【作者】 杨腾；

【导师】 王玮；

【作者基本信息】 福建医科大学 ， 人体解剖与组织胚胎学， 2007， 硕士

【摘要】 研究背景和实验目的:NeuroD是一种bHLH(basic helix-loop-helix,碱性螺旋-环-螺旋)转录因子。在爪蟾的胚胎中,过表达外源NeuroD可以将外胚层中所有类型的细胞分化成为成熟的神经元。在胰岛素瘤细胞中,NeuroD可以结合并激活胰岛素启动子。NeuroD基因敲除的小鼠,位于早期分化中的胰腺内分泌细胞以及神经系统的某些品系的细胞死亡,结果导致胰腺中的胰岛细胞、小脑、海马结构及内耳感觉神经节细胞的缺失,产生糖尿病及包括共济失调、耳聋等神经系统方面的功能障碍。一系列功能获得和丧失的实验证实NeuroD调控不同组织中细胞的存活和分化。了解NeuroD作为转录因子在胰腺组织和神经系统的发育及其功能,为今后诊断和治疗相关的疾病提供有价值的帮助,我们拟重组大鼠NeuroD基因真核质粒表达载体。实验方法:1.NeuroD cDNA克隆从新生大鼠小脑中提取总RNA,巢式PCR扩增得到NeuroD基因,回收并纯化;Ligation Mix连接酶连接pMD19-T载体,转化感受态细菌JM109后经蓝白筛选;小量抽提重组克隆质粒DNA,应用限制性内切酶NheI和XhoI酶切鉴定阳性克隆并对阳性重组质粒。2.构建NeuroD基因真核质粒表达载体和序列分析用限制性内切酶NheI和XhoI对pMD19-T-NeuroD和pcDNA3.1A双酶切,分别回收纯化,以T4连接酶将二者连接,转化感受态DH5a。小量抽提重组质粒DNA,NheI和XhoI筛选鉴定重组质粒pcDNA3.1A-NeuroD并进行基因测序。结果:1. NeuroD基因克隆RT-PCR扩增得到的cDNA为1kb,与GenBank中NeuroD cDNA的长度一致。NeuroD基因片段与克隆载体pMD19-T连接后转化,经蓝白筛选得到大量菌落,PCR扩增鉴定和双酶切鉴定得到阳性重组克隆pMD19-T-NeuroD。2. NeuroD基因质粒表达载体构建双酶切重组质粒pMD19-T-NeuroD和pcDNA3.1A得到基因双粘片段连接后转化,得到大量菌落,通过双酶切鉴定得到阳性重组质粒pcDNA3.1A-NeuroD。序列分析结果与Genbank收录的大鼠NeuroD cDNA全长序列一致。结论:本研究成功克隆了大鼠NeuroD cDNA;成功构建了pcDNA3.1-NeuroD重组表达质粒。更多还原

【Abstract】 NeuroD (otherwise known as BETA2) is a basic helix-loop-helix (bHLH) transcription factor that is capable of converting embryonic epidermal cells into fully differentiated neurons in Xenopus embryos. In insulinoma cells, NeuroD can bind and activate the insulin promoter. When NeuroD is deleted in mice, the early differentiating pancreatic endocrine cells and a subset of the neurons in the central and peripheral nervous systems die, resulting in cellular deficits in the pancreatic islets, cerebellum, hippocampus and inner ear sensory ganglia. As a consequence, mice become diabetic and display neurological defects including ataxia and deafness. These gain-of-function and loss-of-function phenotypes suggest that NeuroD controls both common and distinct sets of molecules involved in cell survival and differentiation in different tissue types. Understanding the primary function of NeuroD will be extremely valuable in the diagnosis and cure of the diseases that involve this transcription factor, which plays essential roles in the development and function of the pancreas and the nervous system. We constructed the plasmid expression vector of rat NeuroD gene.Methods:1. Cloning of NeuroD cDNAThe total RNA was extracted from the newly born rat cerebellum. Reverse transcript reaction and PCR were used to obtain the entire NeuroD cDNA. The fragment of neurod cDNA and pMD19-T were ligated by Ligation Mix. After transferring them into the JM109 competent cells, the recombinant clones were selected and identified through blue and white screening and PCR. Extracted the recombinant plasmids DNA and digested it by restriction endonuclease NheI and XhoI for further identification.2. Construction the palsmid expressing vector of NeuroD and sequencing analysisThe DNA of pMD19-T-NeuroD and pcDNA3.1 A with restriction enzymes XhoI and NheI was digested and purified respectively, then the fragements with sticky ends of NeuroD gene and pcDNA3.1A were obtained. Ligated them with T4DNA ligase and transformed them into the DH5a competent cells. Extracted the recombinant DNA and identified the positive clones with XhoI and NheI. Then the positive recombinant pcDNA3.1 A-NeuroD was sequenced. Blast the gene in gene bank in the Internet. Results:1 .Cloning of NeuroD cDNAThe full length of NeuroD cDNA amplified by RT-PCR was 1.0kb, which was corresponded with its length provided by GenBank. After transformation of the ligated product as well as blue and white screening, many colonies were obtained. Through PCR with the primers and certain restricton endonuclease digestion, the positive transformed clones were identified.2. Construction of the plasmid expressing vector of neuroDAfter restriction endonuclease digestion of recombinant plasmids, two segments of about 5.5kb and 1.0kb appeared clear and bright in agarose gel electrophoresis. The sequence of cloned NeuroD gene was conformity with the sequence deposited in GenBank. Conclusions:Rat NeuroD cDNA was correctly cloned and the recombinant plasmid expression vector pcDNA3.1A-NeuroD was constructed successfully. This research has provided one of the most practical tools to study the role of NeuroD during the neural differentiation and application of gene therapy for the diseases of nerve injury and nerve degeneration.更多还原