非洲爪蟾Brunol和MCPH基因在早期胚胎发育过程中表达的研究及随机定向酵母双杂交cDNA文库的构建
文献类型：学位论文
| 作者 | 武景阳 |
| 学位类别 | 博士 |
| 答辩日期 | 2008-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 毛炳宇 |
| 关键词 | 非洲爪蟾 Brunol/CELF MCPH 表达模式 定向cDNA文库 |
| 其他题名 | Developmental expression of Xenopus Brunol and MCPH genes and the construction of a Xenopus directional two hybrid cDNA library |
| 学位专业 | 发育生物学 |
| 中文摘要 | BRUNOL/CELF家族RNA结合蛋白在转录后调控（post-transcriptional regulation）中起着至关重要的作用，参与多种组织的发育过程。本研究中，我们描述了非洲爪蟾5个Brunol 基因的克隆与表达。其中只有Brunol2是母源性以及合子表达的，其它的4个Brunol基因都是合子表达的，但起始表达的发育时期有所不同。爪蟾发育过程中，Brunol1、4和5基因特异性地在神经系统中表达，包括脑、脊髓、眼泡和耳泡。Brunol2和3基因在体节中胚层与神经系统表达。Brunol2也在很晶状体有非常高的表达。在转染的Hela细胞中，BRUNOL1、2和3蛋白定位于细胞质和细胞核中，BRUNOL4和5只是定位于细胞质中，显示它们具有不同的功能。 人的microcephalin1（MCPH1）基因的遗传突变产生原发性小头症，而在人类的进化过程中，这个基因的变化可能对人脑体积的增加和认知能力的增强也起到重要的作用。但是对于MCPH基因在其它物种中功能的研究才刚刚开始。我们克隆了非洲爪蟾MCPH基因，发现非洲爪蟾具有A，B两个同源基因，其功能域的保守性较高，暗示非洲爪蟾MCPH基因仍然执行一些保守的功能，但MCPHB由于突变只编码一种截短的蛋白，目前尚不清楚它是否是有功能的。胚胎原位杂交的结果显示MCPHA，B基因在胚胎发育中的表达图式相似，但MCPHB的表达水平较低。在神经胚期，二者均表达于头部基板区，在尾芽期主要表达于咽鳃区，而在脑区的表达并不显著，与小鼠中的表达模式不同，提示在爪蟾中MCPH基因可能主要参与咽鳃区而不是脑的发育。 为了进一步筛选这些蛋白可能的结合因子，我们构建了非洲爪蟾双杂交cDNA文库。其中利用修饰的随机引物和特别设计的连接头在合成双链cDNA时，在下游合成一个SalI限制性酶切位点，可以将cDNA定向插入载体。通过对于空克隆率和插入片段长度等一系列参数的分析，表明这个定向cDNA文库的构建是成功的。 |
| 英文摘要 | The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus Laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes start at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed both in the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 only in the cytoplasm, indicating their different functions. Genetic mutations in human gene microcephalin1 (MCPH1) cause primary microcephaly, and during evolution of human being, the fast changes in this gene play important roles in the enlargement of brain size and the increase of cognitive ability. But the functions of MCPH gene in other species are still not clear. We cloned the Xenopus laevis MCPH A and B genes. Paralog B encodes a truncated form of MCPH and it is not known whether it is functional or not. The domain structure of MCPHA and the BRCT domains are well conserved suggesting its conserved role in development. However, in situ hybridization study showed that Xenopus MCPH genes are expressed in neural placode region at neurula stage and later in the branchial arch regions, but not clear in the brain. In the mouse, MCPH is reported to be strongly expressed in the brain. This clear difference might suggest different roles for MCPH during evolution. To screen for interacting factors of these proteins, we constructed a Xenopus yeast two-hybrid cDNA library. When synthesizing the double-strand cDNA, we used modified random primers and specifically designed adaptors. A complete SalI site formed at the 3’-end of the cDNA when the primer, and the adaptor ligated properly. The cDNAs were then introduced into the linearized vector directionally. We checked the ratio of empty colony, the average size of inserts, and the presence of several known gene in the library, the results showed that the construction of the cDNA library was successful. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6095]  |
| 专题 | 昆明动物研究所_发育生物学 |
| 推荐引用方式 GB/T 7714 | 武景阳. 非洲爪蟾Brunol和MCPH基因在早期胚胎发育过程中表达的研究及随机定向酵母双杂交cDNA文库的构建[D]. 北京. 中国科学院研究生院. 2008. |
入库方式： OAI收割
来源：昆明动物研究所
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