【作者】 谭效忠；

【导师】 安利国；

【作者基本信息】 山东师范大学 ， 动物学， 2007， 硕士

【摘要】 斑马鱼是一种新兴的模式生物，近十几年来，以斑马鱼为模式生物的研究在脊椎动物胚胎发育的研究领域获得了长足进展。斑马鱼作为模式生物有着不可替代的优势，它价格便宜，容易获得，体积小、易密集饲养。斑马鱼的胎间距仅为3个月，雌性斑马鱼每周可产生大量后代，其胚胎的受精及发育都在母体外进行，受精卵大，早期胚胎完全透明，即使在发育的高级阶段，仍然可以观察到全部的细胞。斑马鱼胚胎发育非常迅速，从受精到孵出大约3d，主要器官都在受精后24h内形成，在胚胎发育时期，其体型不会发生多大变化；而且，来自于同一母体的胚胎是同步发育的，易于大量收集特定阶段的同期胚胎材料。目前一种新的手段——注射反义吗啉环寡聚核苷酸以抑制某一特定基因的表达的方法已经在斑马鱼上得到了很好的应用。此外，斑马鱼基因组的测序工作也已经完成。因此，我们也以斑马鱼作为模式生物，对其胚胎发育过程中重要的蛋白因子Spr2进行分子生物学方面的研究工作。spr2是斑马鱼胚胎发育过程中在中胚层和外胚层中特异表达的基因，基因全长为1981bp，最大编码框为1075bp。它可编码一种Spl-like转录因子家族成员。Spl-like转录因子家族与锌指蛋白密切相关，它们可以调控大量的复含GC-启动子的基因，在细胞增殖、细胞凋亡、细胞分化和细胞转化中都可能发挥作用。独立的Spl-like转录因子可与不同的启动子和互作蛋白结合，发挥增强子或抑制子的作用。最新研究发现在斑马鱼胚胎发育的后囊胚期和前原肠胚阶段，Spr2在上胚层和下胚层中表达；其后在中胚层和一些神经组织中表达。通过spr2基因敲除实验和过量表达实验，可知Spr2的活性受到Fgf、Nodal、Wnt信号通路的协同控制。在中胚层发生早期，Spr2可以通过调控Fgf信号通路调控中胚层的发生，从而表达一些活性蛋白因子来调控中胚层相关组织器官的发育。由于中胚层是器官发生的重要基地，这一发现对研究脊椎动物早期胚胎的发育机理非常重要。因此，我们以Spr2为诱饵蛋白，通过酵母双杂交系统从斑马鱼早期胚胎cDNA文库中筛选可能与Spr2蛋白相互作用的蛋白，进一步探索其在Fgf信号通路中的作用机制。本论文通过酵母双杂交系统在斑马鱼早期胚胎cDNA文库中筛选可能与Spr2相互作用的蛋白，以酵母融合的方法在6.3×10~6个转化子中筛选到78个阳性克隆，测序鉴定后最终得到11种可能与Spr2蛋白相互作用的蛋白，它们是Pairedbox gene 6a、mSin3A-associated protein 130等，通过结果分析推测Spr2可能通过fgf8参与Fgf信号通路的调控。在所得的11种同源蛋白中，我们经过分析发现重复克隆数最多的1号蛋白为斑马鱼眼睛发生的标志基因，它可以特异的在眼睛和中枢神经系统中表达，最新研究成果显示pax6基因的表达与fgf8有关，这提示了Spr2可能会通过fgf8与Fgf通路产生联系；2号mSin3A-associated protein 130(SAP130)也是我们感兴趣的一个蛋白，它是一种胞质蛋白，其C端含有一个repression区域，N端含有一个参与转录激活功能的区域，SAP130可以通过C端功能区与mSin3A复合物相互作用。mSin3A复合物含有多个蛋白与蛋白相互作用的功能区，它可以作为辅阻遏复合物的支架；而它的辅阻遏复合物可以特异的与大量不同的转录抑制物相互作用，在哺乳动物胚胎发育中起着至关重要的作用，例如Sin3a可以作为BMP中介的转录调控抑制剂影响BMP信号通路；因此，我们认为该蛋白很有研究价值。另外，3号和5号蛋白都与肌肉发生有关；4号、8号、9号蛋白与磷酸化作用有关，提示它们可能也是蛋白信号通路中的重要调控分子；6号、7号都与体内造血作用有关；而Fgf通路主要调控血管生成、中胚层的形成及神经轴突的延伸，因此这些蛋白因子也是很有意义的筛选结果。我们将通过细胞生化和胚胎实验进一步研究它们与Spr2的互作机制和互作功能区域，期待破解Spr2受Fgf信号通路调控的分子机制，提出脊椎动物胚胎中胚层发生的新理论。更多还原

【Abstract】 Zebrafish (Brachydanio rerio), as a new model animal wildly used on embryo development study of vertebrate, became popular and useful. In recent ten years, the study of vertebrate embryo development using this model has developed rapidly. There are several advantages of zebrafish to be a model animal. Firstly, they are small and easy to be raised. The female can lay more than 200 eggs once a week and the embryos need only 3 months to be hatched. Secondly, the zygotes of zebrafish are big and limpid, so it is quite convenient to observe the whole developmental processes of zebrafish embryo and analyse the feasible endpoints in embryotoxicological testing. Finally, the development of zebrafish embryo is quite rapid, only need 3 days from impregnated to incubated. Recently a new method which microinject Morpholino into the embryo of zebrafish to inhibit special genes activity has effectively to be used. As a result, we decided to use zebrafish as a model to study an important protein named zebrafish Spr2.spr2 is a restrictly expressed gene in both mesoderm and ectoderm during the development of zebrofish embryo. Spl-like proteins and Kruppel-like factors (KLFs) are highly related zinc-finger proteins that are important components of the eukaryotic cellular transcriptional machinery. By regulating the expression of a large number of genes that have GC-rich promoters, Spl-like/KLF transcription regulators may take part in virtually all facets of cellular function, including cell proliferation, apoptosis, differentiation, and neoplastic transformation. Individual members of the Spl-like/KLF family can function as activators or repressors depending on which promoter they bind and the coregulators with which they interact.spr2 is expressed in both hypoblast and epiblast cells during late blastulation/early gastrulation, and in some mesodermal andneural tissues at later stages. The spr2 knockdown and overexpression data shows that spr2 expression is dependent on Fgf, Nodal and Wnt signals, and it is implicated in mesoderm induction during early development in zebrafish embryos. Because of the importance of mesoderm development during organgenesis, We use yeast two hybrid system to search for proteins interacting with spr2 from the early embryos of zebrafish cDNA library and explore the mechanism of spr2 function in Fgfs signal pathway.In this study, the yeast two-hybrids system is used to searched for proteins interacting with Spr2. From the early embryos of zebrafish cDNA library, 78 positive clonies from 6.3×106 transformants were found by yeast mating. 11 kinds of proteins are identified after sequencing the plasmids of the positive clonies such as Paired box gene 6a, mSin3A-associated protein 130. We suggest that spr2 can participate in Fgfs signal pathway by the interaction with fgf8.更多还原