牙鲆肌肉发育调节基因的克隆、表达及功能分析

IOCAS-IR > 海洋环流与波动重点实验室

	牙鲆肌肉发育调节基因的克隆、表达及功能分析
其他题名	Cloning, expression and functional analysis of genes regulating musle development from flounder (Paralichthys olivaceus)
	张玉青
学位类型	博士
	2008-06-15
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 Paralichthys Olivaceus Myf5 Myod Foxd1 Foxd3 Foxd5 Rt-pcr 原位杂交显微注射过量表达
摘要	本文克隆了牙鲆的成肌因子MyoD和Myf5，以及牙鲆的Forkhead基因FoxD1、 FoxD3和FoxD5，并对其在牙鲆肌肉发育中的功能进行了分析。牙鲆MyoD和Myf5基因都具有三个外显子，两个内含子。其编码的氨基酸序列都含有保守的bHLH；牙鲆FoxD1，FoxD3，FoxD5基因都只有一个外显子，编码的氨基酸序列都含有保守的翼状螺旋DNA结合结构域。在胚胎发育早期，Myf5在近轴中胚层中表达，体节发生过程中，Myf5在体节中表达，MyoD基因最早在分节板的体节前细胞中表达，随后在近轴细胞、体节中表达；随着胚胎的发育，Myf5在成熟体节中表达量降低，在新生体节中表达较强；MyoD自30个体节时期后只在新生的尾部体节中表达，在成熟的体节中表达量降低；在孵化期，MyoD和 Myf5在头部及鳍的肌肉、尾部的体节中表达；生长期的牙鲆中，Myf5在骨骼肌和肠中表达，成体牙鲆中，Myf5只在肌肉中表达；生长期的牙鲆及成体牙鲆中，MyoD只在肌肉组织中表达。牙鲆MyoD和Myf5的启动子可以驱动绿色荧光蛋白在斑马鱼肌肉纤维中表达，其包含了这两个基因正常表达所需的核心区域，并可以跨物种行使功能。在胚胎发育早期，FoxD3在未迁移神经嵴前体细胞、体节、耳后的基板、头部和躯干的神经嵴细胞、松果体中表达。牙鲆FoxD1主要在脑，体节，肾脏及肠中表达。牙鲆FoxD5主要在体节、尾芽、前脑、耳泡中表达。在斑马鱼中过量表达牙鲆FoxD3，并与斑马鱼的同源基因进行比较，结果表明注射牙鲆和斑马鱼FoxD3的斑马鱼胚胎表型一致，它们在中轴两侧的发育出现了不同步现象，MyoD和Myf5在近轴中胚层中的表达受到不同程度抑制。因此，FoxD3在不同物种之间保守，并且FoxD3在肌肉发育的调控通路中可能通过与MyoD和Myf5相互作用而行使功能。在斑马鱼中过量表达FoxD1后，MyoD在一侧体节中的表达受到了严重抑制，而在近轴细胞中的表达未受影响，Myf5在体节前中胚层，近轴细胞，及体节中的表达都受到了抑制。FoxD1在胚胎发育的早期可能通过调控MyoD和Myf5的表达而参与肌肉发育的调控。将牙鲆FoxD5在斑马鱼中过量表达，MyoD在一侧体节中的表达量有所升高，而在近轴细胞中的表达未受影响；Myf5在一侧体节和体节前中胚层中的表达也有所升高，表明牙鲆FoxD5可以调控肌肉调节因子MyoD和Myf5的表达而参与早期肌肉发育的调控。
其他摘要	This study focused on the cloning and expression of flounder muscle regulatory genes, MyoD and Myf5, and Forkhead domain genes, FoxD1, FoxD3 and FoxD5, and the analysis of their functions during flounder muscle development. The genomic sequences of both flounder MyoD and Myf5 contain three exons and two introns, and a highly conserved bHLH domain. The flounder FoxD1, FoxD3 and FoxD5 contain only one exon and a conserved winged-helix DNA binding domain. In situ hybridization indicated that, Myf5 transcripts were first detected in the paraxial mesoderm, during somitogenesis, Myf5 expression was found in developing somites; MyoD expression was first detected as two rows of presomitic cells in the segmental plate, and then was present in the adaxial cells and the lateral somitic cells. With the development of embryo, Myf5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites; After 30 somites formed, MyoD expression decreased in the somites except the caudal somites. At the hatching stage, MyoD and Myf5 were expressed in head muscle cells and fin muscle cells. In the growing fish, Myf5 was expressed in the skeletal muscle and intestine, and in adult flounder, Myf5 was only expressed in muscle. In the growing fish and adult fish, MyoD was only expressed in muscle. The MyoD promoter and Myf5 promoter could drive GFP specifically expressed in zebrafish muscle fibers, indicating that the two promoters contained the core sequences required for the normal expression of the two genes, also, the two promoters were conserved because they could function across species. During the early stage of embryogenesis, FoxD1 was mainly expressed in the somites, kidney progenitor cells, brain progenitor cells and intestine. FoxD3 was expressed in premigratory neural crest cells, somites, post otic placodes, cranial and trunk neural crest cells, and pineal gland. FoxD5 was mainly expressed in the somites, tail bud, forebrain and otic vesicle etc. Flounder FoxD3 mRNA was over expressed in one cell of the two-cell stage zebrafish by microinjection, zebrafish homologue FoxD3 was used to confirm the effect of flounder FoxD3. After over expression, the two sides separated by the anterior-posterior axis of zebrafish embryo showed an asymmetrical development, the expression of MyoD and Myf5 in the paraxial mesoderm was inhibited. So the flounder FoxD3 might function in the muscle development regulatory network by interacting with MyoD and Myf5. Flounder FoxD1 mRNA was over expressed in one cell of the two-cell stage zebrafish, MyoD expression in the somites on one side was reduced, but the expression in the adaxial cells was not affected; Myf5 expression in the presomitic mesoderm, adaxial cells and somites on one side was reduced. It is indicated that FoxD1 might play a role in the muscle development by regulating MyoD and Myf5. Flounder FoxD5 mRNA was over expressed in one cell of the two-cell stage zebrafish, MyoD expression in the somites on one side was enhanced, but the expression in the adaxial cells was not affected; Myf5 expression in the somites and presomitic mesoderm on one side was also enhanced. FoxD5 might regulate the muscle development by regulating the expression of MyoD and Myf5.
页数	145
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/591
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张玉青. 牙鲆肌肉发育调节基因的克隆、表达及功能分析[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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