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牙鲆成肌因子myogenin和MRF4的研究
其他题名Studies on flounder muscle regulatory factors: myogenin and MRF
徐芃
学位类型博士
2007-06-01
学位授予单位中国科学院海洋研究所
学位授予地点海洋研究所
关键词牙鲆 Myogenin Mrf4 Rt-pcr 原位杂交 显微注射 原核表达 多克隆抗体
摘要本文从牙鲆中克隆到其肌肉调节因子myogenin和MRF4的基因,并对它们的表达、启动子活性及功能进行分析。 研究结果表明牙鲆myogenin和MRF4都由三个外显子和两个内含子组成,其cDNA编码的氨基酸序列分别与几种鱼类的同源基因亲缘关系较近。 原位杂交显示最早在胚胎5-6个体节时体节中部的细胞检测到myogenin的表达,随着发育的进行,它的表达向体节两侧和体后延伸,最后在整个体节表达。当发育到30个体节的时候,在躯干的表达迅速下降,只在胚胎的尾部能检测到信号。 RT-PCR的结果显示,MyoD和Myf5的表达要早于myogenin,而MRF4的表达晚于myogenin,并且myogenin、MRF4只在成鱼的肌肉中表达,这进一步证明其在肌肉发育过程中的作用。 myogenin和MRF4的启动子中含有保守的肌肉特异调控序列,瞬时表达分析的结果证实两段启动子序列足以驱动GFP在斑马鱼胚胎肌肉中的特异表达。首次对鱼类MRF4启动子进行了调控序列的分析,发现在启动子的-181到-506的序列中含有调节MRF4表达的必需位点。 首次应用原核表达系统对MRF4蛋白进行重组表达,并利用纯化的重组蛋白制备多克隆抗体,通过western杂交分析显示所得到的MRF4多克隆抗体能够识别牙鲆内源性的MRF4蛋白。
其他摘要The present study focused on the cloning and expression of myogenin and MRF4 and the analysis of their promoter and function during flounder embryogenesis. The whole genomic sequence of flounder myogenin and MRF4 both contain 3 exons and 2 introns, and the identity of their deduced amino acid sequences were higher with those of several fish species. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites when 5-6 somites were formed, and expanded later to the lateral region of the somite and expressed in the whole somite. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region, but remained strong within the tail region. The result of RT-PCR demonstrated that Myf-5 and MyoD was expressed earlier than myogenin, while the expression of MRF4 was later than that of myogenin. And myogenin and MRF4 were detected only in skeletal muscle by RT-PCR, which further indicated that they play roles in regulating muscle development. Transient expression analysis showed that the promoters of flounder myogenin and MRF4 were sufficient to direct muscle-specific GFP expression in zebrafish embryos, which indicated that there were conserved regulatory sites in the promoter regions and these elements could act across species. Further study on the promoter MRF4 showed that there must be necessary sites for regulating the expression of MRF4 in the promoter between -181 and -506. Flounder MRF4 recombinant protein was expressed and purified, its polyclonal antibodies was prepared and analyzed. The endogenous MRF4 was detected by the polyclonal antibody. Thus, further MRF4 function studies on protein level can be performed.
页数115
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/809
专题海洋环流与波动重点实验室
推荐引用方式
GB/T 7714
徐芃. 牙鲆成肌因子myogenin和MRF4的研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.
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