mtr-miR156a在奶牛乳腺上皮细胞的靶基因筛选鉴定及其调控奶牛乳蛋白生物合成的研究  

Screening and Identification of Target Genes of mtr-miR156a in Bovine Mammary Gland Epithelial Cells and Its Regulation on Milk Protein Biosynthesis in Dairy Cows

作　　者：刘宝宝 贾晶莹 孟桂智 刘祖江 马云 蔡小艳 LIU Baobao;JIA Jingying;MENG Guizhi;LIU Zujiang;MA Yun;CAI Xiaoyan(School of Agricultural,Ningxia University,Yinchuan 750021,China;Key Laboratory of Ruminant Molecular Cell Breeding,Ningxia Hui Autonomous Region,Yinchuan 750021,China)

机构地区：[1]宁夏大学农学院,银川750021 [2]宁夏回族自治区反刍动物分子细胞育种重点实验室,银川750021

出　　处：《动物营养学报》2023年第11期7355-7367,共13页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金项目(31960680);宁夏科技厅优秀青年项目(2022AAC05012)。

摘　　要：本试验旨在探究苜蓿miR156a(mtr-miR156a)在奶牛乳腺上皮细胞(BMECs)的靶基因及其对乳蛋白合成的调控作用,为进一步研究外源性植物微小RNA(miRNA)调控奶牛乳蛋白合成提供研究基础。首先通过联川生物云平台、TargetScan和RNAhybrid在线网站预测和筛选出mtr-miR156a调控蛋白合成的候选靶基因,然后构建候选靶基因丝氨酸/苏氨酸蛋白磷酸酶2A 56 kDa调节亚基delta亚型(PPP2R5D)和磷酸肌醇3激酶调控亚单位2(PIK3R2)的重组质粒,并通过双荧光素酶验证鉴定出靶向基因,最后运用实时荧光定量PCR(qRT-PCR)和酶联免疫吸附试验(ELISA)检测过表达mtr-miR156a对BMECs乳蛋白合成相关基因表达以及酪蛋白编码基因mRNA和蛋白表达的影响。结果表明:1)双荧光素酶报告基因和qRT-PCR鉴定了mtr-miR156a可以靶向结合PPP2R5D和PIK3R2并极显著降低其表达水平(P<0.01)。2)与mimic NC相比,过表达mtr-miR156a极显著降低丙酮酸脱氢酶激酶1(PDK1)、核糖体蛋白S6激酶beta-1(S6K1)、真核翻译起始因子4B(eIF4B)、核糖体蛋白S6(RPS6)、结节性硬化症复合物亚基2(TSC2)、脑富集Ras同源物(RHEB)、哺乳动物雷帕霉素靶蛋白(mTOR)和真核翻译起始因子4E(eIF4E)相对表达量(P<0.01),极显著提高蛋白激酶B1(Akt1)和真核翻译起始因子4E结合蛋白1(EIF4EBP1)相对表达量(P<0.01)。3)与mimic NC处理相比,过表达mtr-miR156a极显著提高酪蛋白alpha S1(CSN1S1)、酪蛋白alpha S2(CSN1S2)、酪蛋白beta(CSN2)和酪蛋白kappa(CSN3)相对表达量(P<0.01)。4)ELISA结果表明,与mimic NC处理相比,过表达mtr-miR156a显著提高BMECs培养液上清液中α酪蛋白和β酪蛋白含量(P<0.05),极显著提高上清液中κ酪蛋白含量(P<0.01)。综上所述,mtr-miR156a通过靶向结合位于磷脂酰肌醇-3-羟激酶(PI3K)/蛋白激酶B(Akt)/mTOR信号通路的PPP2R5D和PIK3R2,调控PI3K/Akt/mTOR信号通路PDK1、S6K1、eIF4B、RPS6、Akt1、TSC2、RHEB、mTOR、eIF4E和EIF4EBP1等基因表达,�The aim of this experiment was to explore the target genes of alfalfa miR156a(mtr-miR156a)in bovine mammary gland epithelial cells(BMECs)and its regulation on milk protein synthesis,so as to provide a research basis for further study on the regulation of milk protein synthesis by exogenous plant microRNA(miRNA).Firstly,the candidate target genes of mtr-miR156a regulating protein synthesis were predicted and screened through the online websites of Lianchuan Bio-Cloud Platform,TargetScan and RNAhybrid,then the recombinant plasmids of the candidate target genes serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform(PPP2R5D)and phosphoinositide-3-kinase regulatory subunit 2(PIK3R2)were constructed,and the target genes were identified by dual-luciferase verification.Finally,the real-time fluorescence quantitative PCR(qRT-PCR)and enzyme linked immunosorbent assay(ELISA)were used to detect the effects of overexpression of mtr-miR156a on milk protein synthesis-related gene expression and casein-coding gene mRNA and protein expression in BMECs.The results showed as follows:1)dual-luciferase reporter gene and qRT-PCR identified that mtr-miR156a could target PPP2R5D and PIK3R2 and extremely significantly reduced their expression levels(P<0.01).2)Compared with mimic NC,overexpression of mtr-miR156a extremely significantly decreased the relative expression levels of pyruvate dehydrogenase kinase 1(PDK1),ribosomal protein S6 kinase beta-1(S6K1),eukaryotic translation initiation factor 4B(eIF4B),ribosomal protein S6(RPS6),tuberous sclerosis complex 2(TSC2),Ras homolog enriched in brain(RHEB),mammalian target of rapamycin(mTOR)and eukaryotic translation initiation factor 4E(eIF4E)(P<0.01),while extremely significantly increased the relative expression levels of protein kinase B1(Akt1)and eukaryotic translation initiation factor 4E binding protein 1(EIF4EBP1)(P<0.01).3)Compared with mimic NC,overexpression of mtr-miR156a extremely increased the relative expression levels of casein alpha S1(CSN1S1),casein

关 键 词：mtr-miR156a BMECs PPP2R5D PIK3R2 跨界调控 酪蛋白 

分 类 号：S811[农业科学—畜牧学]