected by the double luciferase activity assays.The results indicated that oar-mir-150 had a total number of 540 target genes,and 8 target genes were differently expressed between follicular and luteal phase.STAR gene as a target gene was up-regulated when the follicular phase compare to luteal phase that had the target gene binding site (UUGGGAG) of the oar-mir-150 which located in the 3'UTR.The relative quantitative results illustrated that the relative expression of oar-mir-150 was 0.73 which is the significant decrease (P＜0.05) and STAR mRNA is 1.90 which is the significantly increase(P＜0.05) in the Turpan black sheep ovaries when the follicular phase compare to the luteal phase.The wild,mutant and deletant-type luciferase reporter gene vectors were all successfully constructed.The transfection ratio of transfection reagent and green fluorescent protein vector was 1 ∶ 1 which was determined as the best transfection efficiency and the transfection ratio of PGL3-control and PRL-TK was 200:1 which was determined as the optimal transfection ratio.The double luciferase activity assays demonstrated that oar-mir-150 effectively negatively regulated the expression of STAR.Studies have shown that there was a definite relationship of target regulation between oar-mir-150 and STAR,and oar-mir-150 regulated the expression of STAR gene negatively,which would provide a theoretical basis for the study of microRNA in regulating the reproduction process of Turpan black sheep at the different stages of estrus cycle.关键词
吐鲁番黑羊/类固醇激素合成急性调节蛋白基因/绵羊微小RNA-150(oar-mir-150)/发情周期Key words
Turpan black sheep/Steroidogenic acute regulatory protein gene/Ovis aries microRNA-150(oar-mir-150)/Estrus cycle分类
农业科技引用本文复制引用
苗艳平,刘若男,魏彦辉,周荣艳,李相运,锡建中,张振红..绵羊卵巢oar-mir-150靶向调节类固醇激素合成急性调节蛋白基因的表达[J].农业生物技术学报,2018,26(2):234-245,12.基金项目
河北省高等学校科学技术研究青年基金项目(QN2015162)、河北省首批青年拔尖人才支持计划(2016-2018)和河北农业大学青年学术带头人支持项目(2015-2017) （QN2015162）
