斑马鱼肌肉发育相关基因克隆、表达与功能分析

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	斑马鱼肌肉发育相关基因克隆、表达与功能分析
其他题名	Cloning, expression and functional analysis of genes regulating muscle development from zebrafish
	孙威
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	斑马鱼原位杂交显微注射过量表达
摘要	本文主要研究在斑马鱼胚胎发育过程中参与肌肉发育的相关基因，克隆了四个在体节和肌肉中表达的基因全长，分析了基因的时空表达特征，并对其中两个基因进行了过表达，分析其在体节形成和肌肉发生过程中的功能。从斑马鱼中克隆到甲状腺激素受体相关蛋白基因TRAP150。原位杂交分析TRAP150表达在近轴细胞的慢肌和体节的快肌，表达模式与MyoD的模式相近；并表现出肌肉特异性表达，TRAP150在体节形成和肌肉发生早期高水平说明TRAP150在肌肉分化过程发挥着重要的作用。此外，在胚胎的心脏中也检测到TRAP150的表达。在斑马鱼胚胎过量表达TRAP150造成MyoD在近轴中胚层的过量表达，而对MyoD在近轴细胞的表达影响不明显；由于MyoD在近轴中胚层的表达将诱导快肌的形成，因此过量表达TRAP150将可能导致快肌的增多；从过量表达的结果分析，TRAP150在MyoD的上游正向调控MyoD的表达，是诱导快肌的分化的重要基因。从斑马鱼中克隆到双特性酪氨酸调控激酶新基因DYRK2。RT-PCR结果表明DYRK2具有母源性表达的特点，并且在36小时前各个时期都有表达。DYRK2在体节形成后表达在体节的近轴细胞中，大约在15个体节时检测到在快肌部位表达，18小时后在胚胎肌肉组织、脑部以及眼睛表达。DYRK2在斑马鱼胚胎发育过程中与体节和肌肉发生相关的基因之一，并可能参与脑和眼睛的发生。在斑马鱼胚胎中过量表达DYRK2导致肌肉标记基因MyoD的表达出现了很大的变化，注射侧MyoD在近轴细胞和近轴中胚层过量表达，尤其是在未形成体节的体节前体中胚层，注射侧的MyoD有大范围的高表达，而在正常一侧没有表达。MyoD的过量表达说明斑马鱼胚胎早期DYRK2通过调控MyoD的表达影响慢肌的分化。克隆得到斑马鱼的血细胞生成的PBX1互作蛋白基因HPIP1。RT-PCR结果表明HPIP1在斑马鱼胚胎表达具有母源性，但是原位杂交的检测一直到10个体节时才检测到，说明HPIP1一直到肌肉分化后才大量表达，可能在肌肉的成熟阶段起作用。当胚胎发育到18小时，HPIP1表达在所有体节中，前端的较早形成的体节中表达量比晚形成的体节表达量高，也符合HPIP1参与肌肉成熟过程的判断。HPIP1还表达在胚胎的眼部周围，说明HPIP1可能参与到眼部肌肉的形成。在斑马鱼中克隆的Chp-1相似蛋白基因CHORDC1。在3个体节时，CHORDC1表达在脊索两侧的近轴细胞中，而在5个体节时CHORDC1在表达在体节中，这个特点与MyoD的表达很相似，与MyoD在体节中表达不同的是，CHORDC1也在体节前体中胚层中表达，这与Myf5的表达特点相似，CHORDC1紧随着MyoD，Myf5的高表达说明CHORDC1在肌肉细胞的分化的早期即参与肌肉的发育，而其高表达量也说明CHORDC1在这个过程中可能起到非常重要的作用。CHORDC1在近轴中胚层的表达与MyoD，Myf5有不同的特点，这种不同表现在其在近轴中胚层的表达不仅仅限于快肌和慢肌，而且由后到前的逐渐扩展。而且，CHORDC1在心肌中也表达说明其不仅在骨骼肌中发挥作用。综合CHORDC1的表达特点可以认为其对肌肉的作用不限于特定肌肉类型，广泛参与到各种肌肉的发育过程
其他摘要	This study focuses on the genes relating to muscle development during embrogenesis in zebrafish. Full lengths of four genes are cloned and their expession patterns are analyzed by in situ hybridization and RT-PCR. Overexpession experiments are done in two genes to analyze their function on somite and muscle during embyo development in zebrafish. Thyroid hormone receptor associated protein 150 (TRAP150) is cloned from zebrafish cDNA library. Results of in situ hybridizaton show that TRAP150 is detected in slow muscle of adaxial cells and in fast muscle of paraxial mesoderm at early stage of embyogenesis. The expression pattern of TRAP150 is similar to MyoD and exhibits the specific character related to muscle. The high level of expession of TRAP150 in somites and muscle indicates that TRAP150 probably plays an important role in muscle development. Furthermore, TRAP150 is detected in cardiac muscle. Overexpession analysis of TRAP150 results in overexpression of MyoD in paraxial mesoderm. Since fast muscle is induced by MyoD in paraxial mesoderm, overexpression of TRAP150 can up-regulates fast muscle development. It is indicated that TRAP150 might be a positive upstream regulator in regulating MyoD expression and functions in fast muscle development.. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 isoform 2 (DYRK2) is cloned from zebrafish cDNA library. The result of RT-PCR shows this gene is maternal expression and can be expressed in every stage before 36h. Results of in situ hybridizaton showed that DYRK2 is detected in adaxial cells at early stage of embyogenesis, and detected in fast muscle at 15 somites stage. The expession pattern of DYRK2 performs muscle specific character. In 18 hours stage, DYRK2 is also detected in brains and eyes. Overexpession of DYRK2 results in overexpression of MyoD in adaxial cells and paraxial mesoderm in the injected side at early stage of embyogenesis, paticularly in presomite mesoderm. Overexpession analysis of DYRK2 showed that MyoD expression in slow muscle is up-regulated, indicating that DYRK2 is involved in slow muscle development by the way of regulating expression of MyoD. Hematopoietic Pre-B-cell leukemia transcription factor interacting protein 1 (HPIP1) is cloned from zebrafish cDNA library.Results of RT-PCR indicated that Pbxip1 has maternal resourse, but HPIP1 is firstly detected at 10 somites stage of embyogenesisby in situi hybridization, which indicates that HPIP1 is expressed after muscle begin to differentiate and HPIP1 plays an important role in muscle differentiation and maturation. Furthermore, HPIP1 is detected in eyes, indicateing that HPIP1 probably is involved in eye muscle development. CHORDC1 is cloned from zebrafish cDNA library. At 3 somites stage, CHORDC1 is detected in adaxial cells and is found in paraxial mesoderm at 5 somites stage. The expression character of CHORDC1 is similar to MyoD. The difference is that CHORDC1 is also detected in presomitic mesoderm which is similar to Myf5. CHORDC1 expression followed the expression of MyoD，Myf5, which indicates that CHORDC1 might play an important role in muscle differentiation and maturation. The expression of CHORDC1 differs from MyoD and Myf5 in the presomitic mesoderm.The differences display not only in the slow muscle and fast muscle; but also CHORDC1 expression dislays a gradual expansion from posterior to anterior. Furthermore, CHORDC1 is detected in heart muscle as well. Therefore, CHORDC1 performs the specific expression in muscle but not specific muscle type.
页数	112
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/841
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	孙威. 斑马鱼肌肉发育相关基因克隆、表达与功能分析[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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