利用同源重组技术构建Hoxa5、BMP6真核表达载体及共转染成纤维细胞表达  

Construction of Eukaryotic Expression Vector of Hoxa5,BMP6 Gene by Homologous Recombination and Its Expression in Fibroblasts

作　　者：李晶[1,2] 张梦瑶 刘开东 柳楠 贺建宁[1] LI Jing;ZHANG Mengyao;LIU Kaidong;LIU Nan;HE Jianning(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China;Qufu Animal Husbandry and Veterinary Technical Service Center,Qufu 273100,China;Qingdao Institute of Animal Husbangdry and Vetrrinary, Qingdao 266121, China)

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]曲阜市畜牧兽医技术服务中心,山东曲阜273100 [3]青岛畜牧兽医研究所,山东青岛266121

出　　处：《华北农学报》2020年第4期187-194,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：旨在利用同源重组构建敖汉细毛羊Hoxa5、BMP6基因表达载体及共转染成纤维细胞后通过基因表达量变化研究两基因之间的相互作用关系。以敖汉细毛羊为研究对象,采集30日龄的胎羊。首先,通过RNA的提取反转录成cDNA参照GenBank中Hoxa5、BMP6基因序列和pcDNA3.1序列分别设计1对含有同源臂的引物,通过PCR反应扩增获得Hoxa5、BMP6基因片段、利用SoSo试剂盒将目的片段连接到pcDNA3.1真核表达载体上。鉴定构建pcDNA3.1-Hoxa5、pcDNA3.1-BMP6重组表达载体,转化大肠杆菌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,并将构建的质粒pcDNA3.1-Hoxa5、pcDNA3.1-BMP6共转染至成纤维细胞,采用RT-PCR技术和Western Blot技术检测Hoxa5、BMP6基因单转染和共转染在成纤维细胞中表达量的变化。结果显示:经酶切、测序鉴定质粒pcDNA3.1-Hoxa5、pcDNA3.1-BMP6构建成功,并且共转染成纤维细胞Hoxa5基因的表达量极显著下降,BMP6基因的表达量极显著升高且共转染组的表达量极显著地高于单转染组(P<0.01)。利用同源重组技术成功构建了敖汉细毛羊Hoxa5、BMP6基因的表达载体,并且成功共转染成纤维细胞,BMP6基因的表达量升高,Hoxa5基因的表达量极显著下降,因而初步推断基因BMP6抑制了Hoxa5基因的表达,相反地Hoxa5基因促进了BMP6基因的表达,结果可为进一步研究其功能奠定基础。The study aimed to use homologous recombination build Hoxa5 and BMP6 genes expression vector study the interaction between Hoxa5 and BMP6 genes by change of expression.Firstly,a pair of primers that have homologous arm were desigened by RNA extraction and referred Hoxa5 and BMP6 genes sequence information of Aohan Fine Wool Sheep in GenBank,the Hoxa5 and BMP6 genes fragment were amplified by PCR.We used SoSo kit connect the target fragment to pcDNA3.1 eukaryotic expression vector.Recombined plasimd pcDNA3.1-Hoxa5 and pcDNA3.1-BMP6 were constructed and transformed into E.coli DH5αcompetent cell.Then plasmid pcDNA3.1-Hoxa5,pcDNA3.1-BMP6 was cotransfected into fibroblasts,and used RT-PCR to detect the expression level of these genes.As the results showed that,pcDNA3.1-Hoxa5,pcDNA3.1-BMP6 plasmid cotransfected successfully and identified by enzyme and sequencing.The expression level of Hoxa5 gene in fibroblasts was lower than the control group and the expression level of BMP6 gene in fibroblasts was higher than the control group.The plasmid was constructed and cotransfected into fibroblasts successfully by homologous recombination technique.The expression level of BMP6 gene increased significantly,and the expression of Hoxa5 gene decreased.Therefore,we can concluded that BMP6 gene inhibited the expression of Hoxa5 gene,and Hoxa5 gene promoted the expression of BMP6 gene.These results would lay a foundation for the further research.

关 键 词：同源重组 成纤维细胞 Hoxa5、BMP6质粒共转染 RT-PCR Western Blot 

分 类 号：S826[农业科学—畜牧学] Q78[农业科学—畜牧兽医]