广西巴马小型猪miR-148b慢病毒制备及靶基因通路预测分析  

Construction of miR-148b lentivirus vector and prediction of target gene pathways in Guangxi Bama mini-pig

作　　者：张名媛 郭晓萍[1,2] 孙晴超 司景磊[2] 兰干球 郭亚芬 蒋钦杨[2] ZHANG Ming-yuan;GUO Xiao-ping;SUN Qing-chao;SI Jing-lei;LAN Gan-qiu;GUO Ya-fen;JIANG Qin-yang(Laboratory Animal Center,Guangxi Medical University,Nanning 530021,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西医科大学实验动物中心,南宁530021 [2]广西大学动物科学技术学院,南宁530004

出　　处：《南方农业学报》2019年第7期1596-1604,共9页Journal of Southern Agriculture

基　　金：国家重点研发计划项目（2017YFD0501600）;广西青年科学基金项目（2018GXNSFBA281066）;广西高校中青年教师科研基础能力提升项目（2019KY0151)

摘　　要：【目的】构建广西巴马小型猪miR-148b慢病毒表达载体并对其靶基因进行预测及相关通路分析,为揭示miR-148b的作用机制打下基础。【方法】以广西巴马小型猪血液基因组DNA为模板,扩增miR-148b的前体及部分侧翼序列,使用T4连接酶将目的片段连接至pLV-CMV-MCS-EF1载体上,构建获得pLV-miR-148b慢病毒表达载体;以pLVmiR-148b慢病毒表达载体转染293T细胞48 h后进行表达验证;利用miRDB、miRWalk 2.0和TargetScan 7.2三大数据库预测分析广西巴马小型猪miR-148b的靶基因,并以MirPath v.3分析miR-148b靶基因相关通路。【结果】广西巴马小型猪miR-148b片段大小为385 bp,与miRBase 21.0网站上已发布猪miR-148b成熟序列(前体和侧翼序列)的同源性达100%。构建获得的pLV-miR-148b慢病毒表达载体能在293T细胞中成功表达,且相对表达量极显著高于pLV-GFP慢病毒空载质粒转染组(P<0.01)。数据库预测分析得到18个广西巴马小型猪miR-148b的靶基因,分别为CLOCK(时间昼夜调控因子)、MIER1(转录调控因子)、MECP2(甲基化CpG结合蛋白)、ZNF704(锌指蛋白)、SPTY2D1(SPT2染色质蛋白)、QKI(RNA结合蛋白)、ZBTB20(锌指蛋白)、MGAT4A(钠/磷酸转运蛋白)、UHMK1(编码蛋白激酶)、PIK3C2A(磷酸肌醇-3-激酶2α肽)、C6orf62(6号染色体开放阅读框62抗体)、HECW2(E3泛素蛋白连接酶)、MAP1B(微管相关蛋白)、NHS(NHS肌动蛋白调节因子)、B4GALT6(β-1,4-半乳糖基转移酶)、ATP2A2(肌浆ATP酶/内质网Ca2+转运酶)、AP4E1(蛋白复合物接头分子)和BBX(锌指蛋白);且发现有11条靶基因相关通路,其中与疾病相关的通路有脂肪酸合成通路、细胞外基质受体相互作用通路、癌症的蛋白聚糖通路、神经胶质瘤通路、癌症的小分子核糖核酸通路、肾细胞癌通路、N-聚糖生物合成通路和致心律失常性右室心肌病通路。【结论】广西巴马小型猪miR-148b慢病毒表达载体能在293T细胞中成功表达,miR-148b存【Objective】The aims of this study was to construct the lentiviral vector of miR-148b and predict the target gene of miR-148b and relevant pathways,which would lay a foundation for revealing the mechanism of miR-148b.【Method】miR-148b precursor and partial flanking sequences were amplified using Guangxi Bama mini-pig blood genomic DNA as template,and then miR-148b sequence was inserted into pLV-CMV-MCS-EF1 vector by T4 ligase to construct the lentiviral expression vector of pLV-miR-148b.The pLV-miR-148b lentiviral expression vector was transfected into 293T cells for 48 h for expression verification.Three major databases,miRDB,miRWalk 2.0 and TargetScan 7.2,were used to analyze and predict the target genes of mirR148b,and MirPath v.3 was used to analyze the pathways related to miR-148b target gene.【Result】The fragment size of miR-148b of Guangxi Bama mini-pig was 385 bp,and its homology of miR-148b mature sequence(precursor and flank sequence)of pig published on miRBase 21.0 website was up to 100%.The constructed pLV-miR-148b lentivirus expression vector was successfully expressed in 293T cells,and the relative expression level was extremely higher than that of the pLV-GFP lentivirus no-load plasmid transfection group(P<0.01).There were 18 target genes of miR-148b in Guangxi Bama mini-pig obtained by database prediction analysis,which were CLOCK(day and night time control factors),MIER1(transcription regulatory factors),MECP2(methylation CpG binding protein),ZNF704(zinc finger protein),SPTY2D1(SPT2 chromatin proteins),QKI(RNA binding protein),ZBTB20(zinc finger protein),MGAT4A(sodium/phosphate transporters),UHMK1(encoding protein kinase),PIK3C2A(phosphoinositide-3-kinase 2 alpha peptide),C6orf62(chromosome 6 open reading frame 62 antibodies),HECW2(E3 ubiquitin protein connection enzyme),MAP1B(microtubule-related protein),NHS(NHS actin regulatory factor),B4GALT6(beta-1,4-galactosyl transferase),ATP2A2(sarcoplasmic atpase/endoplasmic reticulum Ca2+transporter),AP4E1(protein complex connector molecule)and

关 键 词：广西巴马小型猪 miR-148b 慢病毒载体 靶基因 相关通路预测 

分 类 号：S828.89[农业科学—畜牧学]