oar-miR-143在绵羊卵巢不同发育时期表达研究及靶基因CDK2的验证

The expression of oar-miR-143 in different developmental stages of sheep ovaries and the verification of target gene CDK2

本研究前期通过高通量测序鉴定筛选出oar-miR-143在小尾寒羊卵巢不同发育时期发生差异表达并对其靶基因进行预测,通过GO和KEGG分析,推测其可能在卵巢发育过程中起到重要作用。本研究旨在对oar-miR-143预测到的靶基因进行验证,探究其影响和调控卵巢发育的可能分子机制。采用双荧光素酶检测系统,对筛选出的oar-miR-143与其候选靶基因CDK2的作用关系进行验证,首先进行miRNA与其候选靶基因结合位点的预测分析,然后构建含有靶位点的候选靶基因CDK2的野生型载体及突变型表达质粒载体,最后通过与oar-miR-143 minicis共转染293T细胞,进行双荧光素酶相对活性的检测。结果显示,oar-miR-143可抑制野生型CDK2-3’UTR载体的荧光素酶相对活性,而不影响突变型CDK2-3’UTR载体的荧光素酶相对活性,表明oar-miR-143可结合CDK2基因种子序列抑制CDK2的表达,CDK2被证实为oar-miR-143的靶向基因。这些数据将为阐明绵羊卵巢发育的分子调控机制,确定影响小尾寒羊繁殖性能的生物标志物提供了重要理论依据。

In the early stage of this study,high-throughput sequencing was used to screen out the differential expression of oar-miR-143 in different stages of ovarian development of small-tailed Han sheep,and its target genes were predicted.GO and KEGG analysis suggested that oar-miR-143 might play an important role in ovarian development.The purpose of this study was to verify the target genes pre-dicted for oar-miR-143 and explore the possible molecular mechanisms of its influence and regulation on ovarian development.The relationship between the screened oar-miR-143 and its candidate target gene CDK2 was verified by dual luciferase detection system.First,the binding sites of miRNA and its candidate target genes were predicted,and then wild-type and mutant vectors of candidate target gene CDK2 containing target sites were constructed.After co-transfection with oar-miR-143 mimics into 293T cells,dual luciferase activity was detected.The results showed that oar-miR-143 could inhibit the relative luciferase activity of wild-type CDK23'UTR vector,but had no effect on the relative luciferase activity of mutant CDK23'UTR vector,indicating that oar-miR-143 could bind to the CDK2 gene seed sequence to inhibit the expression of CDK2.CDK2 was confirmed as the target genes of oar-miR-143.These data will provide an important theoretical basis for elucidating the molecular regulatory mechanism of ovarian development in sheep.It can provide an important theoretical basis for determining the biomarkers affecting the reproductive performance of small-tailed Han sheep.