DNMT1基因干扰对猪体细胞克隆胚胎发育、基因转录和DNA甲基化的影响  

Effect of DNMT1 Interference on Development, Gene Transcription and DNA Methylation of Porcine(Sus scrofa) Cloned Embryos

作　　者：许卫华[1,2] 李紫聪 吴珍芳 石俊松 XU Wei-Hua;LI Zi-Cong;WU Zhen-Fang;SHI Jun-Song(College of Life Science,Longyan University,Longyan 364012,China;Fujian Provincial Key Laboratory of Preventive Veterinary Medicine and Biotechnology,Longyan 364012,China;National Engineering Research Center for Breeding Swine Industry,Guangzhou 510642,China;Institute of Wen's Group,Xinxing 527400,China)

机构地区：[1]龙岩学院生命科学学院,龙岩364012 [2]福建省家畜传染病防治与生物技术重点实验室,龙岩364012 [3]国家生猪种业工程技术研究中心,广州510642 [4]广东省温氏集团研究院,新兴527400

出　　处：《农业生物技术学报》2018年第12期2075-2083,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31772554);福建省自然科学基金资助项目(No.2015J01620);福建省中青年教师教育科研项目(No.JA14306);龙岩学院引进人才科研启动经费(No.LB2014025)

摘　　要：相对于体外受精胚胎,猪(Sus scrofa)体细胞克隆胚胎DNA甲基转移酶1(DNA (cytosine-5)-methyltransferase 1, DNMT1)呈现延迟降解的现象。本研究通过向单细胞期重构胚胎注射抗DNMT1siRNA,记录胚胎卵裂率、囊胚发育率以及囊胚期细胞总数,并提取四细胞期和囊胚期胚胎总RNA和DNA,对发育重要相关基因mRNA水平和DNA甲基化水平进行检测。结果显示,虽然抑制DNMT1未能显著提高猪体细胞克隆胚胎体外发育性能(P>0.05),但50μmol/L干扰组囊胚发育率(30.43%)相对于阴性对照组(23.01%)和未注射组(25.74%)有上升的趋势(P=0.19);在基因转录方面,注射抗DNMT1 siRNA显著提高了四细胞期胚胎中POU结构域5类转录因子1 (POU domain, class 5, transcription factor 1,POU5F1)基因和囊胚中NANOG基因的表达水平(P<0.05),也显著上调了囊胚期DNMT1转录水平(P<0.05);在DNA甲基化方面,注射抗DNMT1 siRNA促进了四细胞期POU5F1基因5'-UTR区域DNA的去甲基化和降低了囊胚期整体甲基化水平;然而DNMT1的过度抑制破坏了非编码RNA H19基因印迹的DNA甲基化状态。本研究表明,温和抑制克隆胚胎中过高的DNMT1 mRNA水平,促进了多能基因的表达和DNA去甲基化重编程,该结果对研究DNMT1在早期胚胎生成过程中的作用机制提供了基础资料。Delay in maternal transcript degradation of DNMT1(DNA(cytosine-5)-methyltransferase1 cytosine-5)-methyltransferase 1) in porcine(Sus scrofa) somatic cell nuclear transfer embryos compared with in vitro fertilized counterparts was confirmed, thereafter anti-DNMT1 siRNA was injected into embryos at onecell stage to evaluate the effect of DNMT1 knockdown on the developmental rate of embryos and the mRNA levels and DNA methylation levels of several developmentally important genes. Though a higher proportion ofblastocysts was obtained in 50 μmol/L siRNA injected embryos(30.43%) compared with mock injected embryos(23.01%) and untreated controls(25.74%), while the difference was not statistically significant(P=0.19). In the case of genes expression, inhibition of DNMT1 significantly increased the mRNA levels of POU5 F1 at four-cell stage and of NANOG at the blastocyst stage(P<0.05). In the aspect of DNA methylation,inhibition of DNMT1 reduced the DNA methylation level of POU5 F1 at four-cell stage and decreased the general methylation to the level of in vivo produced embryos at the blastocyst stage, whereas over-depressed DNMT1 disrupted the imprinted methylation of a long noncoding RNA H19. This study suggests that mild inhibition of over-expressed DNMT1 facilitates pluripotent genes transcription and demethylation reprogramming of porcine cloned embryos, furthermore provides a scientific reference for further research on function mechanisms of DNMT1 during early embryogenesis.

关 键 词：猪体细胞克隆胚胎 DNA甲基转移酶1基因(DNMT1) siRNA 基因转录 DNA甲基化 

分 类 号：S828[农业科学—畜牧学]