gp150蛋白对盘基网柄菌多细胞发育基因表达的影响

发布时间：2018-06-16 14:48

  本文选题：盘基网柄菌 + gp150　； 参考：《华东师范大学》2005年硕士论文

【摘要】：盘基网柄菌(Dietyostelium discoideum)是一种低等的真核原生生物,在营养丰富的条件下以阿米巴单细胞形式吞噬细菌为食,行二分裂方式繁殖生长。一旦食物匮乏,细胞由自由生活状态进入多细胞发育阶段。约100,000个细胞在趋化信号cAMP的诱导下聚集成一个多细胞结构,形态发生和细胞分化的结果是形成孢子来帮助有机体度过恶劣环境。盘基网柄菌与后生动物有许多共同的细胞生理生化反应,如胞浆移动、吞噬作用、趋化性、信号转导以及一些发育事件如细胞分类、模式形成和细胞类型决定等。在其它模式有机体中,这些细胞行为和生化反应并不都能很好表现出来,因此盘基网柄菌已作为一种独特的模式生物来研究细胞运动、细胞类型分化以及众多发育事件的分子机理。 实验以从加拿大多伦多大学引进的盘基网柄菌野生型KAX-3细胞株和突变型AK127(gp150缺失)细胞株为材料。剥夺食物触发多细胞的发育后,野生KAX-3细胞经历趋化性聚集、细胞丘形成和蛞蝓体阶段最后形成子实体,发育顺利完成。而突变AK127细胞由于不能表达gp150蛋白,尽管发育早期细胞趋化性聚集,但到疏松聚集阶段发育停滞下来,细胞的分化被锁住,最后细胞解聚。gp150为细胞丘内的形态发生建立一个粘附微环境,同时它也参与调节细胞类型专一化和分化的信号途径。gp150是一种细胞表面膜蛋白,它作为粘附分子,推测介导细胞之间的信号传递,调节细胞内部的酶促反应,开启发育所需基因的表达,进而推进发育进程。突变AK127多细胞发育停滞,在基因表达上必然与野生型KAX-3细胞存在差异。gp150介导的信号途径影响了哪些发育所需基因的表达,以及这些基因在盘基网柄菌发育中的作用正是本论文的主要研究内容。 采用mRNA差异显示法来分析盘基网柄菌野生型KAX-3细胞与突变型AK127细胞在多细胞发育阶段基因的表达差异。提取多细胞发育重要阶段(发育后12h、14h和16h)的两种细胞总RNA,反转录合成cDNA第一链,进行不同锚定引物与任意引物组合的差异显示。琼脂糖凝胶电泳图谱上观察到两种细胞间存在明显的差异表达条带。选取5条特征的差异条带,进行回收和克隆,酶切鉴定。Northern杂交检测,最终确定3个差异片段:d12b、d14a和d16a。片段测序并运用NCBI数据库系统对这3个片段分别进行核苷酸和氨基酸序列同源性信息查询。分析结果表明:d12b与已知基因同源性低,氨基酸序列比对没有找到同源性高的蛋白;d14a与已知基因同源性低,能在数据库中查找到已翻译完成的d14a氨基酸序列,通过SMART软件进行蛋白质检索,最终得知d14a序列可能对应的蛋白质为IgC2;d16a与盘基网柄菌线粒体DNA有较高同源性,氨基酸序列比对发现它与盘基网柄菌线粒体核糖体蛋白S4(Dd-mtRPS4)有同源性。Dd-mtRPS4在盘基网柄菌单
[Abstract]:Dietyostelium discoideum is a kind of low eukaryotic protozoa which feeds on amoeba single cell phagocytic bacteria and propagates and grows in a dimitotic manner. Once food is scarce, cells move from free living to multicellular development. About 100000 cells were induced by chemotactic signal camp to form a multicellular structure. The result of morphogenesis and cell differentiation was the formation of spores to help the organism through the harsh environment. There are many common cellular physiological and biochemical responses such as cytoplasmic mobility, phagocytosis, chemotaxis, signal transduction and some developmental events such as cell classification, pattern formation and cell type determination. In other model organisms, these cell behaviors and biochemical responses are not well demonstrated, so the Phaeopsis discus has been used as a unique model organism to study cell movement. Cell type differentiation and the molecular mechanism of many developmental events. The wild-type KAX-3 cell lines and mutant AK127Gp150 deletion cell lines from the University of Toronto, Canada, were used as materials. After deprivation of food triggered the development of many cells, the wild KAX-3 cells experienced chemotactic aggregation, cell mound formation and final fruiting body formation in slug stage, and the development of wild KAX-3 cells was completed successfully. But the mutant AK127 cells could not express gp150 protein, although the chemotactic aggregation of the cells at the early stage of development, but at the stage of loose aggregation, the differentiation of the cells was blocked. Finally, cell deaggregation.gp150 establishes an adhesion microenvironment for morphogenesis in the cell colliculus. At the same time, it is involved in regulating the signal pathway of cell type specificity and differentiation. Gp150 is a cell surface membrane protein, which acts as an adhesion molecule. We speculated to mediate the signal transduction between cells, regulate the enzymatic reaction in the cells, turn on the expression of genes needed for development, and then promote the development process. The gene expression of the mutant AK127 cells is stagnant, and the signaling pathway mediated by gp150 may influence the expression of which genes are needed for development, and the gene expression is different from that of the wild-type KAX-3 cells. The role of these genes in the development of Staphylococcus discus is the main research content in this paper. MRNA differential display was used to analyze the difference of gene expression between wild-type KAX-3 cells and mutant AK127 cells. Two kinds of cell total RNAs were extracted at the important stage of multi-cell development (12h ~ 14h and 16h after development). The first strand of cDNA was synthesized by reverse transcription and the difference between different anchored primers and arbitrary primer combinations was displayed. There were obvious differentially expressed bands between the two kinds of cells on agarose gel electrophoresis. Five characteristic differential bands were selected for recovery and cloning. Northern blot was used to identify the three differential fragments: d12bnd14a and d16a. The nucleotide and amino acid sequence homology information of the three fragments were queried by NCBI database system. The results showed that the homology of the two genes was low, and the amino acid sequence alignment did not show that the protein with high homology was of low homology with the known gene, and the translated d14a amino acid sequence could be found in the database. The result of protein retrieval by smart software showed that the possible protein corresponding to d14a sequence was IgC2D16a, which had high homology with mitochondrial DNA of P. dissecta. Amino acid sequence alignment revealed that it was homologous to S4Dd-mtRPS4. Dd-mtRPS4 was found to be homologous to S4Dd-mtRPS4.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q75

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