RNA的m6A甲基化修饰调控猪卵母细胞减数分裂恢复及核移植胚胎发育的机制

N6-methyl-adenosine(m6A) is an ubiquitous RNA methylation pattern in tRNA and rRNA. Recently, it was found in mammalian mRNA and non-coding RNA, and methylase and demethylase of m6A were identified also, which raise the possibility that m6A methylation in mRNA may as important as 5-methylcytosine modification and histone modifications on DNA. Oocytes in vitro maturation of is an upstream step of in vitro embryo production. Porcine oocytes after in vitro maturation is required by research on cloning pigs and transgenic pigs, and its quality determines efficiency of nuclear transfer. Nuclear transfer is an important means for research on cloning and reprogramming, but no predecessors combined RNA methylation studies with oocyte maturation, reprogramming and embryonic development. The project was designed to use RNA interfering method to knock-down m6A methylase and demethylase, by immunofluorescence, immunoprecipitation coupled RT-PCR, RNA hybridization to study its effects on porcine oocytes meiotic resumption, fertilizaiton, reprogramming and embryonic development after nuclear transfer. The project not only opens a a window for RNA epigenetics, but also combines transcriptome with epigenetics in pigs. while the research on the mechanism of reprogramming, it is meaningful for animal reproduction and infertility.

6-甲基腺嘌呤（m6A）是普遍存在的tRNA、rRNA甲基化形式。最近发现其也存在于哺乳动物mRNA和非编码RNA，并且鉴定了m6A的甲基化酶和去甲基化酶，证明mRNA的m6A甲基化修饰与DNA的5甲基胞嘧啶修饰及组蛋白的修饰同等重要。体外培养成熟的猪卵母细胞是研究克隆猪和转基因猪所需受体卵母细胞的主要来源，其质量决定核移植胚胎发育效率。核移植是研究克隆和重编程的重要手段，但未见前人将RNA甲基化与卵母细胞成熟、重编程、胚胎发育结合起来研究。本项目率先研究RNA的m6A甲基化，拟采用RNA干扰的方法，敲低m6A甲基化酶和去甲基化酶，通过免疫荧光、免疫沉淀结合RT-PCR、RNA杂交等方法研究其对猪卵母细胞减数分裂恢复、受精、核移植后重编程及猪核移植胚胎发育的影响。本项目将猪转录组学与表观遗传学结合，是研究RNA表观遗传学的窗口，在研究重编程机制的同时，对动物繁殖和不孕不育研究也有借鉴意义。