过表达KDM4家族基因对猪克隆胚胎体外发育效率的影响  

Effects of overexpression of KDM4 family genes on developmental efficiency of cloned porcine embryos in vitro

作　　者：梁雅琳 邝哲 李紫聪 王兴旺 LIANG Yalin;KUANG Zhe;LI Zicong;WANG Xingwang(Department of Animal Genetics Breeding and Reproduction,College of Animal Science,South China Agricultural University,Guangzhou 510642,China;National Engineering Research Center for Breeding Swine Industry,Guangzhou 510642,China;National and Local Joint Engineering Research Center for Breeding Animal Industry,Guangzhou 510642,China;Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding,Guangzhou 510642,China;Department of Animal Science,Guangdong Maoming Agricultural&Forestry Technical College,Maoming 525000,China)

机构地区：[1]华南农业大学动物科学学院动物遗传育种与繁殖系,广州510642 [2]国家生猪种业工程技术研究中心,广州510642 [3]畜禽育种国家地方联合工程研究中心,广州510642 [4]广东省农业动物基因组学与分子育种重点实验室,广州510642 [5]广东茂名农林科技职业学院动物科学系,广东茂名525000

出　　处：《黑龙江畜牧兽医》2023年第13期55-62,135,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：广东省云浮市科技计划项目(2021020601);广东省科技厅“广东特支计划”本土创新创业团队项目(2019BT02N630)。

摘　　要：为了探究过表达H3K9me3的组蛋白去甲基化酶4(KDM4)家族基因对猪克隆胚胎体外发育效率的影响,试验采用pc-KDM4A~D质粒构建、酶切鉴定、细胞转染、定量PCR扩增及体外转录等方法制备过表达KDM4A~D的猪胎儿成纤维细胞和KDM4A~D mRNAs,通过体细胞核移植和显微注射的方式制备过表达KDM4A~D的猪克隆胚胎。将供体细胞过表达KDM4A~D的猪克隆胚胎分为1个对照组和5个试验组,分别为pcDNA3.1(+)组、pc-KDM4A组、pc-KDM4B组、pc-KDM4C组、pc-KDM4D组及4质粒组;将注射KDM4A~D mRNA的猪克隆胚胎分为1个对照组和5个试验组,分别为H_(2)O组、KDM4A组、KDM4B组、KDM4C组、KDM4D组及4mRNA组。统计各组的卵裂数、囊胚数和囊胚细胞数;利用免疫荧光检测4质粒组和4mRNA组4-细胞期猪克隆胚胎中H3K9me3的表达水平。结果表明:pc-KDM4A~D质粒构建成功,质粒转染的猪胎儿成纤维细胞可过表达KDM4A~D和降低细胞内H3K9me3水平。与pcDNA3.1(+)组比较,4质粒组可显著提高猪克隆胚胎的卵裂率和囊胚率(P<0.05),并显著降低4-细胞期猪克隆胚胎的H3K9me3水平(P<0.05);与H_(2)O组比较,4mRNA组可显著提高猪克隆胚胎的囊胚率(P<0.05),并显著降低4-细胞期猪克隆胚胎的H3K9me3水平(P<0.05)。说明过表达KDM4A~D可有效降低猪克隆胚胎中的H3K9me3水平并提高猪克隆胚胎的体外发育能力。To investigate the effects of overexpression of H3K9me3 demethylase KDM4 family genes on development competence of porcine cloned embryos in vitro,the porcine fetal fibroblasts overexpressing KDM4A-D and the KDM4A-D mRNAs were prepared by pc-KDM4A-D plasmid construction,enzyme digestion identification,cell transfection,quantitative PCR and in vitro transcription,and the cloned porcine embryos overexpressing KDM4A-D were prepared by somatic cell nuclear transfer and microinjection.The cloned porcine embryos with donor cells overexpressing KDM4A-D were divided into 1 control group and 5 experimental groups,namely pcDNA3.1(+) group,pc-KDM4A,pc-KDM4B group,pc-KDM4C group,pc-KDM4D group and 4 plasmid group;the cloned porcine embryos injected with KDM4A-D mRNAs were divided into 1 control group and 5 experimental groups,namely H_(2O) group,KDM4A group,KDM4B group,KDM4C group,KDM4D group and 4mRNA group.The number of cleavage embryo,blastocyst and blastocyst cell in each group were measured.Then the expression of H3K9me3 in 4-cell stage cloned porcine embryos in the 4 plasmid group and 4mRNA group were detected by immunofluorescence.The results showed that pc-KDM4A-D plasmids were constructed successfully and the fetal fibroblasts transfected with pc-KDM4A-D plasmids could overexpress KDM4A-D and reduce the expression of H3K9me3.Compared with pcDNA3.1(+) group,the cleavage rate and blastocyst rate of cloned porcine embryos in the 4 plasmid group were significantly increased(P<0.05),and the expression of H3K9me3 in the 4-cell stage cloned porcine embryos was significantly decreased(P<0.05).Compared with H_(2O) group,the blastocyst rate in the 4 mRNA group was significantly increased(P<0.05),and the expression of H3K9me3 in the 4-cell stage cloned porcine embryos was significantly decreased(P<0.05).The study indicated that the overexpression of KDM4A-D could effectively remove H3K9me3 in cloned porcine embryos and improve development competence of cloned porcine embryos in vitro.

关 键 词：组蛋白去甲基化酶4(KDM4) H3K9me3 克隆胚胎 重编程 猪 

分 类 号：S813.3[农业科学—畜牧学]