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| Target Regulation of the Expression of Steroidogenic Acute Regulatory Protein Gene in Sheep (Ovis aries) Ovaries by oar-mir-150 |
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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.
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Received: 05 May 2017
Published: 04 February 2018
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