长链非编码RNA调控山羊体细胞核移植胚胎合子基因激活期重编程的研究

Long noncoding RNA (lncRNA) are a group of endogenous RNA molecules that exceed 200nt in length and lack the ability to encode proteins. Generally, they regulate gene expression through DNA methylation and histone modification. Current studies revealed that lncRNAs play vital roles in zygotic gene expression regulation in mice zygote as well as in induced pluripotent stem cell reprogramming processes. Therefore it is hypothesized that lncRNA may also involve in reprogramming by somatic cell nuclear transfer（SCNT）induced zygotic genome activation (ZGA). To confirm this hypothesize and further investigate the mechanism of reprogramming by SCNT, first, single cell transcriptome analysis will be used to pool differentially expressed lncRNA at goat ZGA stage. Then, key differentially expressed lncRNA will be selected using RNA immunoprecipitation. Furthermore, overexpress or loss of function of the selected lncRNA will be introduced to somatic cell nuclear transferred embryos. Embryo development and their epigenetic modification will be studied and DNA microarray will be used to investigate target genes of lncRNA during reprogramming processes. Taken together, the aim of this study is to nail down lncRNAs that involve in somatic cell nuclear transfer induced reprogramming process and to investigate their regulatory mechanism on reprogramming process. This research will build a foundation to overcome SCNT generated developmental defects, it also will increase the efficiency of embryo development and transgenic animal production. Moreover, this study will be a valuable addiction to embryo development biology theory.

长链非编码RNA（lncRNA）是一类长度超过200nt、不编码蛋白的RNA，可通过DNA甲基化和组蛋白修饰影响基因表达，在调控胚胎合子基因表达和诱导多能干细胞的重编程过程中具有重要作用。然而，lncRNA在山羊核移植胚胎合子基因激活期重编程的作用及其机制尚无报道。因此，本研究利用单细胞转录组测序和RNA免疫沉淀手段寻找与山羊核移植胚胎合子基因激活期重编程有关的差异表达lncRNA，利用显微注射技术将关键差异表达lncRNA的过表达载体或反义寡核苷酸注入核移植胚胎中观察胚胎发育状态，分析表观遗传修饰改变，并利用基因芯片找出lncRNA的靶基因。通过这些研究，找出调控核移植胚胎合子基因激活期重编程的关键lncRNA，探讨lncRNA调控核移植胚胎重编程的机制，不仅可以改善核移植胚胎不完全重编程的缺陷，提高核移植胚胎发育能力和克隆动物生产效率，而且可以丰富动物胚胎发育生物学的相关理论。

长链非编码RNA（lncRNA）是一类长度超过200nt、不编码蛋白的RNA，可通过DNA甲基化和组蛋白修饰影响基因表达，在调控胚胎合子基因表达和诱导多能干细胞的重编程过程中具有重要作用。然而，lncRNA在山羊核移植胚胎重编程中的作用及其调控机制仍不清楚。本项目以山羊为试验动物，采用单细胞转录组测序等手段筛选出核移植胚胎合子基因激活期调控重编程的关键lncRNA，并利用干扰试验和转录组测序技术深入探讨lncRNA调控体细胞核移植胚胎重编程的机制。项目（1）确定了山羊合子基因组激活发生时期为8细胞期；（2）阐明了lncRNA在山羊IVF和核移植胚胎合子基因组激活过程中的表达规律，筛选到差异表达lncRNA并研究了lnc_137和lncRNA Xist在山羊胚胎发育过程中的作用；（3）解析了组蛋白甲基化和DNA甲基化在山羊合子基因组激活期核移植胚胎的表观遗传变化；（4）阐明了山羊核移植胚胎转录组重编程的规律，发现Kdm5b促进山羊核移植胚胎重编程；（5）预测了靶向结合Kdm5b的lncRNA，筛选到lnc_3712参与调控山羊核移植胚胎重编程；（6）揭示了lnc_3712靶向Kdm5b调控山羊核移植胚胎重编程的分子机制。项目完成了各研究计划，实现了研究目标，研究结果已正式发表SCI论文11篇、中文期刊1篇，培养博士研究生5名。研究成果不仅丰富了山羊生殖生理学的理论研究，而且对于改善大动物核移植胚胎不完全重编程的缺陷，提高核移植胚胎发育能力和克隆动物生产效率具有重要意义。