敖汉细毛羊BMP4基因质粒的构建及在成纤维细胞中表达量的研究  

BMP4 Gene Plasmid Construction and Its Expression Amount in Fibroblasts of AoHan Fine Wool Sheep

作　　者：张梦瑶 杨峰 刘开东 刘积凤[1] 柳楠[1] 贺建宁[1] 薛明[4] ZHANG Mengyao;YANG Feng;LIU Kaidong;LIU Jifeng;LIU Nan;HE Jianning;XUE Ming(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China;Qingdao Institute of Animal Husbangdry and Vetrrinary,Qingdao 266121,China;Inner Mongolia Agricultural University,Hohhot 010018,China;National Animal Husbandry Station,Beijing 100125,China)

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]青岛畜牧兽医研究所,山东青岛266121 [3]内蒙古农业大学,内蒙古呼和浩特010018 [4]全国畜牧总站,北京100125:

出　　处：《华北农学报》2019年第6期227-234,共8页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31402047);国家绒毛用羊产业技术体系专项资金(CARS-39-05);青岛农业大学高层次人才科研基金项目(631410)

摘　　要：旨在构建敖汉细毛羊BMP4基因的质粒及转染成纤维细胞后研究基因表达量的变化。以30日龄的敖汉细毛羊胚胎为研究对象。首先,通过RNA的提取反转录成cDNA参照GenBank中BMP4基因序列信息设计1对引物,通过PCR反应扩增获得BMP4基因片段、将得到的BMP4基因连接到pEASYTM-T1载体,构建pEASYTM-T1-BMP4重组质粒并转化大肠杆菌(E.coli)DH5α感受态细胞提取质粒进行酶切鉴定。鉴定正确后构建pcDNA3.1-BMP4重组质粒,转化大肠杆菌(E.coli)DH5α感受态细胞;其次对敖汉细毛羊的成纤维细胞进行分离培养,并将重组pcDNA3.1-BMP4表达载体转染成纤维细胞,后检测BMP4基因在mRNA和蛋白水平上表达量的变化。结果显示:pcDNA3.1-BMP4构建成功后转染成纤维细胞BMP4基因的mRNA和蛋白表达量均显著上升,且转染组的表达量极显著地高于对照组(P<0.01)。成功构建了敖汉细毛羊BMP4基因的质粒,并且成功转染成纤维细胞,基因在细胞中过表达,结果可为进一步研究其功能奠定基础。This study aims to construct the expression plasmid of BMP4 gene in AoHan sheep and explore the change of gene expression after transfection of fibroblasts. 30-day old AoHan sheep embryos were selected as the research subject. Firstly, a pair of primers was designed by reverse transcribing RNA into cDNA based on the sequence of BMP4 in GenBank. The BMP4 gene fragment was amplified by PCR reaction, and was ligated into pEASYTM-T1 vector to construct pEASYTM-T1-BMP4 recombinant plasmid, which was transformed into E.coli DH5α competent cells to extract the plasmid for restriction enzyme digestion. Then the fibroblasts of AoHan sheep were isolated and cultured, and the constructed plasmid pcDNA3.1-BMP4 was transfected into fibroblasts. The expression of BMP4 gene in fibroblasts was detected by quantitative PCR and Western Blot. The results showed that plasmid pcDNA3.1-BMP4 was successfully constructed by restriction enzyme digestion and sequencing, and BMP4 gene expression was significantly increased after transfection of fibroblasts. The expression level of transfection group was significantly higher than that of the control group. The plasmid was constructed and transfected into fibroblasts successfully. The result can be a basis for further study of its function.

关 键 词：敖汉细毛羊 成纤维细胞 质粒构建 BMP4基因转染 RT-PCR Western Blot 

分 类 号：Q78[生物学—分子生物学] S826.03[农业科学—畜牧学]