| Cecr2 基因的分子克隆以及其在鸡胚发育中的初步功能探索 |
| Alternative Title | Cecr2 molecular cloning and its pre-paratory functional investigation on chicken embryo
|
| 陈敬臣 |
| Thesis Advisor | 王建林
; Beate Brand-Saberi
|
| 2009-05-24
|
| Degree Grantor | 兰州大学
|
| Place of Conferral | 兰州
|
| Degree Name | 博士
|
| Keyword | Cecr2
肌节
脊髓
神经管形成
鸡胚
RNA干扰
电穿孔
|
| Abstract | Cecr2基因来自于猫眼综合症染色体区域，先前的研究表明Cecr2蛋白可与SNF2L形成染色质重构复合物CERF。Cecr2在小鼠的突变导致露脑畸形，相当于人类的无脑畸形，表明此基因可能参与神经管形成的过程。通过RT-PCR，我们得到了鸡的Cecr2基因的cDNA序列全长并将其投到了基因数据库。其cDNA全长共4419bp。序列分析表明它含有“AT勾”以及“Bromo域”。通过对不同物种之间Cecr2基因序列的对比，发现Cecr2基因的“AT勾”和“Bromo域”在不同物种之间是高度保守的。含有“AT勾”和“Bromo域”是染色质重构蛋白的基本特征，表明Cecr2基因在不同物种之间可能通过保守的染色质重构来发挥其在生物体中的作用。进一步，通过原位杂交研究了Cecr2基因在鸡胚胎发育中的表达模式。除在神经管形成中主要表达在神经组织外，Cecr2还主要表达在发育中的肌节以及脊髓的中间区。表明Cecr2功能可能涉及到肌节的发育以及神经元的分化。为了进一步研究Cecr2基因在肌肉发生中的作用，比较了它与其他肌肉发生标志基因如Pax7、 Frek、MyoD、Myf5、Myogenin在体节中的表达模式。结果发现，Cecr2基因与这些肌肉发生标志基因在体节中有着不同的表达模式。所以，Cecr2基因可能不参与肌肉发生基因网络调控。但同时，Cecr2在肌节的表达模式与肌节发育中第二波细胞的运动动态相吻合，意味着Cecr2基因可能参与第二波肌节发育。为了更多的了解Cecr2在胚胎发育中的作用，设计，制备并测试了基于质粒载体的Cecr2基因的RNA干扰系统。首先把这些RNA干扰质粒利用电穿孔技术导入到鸡胚的神经管或体节中，经过一定时间的孵化后，通过绿色荧光蛋白的指示，发现shRNA成功的表达在手术侧神经管或体节中。对这些手术后的胚胎用Cecr2探针进行原位杂交发现Cecr2基因的表达在手术侧被成功的抑制了。而在对照组，Cecr2基因在对照侧和手术侧的表达没有明显的不同。为了进一步确认Cecr2 RNA干扰的效果，用尼氏兰进行了细胞凋亡的测试，在手术侧并没有发现明显的细胞凋亡。这些结果表明，本论文中所设计的RNA干扰质粒是有效的，可以用于进一步的Cecr2基因功能的研究。 |
| Other Abstract | The full length of the coding sequence of the chicken homologue of cat eye syndrome chromosome region, candidate 2 (Cecr2) was obtained by RT-PCR and submitted to database. The full length is 4419 bp. Sequence analysis and alignment showed that it contained an AT hook and a bromodomain with high conservancy among different species, consistent with its role in chromatin remodeling. The expression pattern of chicken Cecr2 was subsequently investigated during chicken embryo development. Except for the main expression in the neural tissues during neurulation, it was predominantly expressed in the developing myotome and the intermediate zone of the spinal cord, suggesting its roles in myotome and neuronal development as well. To further analyze the role of Cecr2 in myogenesis, its expression pattern was compared with that of myogenic markers such as Pax7, Frek, MyoD, Myf5 and Myogenin in somites. The result showed that Cecr2 had a distinctly different expression from those myogenic markers, thus impossible to relate it with corresponding gene regulatory network during myogenesis. But further analysis revealed that the expression pattern of Cecr2 in the myotome resembled the cell dynamics of the second wave of myotome formation, implying its potential role in the second wave during the formation of the myotome. To better understand the role of Cecr2 during the embryo development, plasmid vector based shRNA constructs for RNA interference (RNAi) were designed and prepared. These constructs were electroporated into the neural tube or somites of the chicken embryo at proper stages. It was observed that shRNA was expressed in the operated side denoted by the expression of enhanced green fluorescent protein (EGFP). In situ hybridization with Cecr2 probe after the electroporation demonstrated that Cecr2 experssion on the operated side was successfully down regulated. In contrast, H1-scramble constructs electroporated embryos showed no obvious difference of expression between control and operated sides. To examine if the apoptosis was induced by the operation, thus leading to the different expression level, nile blue sulfate (NBS) staining was performed. It turned out there was no prominent cell death displayed by the NBS staining. These results indicated that the Cecr2 shRNA constructs were effective in inhibiting the expression of Cecr2 and could be further used for functional study of Cecr2 during embryo development. |
| URL | 查看原文
|
| Language | 中文
|
| Document Type | 学位论文
|
| Identifier | https://ir.lzu.edu.cn/handle/262010/222037
|
| Collection | 生命科学学院
|
Recommended Citation
GB/T 7714 |
陈敬臣. Cecr2 基因的分子克隆以及其在鸡胚发育中的初步功能探索[D]. 兰州. 兰州大学,2009.
|
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.
