关键词: 转录因子, 启动子, MyoD基因家族, 报告基因, 荧光素酶活性

Abstract: 【Objective】The myogenic determination gene (MyoD) family is an important one in the molecular regulation of the muscle generation process. It includes MyoD1, Myf5, MyoG and Myf6 genes. They only express in mature skeletal muscle cells and its early cells, but will be suppressed in other than muscle cells. In this family, MyoD1 is responsible for the activation of early embryo muscle progenitor cells and participates in the adjustment of growth, development and repair of skeletal muscle after the embryonic stage, which in order to maintain the relatively stable of individual skeletal muscle. MyoD1 is an important factor for starting and maintaining skeletal muscle cell differentiation and growth, such that it has become a research hotspot. At present, the research on MyoD1 in polymorphism and correlation analysis is more, the expression and regulation of it are mainly on the formation mechanism of muscle cells in mice, chicken and pig. In cattle, the researches on MyoD1 are mainly in the expression levels of transcription and translation, but the mechanism of MyoD1 on transcriptional regulation is not clear. This study was to research the effect of MyoD family on the promoter of MyoD1 in Guangling Cattle, in order to lay the foundation of probing into the expression regulation mechanism of cattle MyoD1. 【Method】 Specified primers were designed to amplify CDS regions of MyoD family and MyoD1 promoter fragments P1 and P2 of Guangling Cattle. At the same time, the CDS regions and promoter fragments were connected to pcDNA3.1(+)and pGL3-Basic framework by double enzyme digestion, respectively, to construct eukaryotic expression vectors of pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)-MyoD, pcDNA3.1(+)-MyoG and report carrier pGL3-P1, pGL3-P2 which contain the firefly luciferase gene as the reporter gene. After the recombinant plasmids were identified by double enzyme digestion and sequencing, the expression vector and report carrier were transfected into myoblast cell line C2C12 of mice by cotransfection. After 30 hours, we cracked the cells and tested the double luciferase activity of cell lysis buffer. According to the relative luciferase activity to analyze the effect of the MyoD1 promoter activity by the MyoD family. 【Result】The sequences of the cloned CDS regions of MyoD family and MyoD1 promoter of Guangling Cattle were validated by DNA sequencing. Restriction analysis and sequencing confirmed that the vectors pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)-MyoD, pcDNA3.1(+)-MyoG, pGL3-P1 and pGL3-P2 were successfully constructed. Compared with the control group with the same dose of pcDNA3.1 (+), the relative luciferase activities of pGL3-P1 were obviously enhanced after transfected pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)–MyoD. Among them，when transfection concentration was 200 ng, the potentiation was maximal. There were significant difference (P<0.05). Luciferase activity of pGL3-P1 was enhanced after transfected pcDNA3.1(+) –MyoG. but not have significant difference (P＞0.05). The luciferase activity of pGL3-P2 had no obvious change after transfected pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)–MyoD and pcDNA3.1(+)-MyoG (P＞0.05). 【Conclusion】 In myoblast cell line C2C12 of mice, exogenous overexpression transcription factors MyoD, Myf5 or Myf6 could significantly improve the transcription activities of Guanling cattle MyoD1 promoter P1 (P<0.05). But exogenous overexpression transcription MyoD family could not significantly improve the transcription activities of Guanling cattle MyoD1 core promoter P2. This observation helps explain that the action sites of transcription factors MyoD, Myf5 and Myf6 of Guanling cattle with MyoD1 promoter are not on its core promoter region P2.

Key words: transcription factors, promoter, MyoD family, reporter gene, luciferase activity