敖汉细毛羊DKK1基因重组质粒的构建及其在成纤维细胞中表达量的研究  

Study on Construction of DKK1 Gene Recombinant Plasmid in Aohan Fine Wool Sheep and Its Expression in Fibroblasts

作　　者：张梦瑶 杨峰 刘开东 程明 王国义 刘积凤[1] 柳楠[1] 贺建宁[1] 

机构地区：[1]青岛农业大学动物科技学院,青岛266109 [2]青岛畜牧兽医研究所,青岛266100 [3]赤峰市敖汉旗种羊场,赤峰024300

出　　处：《中国畜牧兽医》2018年第1期131-139,共9页China Animal Husbandry & Veterinary Medicine

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-40);青岛农业大学高层次人才科研基金(631410)

摘　　要：本研究旨在构建敖汉细毛羊DKK1基因的重组质粒,并对其转染成纤维细胞后基因的表达量变化进行研究。试验采集敖汉细毛羊肱二头肌提取基因组DNA,参照GenBank中DKK1基因序列设计1对引物,PCR扩增获得DKK1基因片段,连接到pEASYTM-T1载体,构建pEASYTM-T1-DKK1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PmaxGFP-DKK1重组质粒,转化大肠杆菌(E.coli)DH5α感受态细胞;对敖汉细毛羊的成纤维细胞进行分离培养,并将构建的重组质粒PmaxGFP-DKK1转染成纤维细胞,利用实时荧光定量PCR技术检测DKK1基因在成纤维细胞中的表达量变化。结果显示,经酶切、测序鉴定发现,重组质粒PmaxGFP-DKK1构建成功,大小为3 956bp,并成功瞬时转染成纤维细胞,转染细胞后DKK1基因的表达量明显升高,且转染组的表达量极显著高于对照组(P<0.01)。试验成功构建了敖汉细毛羊DKK1基因重组质粒,并成功转染成纤维细胞,为进一步研究DKK1基因的功能奠定基础。This study was aimed to constract the recombiant plasmid of DKK1 gene in Aohan Fine Wool sheep and detect its expression in fibroblasts.The genomic DNA of biceps brachii in Aohan Fine Wool sheep was extracted,apair of primers was designed according to DKK1 gene sequence in GenBank,and the DKK1 gene fragment was amplified by PCR method.DKK1 gene was ligated into pEASYTM-T1 vector to construct pEASYTM-T1-DKK1 recombinant plasmid and transformed into E.coli DH5αcompetent cell,the plasmid was identified by restriction enzyme digestion.The recombinant plasimd PmaxGFP-DKK1 was constructed and transformed into E.coli DH5αcompetent cell.Then PmaxGFP-DKK1 plasmid was transfected into fibroblasts,and Realtime quantitative PCR was used to detect the expression level of DKK1 gene.The results showed that the PmaxGFP-DKK1 plasmid was constructed successfully（3 956 bp）,and was identified by enzyme and sequencing.The expression level of DKK1 gene in fibroblasts was extremely signifi-cantly higher than control group（P〈0.01）.The plasmid was constructed and transfected into fibroblasts successfully,the results would lay a foundation for the further research.

关 键 词：敖汉细毛羊 DKK1基因 成纤维细胞 转染 实时荧光定量PCR 表达 

分 类 号：Q78[生物学—分子生物学]