关岭牛MyoD基因家族对MyoD1启动子活性的影响

Influence of MyoD1 Promoter Activity by MyoD Family of Guangling Cattle

【目的】生肌决定因子(myogenic determination gene,MyoD)家族是肌肉生成过程中参与分子调控作用的一个重要家族。该家族包括MyoD1,Myf5,Myo G和Myf6,只表达在成熟的骨骼肌细胞和其前期细胞中;在其他的非肌细胞中,MyoD基因家族会被抑制。该家族中,MyoD1负责早期胚胎成肌祖细胞的激活并参与胚后骨骼肌的生长、发育和修复等的调节,以维持个体骨骼肌的相对稳定,是启动和维持骨骼肌细胞分化和生长的重要因素,具有尤为重要的作用,已成为研究热点。目前MyoD1在多态性和关联性分析等方面的研究较多,表达调控方面主要是研究小鼠、鸡和猪肌细胞生成机理;在牛上对它的研究主要在转录后和翻译水平的表达,但对MyoD1在转录调控方面的作用机制还不明确。文章研究关岭牛MyoD基因家族对MyoD1启动子活性的影响,为探讨牛MyoD1的表达调控机制奠定基础。【方法】通过设计特异性引物克隆关岭牛MyoD基因家族CDS区和MyoD1启动子片段P1和P2;同时利用双酶切的方法分别将CDS区和克隆的启动子序列连入pcDNA3.1(+)和p GL3-Basic基本骨架,构建真核表达载体pcDNA3.1(+)-Myf5、pcDNA3.1(+)-Myf6、pcDNA3.1(+)-MyoD、pcDNA3.1(+)-Myo G和含萤火虫荧光素酶报告基因的报告载体p GL3-P1、p GL3-P2。重组质粒经酶切和测序鉴定后,利用共转染的方法将真核表达载体和报告载体转染小鼠C2C12细胞,30 h后裂解细胞并检测细胞裂解液的双荧光素酶活性。最后根据荧光素酶的相对活性来分析MyoD基因家族对MyoD1启动子活性的影响。【结果】克隆得到的关岭牛MyoD基因家族CDS区和MyoD1启动子序列测序正确,载体pcDNA3.1(+)-Myf5、pcDNA3.1(+)-Myf6、pcDNA3.1(+)-MyoD、pcDNA3.1(+)-Myo G、p GL3-P1和p GL3-P2经酶切和测序鉴定,证实载体构建成功;与相应剂量的对照组相比,转染pcDNA3.1(+)-Myf5、pcDNA3.1(+)-Myf6、pcDNA3.1(+)-MyoD后,p GL3-P1的相对荧光素酶活性明显增强,其中,在转染量为200 ng时增强作用最强,差异显著(P<0.05);转染pcDNA3.1(+)-Myo G后,虽然对p GL3-P1的相对荧光素酶活性有增强作用,但差异不显著(P>0.05);而转染pcDNA3.1(+)-Myf5、pcDNA3.1(+)-Myf6、pcDNA3.1(+)-MyoD、pcDNA3.1(+)-Myo G后,p GL3-P2的相对荧光素酶活性变化不明显(P>0.05)。【结论】在小鼠C2C12细胞中外源过表达转录因子MyoD、Myf5、Myf6均能显著提高关岭牛MyoD1启动子全长P1的转录活性(P<0.05);而外源过表达转录因子MyoD基因家族不能显著提高关岭牛MyoD1启动子核心区P2的转录活性。说明关岭牛MyoD、Myf5和Myf6转录因子与关岭牛MyoD1启动子的作用位点不在其核心启动子区P2上。

【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 p GL3-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 p GL3-P1, p GL3-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, p GL3-P1 and p GL3-P2 were successfully constructed. Compared with the control group with the same dose of pcDNA3.1（＋）, the relative luciferase activities of p GL3-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 p GL3-P1 was enhanced after transfected pcDNA3.1（＋）-MyoG, but not have significant difference（P〉0.05）. The luciferase activity of p GL3-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.