牛Mfn1基因通过调控线粒体代谢影响克隆胚胎ZGA的研究

线粒体代谢对核移植胚胎发育的影响一直是人们关心的重点，但是未能取得满意的研究结果。已经有研究报道，线粒体代谢产物可以参与细胞染色质表观修饰来影响基因表达。 申请人前期研究发现，牛IVF胚胎中线粒体融合基因（Mfn1）表达水平高于牛SCNT胚胎，并且过表达牛克隆胚胎Mfn1基因可以提高SCNT胚胎囊胚发育效率。但是，Mfn1调控牛SCNT胚胎囊胚发育率的机制还不清楚。本项目拟构建Mfn1基因过表达和敲减载体转染体细胞，同时构建体细胞克隆胚胎，在细胞水平和克隆胚胎水平检测线粒体代谢水平的变化，结合单细胞RNA-seq测序技术手段研究代谢产物对于克隆胚胎基因表达和染色质表观重编程的影响，揭示Mfn1基因如何通过调控线粒体代谢而影响牛SCNT胚胎ZGA发生以及对克隆胚胎发育的影响。本项目对深入了解牛核移植胚胎重编程过程中线粒体代谢物参与表观修饰的作用具有重要意义。

The effect of mitochondrial metabolism on embryo development in somatic cell nuclear transfer(SCNT) has always been the focus of attention, but the results are not satisfactory. Research has been reported that the epigenetic modification of chromatin is regulated by mitochondrial metabolites. Our previous research found that the expression of Mfn1 in bovine IVF embryos was higher than that of SCNT embryos, and bovine SCNT embryo blastocysts rate could be improved by overexpressing of bovine Mfn1 gene. However, the mechanism by which Mfn1 regulates the development of bovine SCNT embryos is still unknown. Here, in this project, we intend to construct the overexpression and knockdown vector of Mfn1 gene to transfect the somatic cells, at the same time construct the somatic cell cloned embryos, detect the difference of mitochondrial metabolism level in cells and cloned embryos. Besides, we intend to use single-cell RNA-seq sequencing, qPCR, and immunofluorescence to explore the mechanism of the chromatin epigenetic modification of bovine SCNT zygotic genome activation stage embryos, thus promoting our understanding on the reprogramming mechanism of bovine somatic cell nuclear transfer embryos.