【作者】 谭训刚；

【导师】 张培军； 杜少军；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物， 2003， 博士

【摘要】 本文主要研究了肌肉发育过程中 Hedgehog信号系统中转录因子Gli2 部分蛋白的制备,纯化以及抗体的制备;鱼类肌肉发育-分化过程内转录因子的特性,以及通过比较文昌鱼的 MyoD 基因进行其进化的分析。并利用酵母双杂交的方法分离到一个对肌肉发育-肌肉分化和成熟过程起关键作用的基因。并对这些基因的启动子的特性进行了分析。 实验中制备得到一个可溶性的蛋白-Gli2N2 蛋白。利用该蛋白注射兔子,得到一个可以检测异位表达的 Gli2 蛋白的抗体。利用该抗体分析了异位表达的 Gli2 的时间图式。在 12hpf 时,外源表达的 Gli2 主要以全长存在,而从 15hpf 以后 Gli2 蛋白以缺失 C-端序列的蛋白出现。这是第一个得到的抗 Gli2 抗体;也是第一次用抗 Gli2 的抗体分析外源的 Gli2 蛋白表达。 从斜纹鲈中分离到 Myf-5 和 myogenin 基因,特征分析表明它们和其它种属的生肌因子-MyoD 家族内基因含有相同的结构:三个外显子和两个内含子。从真鲷中分离到两个 MyoD 基因,它们在真鲷中的表达图谱有不同,其中 MyoD1 的表达受 Hedgehog 的影响。通过和其它种属的 MyoD 基因比较研究 MyoD 基因的进化,结果表明这两个MyoD 可能是由独立的复制产生的或者在进化的过程中整个基因组很有可能发生了复制,而在某些种属内一个 MyoD 基因丢失。 上述 MyoD 家族基因的成员的启动子可以驱动报告基因 GFP 在斑马鱼胚胎肌肉中特异性表达。这表明这些启动子中含有调节肌肉特异性表达的元件;并且这些元件可以在不同种属中保守以及跨越种属起作用。 在进化中,头索动物文昌鱼被认为是接近于脊椎动物的活化石。我们从文昌鱼中分离到一个 MyoD 家族的基因 AmphiMyoD。序列分析表明 AmphiMyoD 与脊椎动物以及无脊椎动物的生肌基因比其它三个早已公布的 MyoD 类似基因更有同一序列。这些数据表明 AmphiMyoD 可能更接近于进化过程中产生四个生肌基因的原始生肌基因。并且,在系统发育分析中文昌鱼中的四个生肌基因独自形成一组而不是分别成为脊椎动物的 MyoD 家族四个基因组中的一位,这表明在文昌鱼中产生多个生肌基因的基因复制与脊椎动物中产生四个生肌基因的基因复制没有关联。其启动子可以驱动报告基因 GFP 在斑马鱼胚胎骨骼肌以及心肌中特异性表达。这表明控制肌肉特异性表达的元件在进化中保守,这些元件可以跨越种属起作用;而且我们发现其在心肌中的表达也是非常有趣的。 用酵母双杂交得到的 Bop 基因在斑马鱼中通过不同的剪切拼接产生至少两种形式。实验分析表明 Bop 特异性地表达在斑马鱼胚胎发更多还原

【Abstract】 In this thesis, the production of partial protein of Gli2 which is one of transcription factors in Hedgehog signaling that involve in muscle development; its purification and the production of its antibody; the characters of transcription factors in muscle development in fish- differentiation step and the analysis of its evolution through comparing MyoD of amphioxus with that of other species was studied.A gene that have critical function in muscle development-differentiation and maturation of muscle cells was got through yeast two-hybrid system. The specific character of these promoters of these genes was also studied. During the process of production of partial Gli2 protein, a soluble protein - -Gli2N2protien was got. A antibody that can recognize ectopic Gli2 was produced from rabbit injected with Gli2N2 protein. The temporal expression of ectopic Gli2 protein was analysed by anti-Gli2 antibody.At 12hpf,ectopic Gli2 protein was full length ,but from 15hpf most Gli2 protein was C-terminal truncated form.This is the first anti-Gli2 antibody and the first time ectopic expression of Gli2 was detected by anti-Gli2 antibody. From Striped bass , Myf-5 and myogenin wasisolated. Analysis of its character show these two gene share the same gene structure(three exon and two intron) with other myogenic factors-MyoD family.Two MyoD was isolated from Seabream.They have different expression pattern in Seabream embryos, the expression of MyoD1was affected by Hedgehog.The studied of evolution history of evolution of MyoD was done by comparing them with other MyoD of other species.The results show these two MyoD was produced through independent gene duplication or duplication of entire genomes but in some species one of myogenic gene was lost . All of the promoters of these genes of MyoD family can drive report gene –GFP muscle specific expression in zebrafish embryos.This suggested that there is muscle specific regulatory elements in the promoters and the elements are conserved between species and have function cross species. In evolution, Amphioxus, a cephalochordate, has been regarded as the living fossil closest to the vertebrates. In this study, we have isolated and characterized a new member of the MyoD family (AmphiMyoD) in amphioxus. Sequence analysis revealed that AmphiMyoD shares more identity with vertebrate and invertebrate myogenic genes compared with other three amphioxus MyoD-like genes. These data suggest that AmphiMyoD may be closely related to the ancestral myogenic gene that gave rise to the four vertebrate myogenic genes during evolution. Moreover, because the four myogenic genes in amphioxus are grouped together in a phylogenetic tree analysis rather than segregated with the four members of the MyoD family in vertebrates, it suggests that gene duplications that generated the multiple myogenic genes in amphioxus were independent from duplications that produced the four vertebrate myogenic genes. Its promoter can drive reporter gene-GFP in skeletal and cardiac muscles in zebrafish embryos. These data indicate that the activity of AmphiMyoD promoter might not be exclusively restricted to skeletal muscles as in vertebrates. The AmphiMyoD promoter may contain regulatory elements for cardiac muscle expression This data suggests that some of the regulatory elements for skeletal muscle-specific expression might be conserved during evolution. The cardiac muscle-specificity of the AmphiMyoD promoter is intriguing. Bop which came from yeast two-hybrid system produce at least two isoforms of Bop in zebrafish by alternative splicing. Expression analysis revealed that Bop is specifically expressed in developing somite and heart in zebrafish embryos. Functional studies by antisense knockdown approach demonstrated that Bop is involved in skeletal muscle maturation and heart formation. Blocking Bop expression in zebrafish embryos resulted in malfunction of motivation of zerbrafish embryos, and moreover the blood circulation. Together, these data suggest that Bop plays a critical role in the differentiation of skel更多还原