摘要
本研究旨在探究关岭牛4个不同长度的MyoD I启动子的启动活性,初步确定该基因核心启动子位置。采用实时荧光定量PCR技术对关岭牛不同组织中MyoD I基因的相对表达量进行检测。以关岭牛血液DNA为模板,设计5'端加磷的引物,PCR扩增获得4个不同长度的关岭牛MyoD I启动子,定向精确替换pEGFP-N3载体的CMV启动子区,构建重组真核表达载体pEGFP-N3-MyoD I。利用脂质体法将重组质粒瞬时转染小鼠C2C12细胞,依据小鼠C2C12细胞发光的强弱判断MyoD I启动子的启动活性。结果显示,MyoD I基因在关岭牛肌肉组织中表达量较高;目的片段成功替换pEGFP-N3载体的CMV区;重组质粒能在小鼠C2C12细胞中成功表达,细胞在激发光下发绿色荧光。本研究成功构建了重组真核表达载体pEGFP-N3-MyoD I,4个启动子均具有启动活性,其中P3启动子在小鼠C2C12细胞中表达量最高,初步推断P3启动子为该基因的核心启动子。
The aim of this study was to analyze promoter activity of 4different lengths MyoD I promoters in Guanling cattle and determine preliminarily the position of the core gene promoter.The relative expression quantity was detected in different tissues of Guanling cattle by real-time quantitative PCR.The DNA extracted from Guanling cattle blood was as a template and the phosphorylated 5'end PCR primers was designed to amplify 4different lengths MyoD I promoters in Guanling cattle,ultimately accurately replaced the CMV region of pEGFP-N3 vector and got eukaryotic expression vector pEGFP-N3-MyoD I.Then the recombinant plasmid was transiently transfected into mouse C2C12 cells by liposome method.The aim was to verify promoter activity of MyoD I promoter by Luminous intensity of mouse C2C12 cells.The expression level ofMyoD I gene was the highest in muscle,the fragment was inserted into an expression vector,the recombinant plasmid could be successfully expressed in mouse C2C12 cells with green fluorescence under the excitation light.In this study,eukaryotic expression vector pEGFP-N3-MyoD Iis successfully constructed,4promoters constructed show promoter activity,expression quantity of P3 promoter is the highest,so P3 promoter is regarded as the core promoter of MyoD I gene.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2015年第5期719-727,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
转基因生物新品种培育重大专项(2013ZX08009-004)
黔科合重大专项(字[2013]6008号)
贵州省科技厅农业攻关项目(黔科合NY字[2012]3008号)
贵州大学研究生创新基金(研农2014021)
