摘要
为了克隆关岭牛MyoDⅠ基因启动子并验证其启动活性,根据GenBank中牛的MyoDⅠ基因序列设计PCR引物,用PCR技术扩增牛MyoDⅠ基因的启动子,构建重组克隆载体pUCmT-MyoDⅠ;并通过PCR扩增、限制性酶切、测序及生物信息学分析对阳性克隆进行鉴定;构建报告质粒pGL3-MyoDⅠ,并将其转染小鼠C2C12、3T3-L1细胞系,检测其24h后的双荧光素酶活性。实验结果获得了关岭牛MyoDⅠ基因启动子,其序列长度为993bp,并成功构建了MyoDⅠ启动子报告质粒;双荧光素酶活性检测表明pGL3-MyoDⅠ在小鼠C2C12细胞中的表达是pGL3空载体40.65倍,在小鼠3T3-L1细胞中的表达是空载体的1.13倍,MyoDⅠ启动子在小鼠C2C12细胞中的表达高于3T3-L1细胞(**p<0.01)。结果表明关岭牛MyoDⅠ基因启动子具有启动活性,在小鼠骨骼肌细胞中特异性表达。
To verify the promoter activity of the MyoD I gene by cloning promoter t^om the Guanling cattle, ac- cording to the MyoD I gene sequence of cattle in GenBank, the PCR primers were designed. The gene promoter was amplified by using PCR technology to construct the recombinant cloning vector pUCmT-MyoD I, and then identi- fied the positive clones by the PCR amplifying, restriction enzymes analysis and bioinformatics analysis. The report plasmid pGL3-MyoD l was constructed and transfected into the C2C12 and 3T3-L1 cells of mouse to detect the du- al luciferase activity after 24 hours. We obtained the promoter ofMyoD I was obtained, and its sequence length was 993 bp. The report plasmid ofMyoD I gene promoter was constructed successfully. The expression ofpGL3-MyoD I was 40.65 times of pGL3 empty vector in mouse C2C12 cell, and was 1.13 times in 3T3-L1 cell through the method of relative activity of luciferase, which indicated that the expression of MyoD I gene promoter in C2C 12 cell was higher than that of 3T3-L1 cell in mouse （＊＊ p〈0.01）. The results showed that the MyoD ][ gene promoter of Guanling cattle contains the promoter activity and can specifically express in skeletal muscle cells.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2013年第4期459-466,共8页
Genomics and Applied Biology
基金
国家转基因生物新品种培育科技重大专项课题《牛组织特异性调控元件克隆和功能验证》(2011ZX0800-9004)
贵州省科技厅农业攻关计划《影响牛脂肪沉积组织特异性调控元件克隆和功能验证》(黔科NY字[2012]3008号)
贵州大学研究生创新基金(校农科2012007)项目共同资助
