【作者】 葛茜；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理学， 2007， 博士

【摘要】 Caenorhabditis elegans(C. elegans,线虫),一种体长仅1 mm的土壤线虫,最早被英国科学家西德尼·布雷纳作为一种模式生物,用于研究发育和遗传学。线虫是一种非常理想的遗传学研究模型。首先,它的基因组已经被完全解析,具有5对常染色体和一对性染色体。其次,它生活史短,个体小,可以自体受精,可以方便快捷地进行正义、反义遗传筛选;第三,易于用各种遗传学技术对其进行操作和研究。基于线虫在多方面的优势,我们希望建立起一套线虫培养、操作的实验平台,尝试在线虫中开展细胞分泌特性和相关蛋白质功能的研究。我们介绍了线虫培养过程中的各项操作方法。在实验室内,线虫可以在琼脂平板上进行培养,以大肠杆菌OP50为食。线虫雌雄同体共有959个体细胞,胚胎发育的细胞谱系已经完整地被描绘出来。而且,关于线虫302个神经元的位置、形态等也了解得比较清楚。因此,我们通过线虫胚胎细胞原代培养技术,成功地在体外培养了多种神经细胞和肌肉细胞,并对其形态特征进行了观察和分析。然后,我们选择了tph-1::GFP融合蛋白特异标记的虫系GR1366进行胚胎细胞的体外培养,得到了有荧光标记的线虫咽部NSM神经元。NSM神经元主要分泌的神经递质是5-HT。利用全细胞膜片钳和膜电容检测技术,记录了NSM神经元上的通道电流,该电流表现出明显的外向整流的特性。利用光解释放钙离子刺激,首次在体外培养的线虫细胞上记录到细胞分泌引起的膜电容增加,增加量平均在10 fF左右,具有良好的信噪比。采用碳纤电极安培测量技术,记录到NSM在高钾刺激下5-HT的电流信号。在本文中,我们还进行了线虫基因突变库的构建,采用TMP和紫外照射,对大量的线虫进行随机突变。突变库建成之后,可以选择感兴趣的基因,特别是在细胞分泌过程中起作用的基因,设计相应的PCR反应的引物,通过一系列的筛选,即有可能从突变库中发现相应的突变等位基因。利用突变的线虫个体,就可以从单细胞水平对分泌特性和动力学过程进行分析,也可以从行为学深入研究基因和蛋白质的关系。更多还原

【Abstract】 The 1 mm large soil nematode Caenorhabditis elegans was first used by Sydney Brenner to study the genetics of development and behavior.C. elegans is an ideal genetic model system. Firstly, its complete genome, consisting of five pairs of autosomal and one pair of sex chromosomes, has been sequenced and annotated. Secondly, due to the short generation time, self-fertilization and small size of C. elegans, forward and reverse genetic screens are fast, easy, cheap and can be easily scaled up. Thirdly, C. elegans is susceptible to manipulation using a range of genetic techniques. Because the advantages of C. elegans are numerous, we attempt to establish a research stage for the culture and manipulation of C. elegans, and study on the characteristic of secretion and protein function related secretory vesicles and membrane.We described all the techiniques using in the project, including maintaining a culture of a worm strain. In the laboratory, C. elegans is cultured on Petri dishes containing buffered agar and OP50 that serves as food.The C. elegans hermaphrodite consists of 959 somatic cells. Of all cells, the complete embryonic lineage has been determined. Furthermore, the positions, morphologies, synapses and gap junctions of all 302 neurons have been described. The primary culture system that allows culture of C. elegans embryonic cells had been described. We observed several types of cells and compared their morphological characterization.And using tph-1::GFP transgenic strain GR1366, we cultured the NSM neurons located on the pharynx in C. elegans. The major neurotramitter of NSM neuron is 5-HT. We undertook a series of patch clamp studies to in vitro electrophysiological analysis. The cultured NSM neuron expressed slowly inactivating, outwardly rectifying currents. By using high time resolution measurements of membrane capacitance, flash photolysis of caged Ca2+ and Carbon-fiber amperometric measurement, we first characterize ~10 fF Cm increasing and the 5-HT release spike current in vitro neurons in C. elegans.In this dissertation, we also present the construction and screening of deletion mutant libraries to generate C. elegans knockouts. This gene knockout strategy used a random mutagen, TMP and UV to mutagenize a very large number of worms. If PCR primers flanking an area of the gene of our interest are used to amplify from the genomic DNA samples, deletions between the primes can be detected. Once a DNA sample containing a deletion is identified, one can work back to identify the subculture of worms. Individual live animals carrying the deletion mutation can be used to study the molecular and cellular basis of secretory, even in the relationship of gene and behavior.更多还原