非洲爪蟾早期发育中的转录后调控机制及眼睛发育相关基因的表达研究
文献类型：学位论文
| 作者 | 马莉 |
| 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 | 转录后调控 Sox2 3’UTR Xfhl3 非洲爪蟾 XSox1 III |
| 其他题名 | The study of post-transcriptional regulation of neural development and eye specific gene expression in Xenopus laevis |
| 学位专业 | 动物学 |
| 中文摘要 | 近来的研究表明，转录后调控对于调节脊椎动物发育过程中的细胞分化，细胞分裂及基因区域特异性表达都具有重要作用。转录后调控包括对mRNA稳定性、翻译效率、细胞内定位及poly(A)水平的调控等。Sox2基因是脊椎动物早期发育中最早表达的神经系统特异性基因之一，是脊椎动物早期神经系统发育的重要调节因子。通过生物信息学分析，我们发现，在脊椎动物Sox2 mRNA 3’非翻译区中存在4段非常保守的富含AU的区域，通过报告基因分析等手段研究发现Sox2 3’非翻译区中的部分元件可显著提高报告基因表达，提示我们Sox2的表达可能受到转录后调控。 我们通过对爪蟾Xfhl3基因序列分析时发现其3’非翻译区存在一段保守的只在两栖类具有的序列，我们克隆并检测了该基因的表达图式，并采用报告基因分析等手段研究了Xfhl3基因 3’非翻译区对报告基因表达的影响。结果发现其3’UTR可抑制报告基因表达水平。由于Xfhl3基因3’UTR中这段序列只在爪蟾基因中高度保守，而在在进化过程中两栖动物最独特的便是变态现象，这提示我们去探索这段爪蟾特有的保守序列是否与两栖类变态发育密切相关。由于甲状腺激素在两栖类的变态中的重要作用，因此我们设想Xfhl3基因的3’UTR中的保守序列可能与甲状腺激素相互作用共同调节爪蟾的变态过程。我们的初步结果表明，在爪蟾胚胎中，甲状腺激素对于正常报告基因表达没有明显的作用，但是在插入Xfhl3基因3’UTR中保守序列后，甲状腺激素处理可显著提高报告基因的表达，表明甲状腺素可能直接或间接通过与该段保守序列参与基因的表达调控。 脊椎动物的眼是一个功能非常特殊的器官，受到复杂的调控网络的调节，众多对神经发生重要的基因在眼中表达并参与了这一调节过程。我们克隆了非洲爪蟾的Sox1基因并研究了它在非洲爪蟾早期发育过程中的时空表达图式，比较了Sox1-3基因在发育的脑和眼中的表达图式，进一步阐明SoxB1基因家族在脊椎动物神经系统发生过程中的作用。此外，我们还克隆了非洲爪蟾MGC85160基因并利用RT-PCR和胚胎整体原位杂交技术探测它在不同胚胎阶段的时空表达图式。结果表明母源性表达的MGC85160基因早期主要在动物极表达；从神经板期开始在发育的中枢神经系统和眼中表达，石蜡切片显示它主要在视网膜和晶状体中表达，说明该基因在爪蟾早期外胚层的模式化以及中枢神经系统的发育过程中可能起到重要作用。 此外，我们还研究了鱇浪白鱼的早期发育分期和眼睛特异基因的表达图式。鱇浪白鱼（Anabarilius grahami ）是云南抚仙湖的特有鱼种。我们首次完成了鱇浪白鱼早期发育的完整分期，主要包括合子期，卵裂期，囊胚期，原肠期，体节期和孵化期六个主要的时期。为了理解鱇浪白鱼眼睛的发育，我们克隆并检测了在眼发育早期起关键作用的基因Sox2, Pax6a, Six3a 和 Rx2的表达图式。结果表明这四个基因全部在尾芽期的前端神经板中表达，随后在视网膜原基细胞中表达明显。在晚期阶段，除Rx2外其它三个基因也在晶状体中表达。其表达模式与斑马鱼中同源基因的表达很相似，说明涉及眼发育的分子网络在鱇浪白鱼中也是高度保守的。 |
| 英文摘要 | Post-transcriptional regulation, including the regulation of pre-mRNA alternative splicing, mRNA stability, localization and translation, play important roles in embryonic development. Post-transcriptional control is often achieved through elements in its 5’- or 3’-untranslated region (UTR) of an mRNA. Recent studies show that post-transcriptional control is also crucial for vertebrate neural development. Sox2 gene encodes one transcriptional factor and is one of the earlist neural determinants of the neural system in vertebrate. Sox2 is expressed strongly in the neuroepithelium of the developing central nervous system and has been shown to function in neural stem cells. Through bioinformatical analysis, we found that the 3’UTR sequences of vertebrate Sox2 genes are highly conserved and contained four conserved AU rich fragments. Through reporter gene analysis, we analyzed the ability of the elements in Sox2 3’UTR to regulate gene expression in Xenopus embryos and cultured cells. Our results showed that two of these elements were able to promote reporter expression significantly, suggesting that the expression of Sox2 might be regulated post-transcriptionally. We also identified one element from Xenopus fhl3 3’UTR that was highly conserved in the 3’UTR of about 20 different genes. Interestingly, so far this element is found only in Xenopus. To study its possibile involvement in post-transcriptional regulation, we compared the expression of reporter genes with or without this element in Xenopus embryos and 293T cells. This element was found able to reduce the expression of reporter genes. Since it is found only in amphibians, we speculated that it might be involved in the regulation of certain amphian specific process, such as metamorphosis. To check this possibility, we checked the responsiveness of the reporter genes to thyroid hormone, the key regulator of metamorphosis in amphibian. Our results showed that thyroid hormone treatment could significantly enhance the expression of the reporter genes with this element, but not the control reporter. The results suggested the possibility for this conserved element to mediate thyroid hormone induced post-transcriptional control of the target genes. Vertebrate eye development is controlled by a complex regulatory network. Several transcription factors that pattern the anterior neural plate of the early vertebrate embryo are essential for normal eye development. We studied the temporal and spatial expression pattern of Sox1 and MGC85160 genes during Xenopus laevis early development, and compared the expression patterns of Sox1-3 in the developing eye and brain. Sox1 was expressed maternally and was detected in the animal pole at cleavage to blastrula stages and in the central nervous system (CNS) and the developing eye from neurula stage on. At neurula to tailbud stages, MGC85160 was also strongly expressed in the CNS and the developing eye. Anabarilius grahami is a cyprinoid fish endemic to Fuxian Lake, Yunnan, China. In this study, we also produced a comprehensive staging series of A. grahami. The embryonic development of A. grahami was divided into six main periods: zygote period, cleavage period, blastula period, gastrula period, segmentation period and hatching period. Its embryonic development is essentially similar to that of zebrafish but relatively slower. We have also cloned and checked the expression patterns of A. grahami Sox2, Pax6a, Six3a and Rx2 genes during eye development. The four genes showed similar expression patterns to their zebrafish homologs, suggesting the evolutionary conservation of the regulatory network of eye development. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6093]  |
| 专题 | 昆明动物研究所_发育生物学 |
| 推荐引用方式 GB/T 7714 | 马莉. 非洲爪蟾早期发育中的转录后调控机制及眼睛发育相关基因的表达研究[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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