NOS2基因DNA甲基化编辑调节NO浓度影响肌肉发育通路基因的表达  

Regulation of NO Concentration by NOS 2 Gene DNA Methylation Editing Affects the Expression of Muscle Development Pathway Genes

作　　者：申琦 王凯 赵真坚 陈栋 余杨 崔晟頔 王俊戈 陈子旸 吴平先 唐国庆[1,2,3] SHEN Qi;WANG Kai;ZHAO Zhenjian;CHEN Dong;YU Yang;CUI Shengdi;WANG Junge;CHEN Ziyang;WU Pingxian;TANG Guoqing(Key Laboratory of Livestock and Poultry Multi-omics of Ministry of Agriculture and Rural Affairs,College of Animal Science and Technology,Sichuan Agricultural University,Chengdu 611130,China;Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province,Sichuan Agricultural University,Chengdu 611130,China;State Key Laboratory of Swine and Poultry Breeding Industry,College of Animal Science and Technology,Sichuan Agricultural University,Chengdu 611130,China;New Hope Liuhe Co.Ltd.,Beijing 100102,China;National Center of Technology Innovation for Pigs,Chongqing 402460,China)

机构地区：[1]四川农业大学动物科技学院农业农村部畜禽生物组学重点实验室,成都611130 [2]四川农业大学畜禽遗传资源发掘与创新利用四川省重点实验室,成都611130 [3]四川农业大学动物科技学院猪禽种业全国重点实验室,成都611130 [4]新希望六和股份有限公司,北京100102 [5]国家生猪技术创新中心,重庆402460

出　　处：《畜牧兽医学报》2024年第3期984-994,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家生猪技术创新中心先导科技项目(NCTIP-XD/B01);四川省科技厅项目(2020YFN0024,2021ZDZX0008,2021YFYZ0030);四川省猪创新团队项目(sccxtd-2022-08)。

摘　　要：旨在探究对一氧化氮合成酶(NOS)2进行DNA甲基化编辑后对肌红蛋白的调控作用机制,并研究其对神经元一氧化氮合酶(nNOS)和内皮细胞一氧化氮合酶(eNOS)的表达水平是否有影响。本试验采用DNA甲基化编辑技术在猪肾细胞系PK-15细胞对基因NOS2进行甲基化编辑,在猪肾细胞中检测NOS2基因启动子DNA甲基化水平、NOS2表达水平、细胞内一氧化氮浓度以及肌纤维和肌红蛋白相关基因的表达。结果发现,NOS2基因启动子前500 bp区域平均DNA甲基化水平降低,但差异不显著,而gRNA2结合位点附近的CpG位点甲基化水平增高。此外,对NOS2进行甲基化编辑后,NOS2基因表达量和其iNOS蛋白质表达量显著降低。随着NOS2表达降低,nNOS和eNOS表达显著增加。MYHC-Ⅱb和MYHC-Ⅱx的表达水平显著下降,Myoglobin蛋白表达也显著下降。综上,NOS2基因的异常表达能够影响细胞中的NO生成,同时也会影响NOS1和NOS3的表达,以及和肌肉发育相关基因的表达。This study aimed to explore the regulation mechanism of DNA methylation editing of nitric oxide synthase(NOS)_(2) on Myoglobin,and study its effect on the expression level of neuronal nitric oxide synthase(nNOS)and endothelial nitric oxide synthase(eNOS).In this experiment,DNA methylation editing technology was used to methylate NOS2 in porcine kidney cell line PK-15 cells,and the DNA methylation level of the NOS_(2) gene promoter,the expression level of NOS_(2),the concentration of intracellular nitric oxide,as well as the expression level of myofibrillar-and myoglobin-related genes were detected in porcine kidney cells.The results revealed that the average DNA methylation level of the first 500 bp region of the NOS_(2) gene promoter was reduced,but the difference was not significant,while the methylation level of the CpG site near the gRNA2 binding site was increased.In addition,after DNA methylation editing of NOS_(2) gene,NOS_(2) gene expression and its iNOS protein expression were significantly reduced.With the decrease of NOS_(2) expression,nNOS and eNOS expression increased significantly,the expression level of MYHC-Ⅱb and MYHC-Ⅱx decreased significantly,and the expression of Myoglobin protein decreased significantly.In conclusion,the abnormal expression of the NOS_(2) gene can affect the generation of NO in cells,as well as the expression of NOS 1 and NOS_(3),and the expression of genes related to muscle development.

关 键 词：NOS2 DNA甲基化 NO 肌纤维 肌红蛋白 

分 类 号：S828.2[农业科学—畜牧学]