过表达Kdm4d对山羊孤雌胚胎基因组激活期Xist表达的影响  

Effect of overexpression of Kdm4d on Xist expression in goat parthenogenetic embryos during embryo genome activation

作　　者：杨瑛楠 蔡玉 刘孜斐 李东旭 万永杰 王锋 邓明田 YANG Yingnan;CAI Yu;LIU Zifei;LI Dongxu;WAN Yongjie;WANG Feng;DENG Mingtian(Livestock Embryo Engineering Laboratory,College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物科技学院江苏省家畜胚胎工程实验室,江苏南京210095

出　　处：《南京农业大学学报》2022年第6期1227-1234,共8页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金青年基金项目(32102546);江苏省基础研究计划青年基金项目(BK20200563)。

摘　　要：[目的]本试验旨在探究过表达赖氨酸特异去甲基化酶4D(Kdm4d)对山羊孤雌胚胎基因组激活期(8细胞期)Xist表达的影响。[方法]通过构建Kdm4d-IRES-EGFP重组质粒,体外转录Kdm4d mRNA,再利用显微注射、免疫荧光和亚硫酸氢盐测序等方法,分析过表达Kdm4d对山羊孤雌胚胎基因组激活期(8细胞期)Xist表达的影响。[结果]与对照组相比,组蛋白H3K9甲基化(H3K9me3)水平在2~8细胞期和囊胚期过表达Kdm4d的山羊孤雌胚胎中均显著降低,过表达Kdm4d山羊的4、8细胞期胚胎和囊胚的发育潜能无显著变化;Xist表达水平在过表达Kdm4d的山羊8细胞期孤雌胚胎中显著升高,但Xist启动子仍维持高甲基化,与对照组相比无显著变化。[结论]Kdm4d能有效移除孤雌胚胎中H3K9me3修饰,促进Xist表达,但不影响Xist启动子DNA甲基化水平和山羊孤雌胚胎发育潜能。本研究为深入理解Kdm4d调控山羊早期胚胎X染色体失活的机制奠定了基础。[Objectives]The experiment was conducted to investigate the effect of Kdm4d mRNA injection on the expression of Xist in goat parthenogenetic embryo(PE).[Methods]Kdm4d mRNA was transcribed in vitro by constructing the recombinant plasmid Kdm4d-IRES-EGFP,and then we investigated the effect of Kdm4d mRNA injection on the expression of Xist in goat parthenogenetic embryos by using in vitro transcription,microinjection,immunostaining,and bisulfite sequencing.[Results]The results revealed that the signal intensity of H3K9me3 significantly decreased at the 2-to 8-cell stage embryos and blastocysts in the Kdm4d mRNA injected PE;the embryonic development rate in the 4-and 8-cell stage embryos and blastocysts was not statistically changed in the Kdm4d mRNA injected PE when compared to that of the control group;the expression level of Xist markedly increased at the 8-cell stage PE in the Kdm4d mRNA injection group.However,the methylation level in the Xist promoter remained statistical un-altered.[Conclusions]In conclusion,Kdm4d can effectively remove H3K9me3 in goat parthenogenetic embryos and significantly increase the expression level of Xist,but does not affect the developmental potential of goat parthenogenetic embryos.This study lays the foundation for an in-depth understanding of the mechanism by which Kdm4d regulates X chromosome inactivation in early goat embryos.

关 键 词：Kdm4d XIST 孤雌胚胎 胚胎基因组激活 山羊 

分 类 号：S827[农业科学—畜牧学]