【作者】 刘伟；

【导师】 侯连生；

【作者基本信息】 华东师范大学 ， 动物细胞与分子生物学， 2009， 硕士

【摘要】 盘基网柄菌(Dictyostelium 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,单个细胞聚集成多细胞体,细胞历经细胞丘、蛤输体、拔顶阶段完成发育和分化过程,最终形成由孢子细胞和柄细胞组成的子实体。在合适条件下,孢子细胞萌发开始新生命周期。为了保持细胞聚集体的完整性和整个发育过程,盘基网柄菌在发育的不同阶段表达了特定的细胞粘附分子。盘基网柄菌一旦饥饿便进入多细胞发育期,在发育早期会表达粘附分子gp150。LagC基因编码的gp150蛋白是一种膜蛋白,一种异嗜性细胞表面粘附分子,通过细胞间异嗜性粘着的相互作用来调节细胞的粘着,以此来调节细胞的分化。gp150缺失的细胞株AK127不能完成多细胞发育,发育只能停留在细胞松散聚集阶段。由此推测,gp150蛋白在盘基网柄菌发育过程中起着重要作用,它可能参与介导细胞分化和凋亡的信号转导途径,但具体的信号通路至今仍未完全清楚。为了研究gp150对盘基网柄菌多细胞发育相关基因的影响,本实验室人员对野生型细胞KAx-3和突变细胞AK127发育14小时的细胞进行了mRNA差异显示分析,结果发现一差异表达片断(GenBank登录号:AY894718)。经blast分析,该片断为尿囊酸酶基因的一个片断。尿囊酸酶是嘌呤代谢中一种重要的酶,在灵长类、鸟类以及昆虫体内,嘌呤经过代谢最终以尿酸的形式排出体外;而在微生物、两栖动物和鱼中,尿酸则会继续水解为尿囊酸。尿囊酸在尿囊酸酶催化下生成脲乙醇酸和尿素,而脲基乙醇酸会最终水解为氨气、二氧化碳和乙醛酸。有报道认为氨通过抑制诱导分化因子(DIF)的累积来抑制柄细胞的发育并促进孢子细胞的形成。所以氨可能是盘基网柄菌细胞发育调控中一种重要的信号分子,但是其具体的调控作用还不是很清楚。为此我们将构建好的以盘基网柄菌尿囊酸酶基因为靶标的RNAi载体pAct15Gal-allCi,转染野生型KAx-3细胞,为研究该基因在盘基网柄菌发育的作用提供依据。本实验将已构建好的以盘基网柄菌尿囊酸酶基因为靶标的RNAi载体pAct15Gal-allCi,通过电击转化的方法转入野生型盘基网病菌KAx-3细胞。利用G418抗性筛选阳性克隆RNAi-allc。同时发育野生型细胞和RNAi-a11c转染细胞,发育20h后发现,转染细胞仅能形成类似细胞丘的小突起,不能完成完整的发育,而野生型细胞则能完成完整的发育,形成子实体。利用Western blot检测尿囊酸酶蛋白的表达,结果发现同野生型细胞比较,转染细胞中几乎检测不到有尿囊酸酶的免疫条带;利用流式细胞术检测转染细胞内尿囊酸酶分子的表达,结果发现表达量由野生型细胞的72.18%降为转染细胞的0.67%。表明本研究设计的核苷酸序列对靶标基因的表达有较明显的干扰作用,说明尿囊酸酶对盘基网柄菌细胞发育有一定的调控作用。更多还原

【Abstract】 The social amoeba Dictyostelium discoideum which phagoeytosis soil bacteria for food is a unieellular eukaryote.Under favorable condition,it multiplies as a unicellular organism.UP on starvation,a pathway involving aggregation,mound,slug, eulmination stages induces the formation of a fruitingbody consisting of a head of spores supported on a stalk of vacuolated cells.Spores await dispersal and germinate in favourable conditions for amoeboid growth.Cells of Dictyostelium discoideum are triggered to undergo development when they have depleted their food souree.During the early hours of development,cell adhesion molecules play an important role in maintaining the integrity of cell aggregation and influence subsequent cell-type differentiation and cell sorting.gp150 is normally first expressed at the loss aggregation stage and this adhesion moleculeis important for development.The membrane glycoprotein gp150 encoded by Lag C,play a role in cell-type specification by heterophilic interactions.A strain in which lagC is disrupted,AK127,undergoes normal chemotaxis,but is arrested at the loose aggregate stage.So we inferred that gp150 play a key role during the whole development of dictyostelium.It may be in the signal transduction way of cell differentiation and apoptosis.However the concrete signal route has not been known.To study the influence of the cell adhesion molecule gp150 on the expression of genes needing for Dictyostelium discoideum development,mRNA differential display was used to analyse the differences of gene expression between the wild strain KAx-3 cells and the mutant strain AK127 cells.Members of my laboratory found a gene fragnlent relating to the regulation of Dictyostelium discoideum development (GeneBanknumber:AY894718).By analysis of BLAST,we knew the fraglnent was part of allantoicase gene.Allantoiease is an important enzyme in purine degradation. In primates,birds,and insects,purine is converted into uric acid and then excreted.But in microorganisms,amphibians,and fish,uric acid is converted into allantioc acid. Allantoicases catalyze the hydrolytic conversion of allantoic acid into ureidoglycolate and urea.Ureidoglycolate is converted into ammonia,carbon dioxide,and glyoxylate. It was reported that ammonia inhibits stalk cell differentiation and promotes differentiation of pre-spore cells to spores by suppressing DIF cumulation.Ammonia may be an important signal molecule which regulate the development of Dictyostelium. However the concrete regulation has not been known.So we transfected RNAi expression vector pAct15Gal-allCi into KAx-3 as to study the gene function in Dictyostelium development.The RNAi expression vector pAct15Gal-allCi was transfected into KAx-3 by electroporation.Positive clones were obtained by stable transfection KAx-3 using pAct15Gal-allCi and G418 screening.Compared with untransfected KAx-3, transfected cells,arrested early stage of mound,could not complete integrative cell development.Western blot analysis showed allantoicase protein in interference sequence transfected cells was significantly down-regulated.The transfection rate was estimated by FACS analysis,indicating the allantoicase expression decreased from72.18%to 0.67%after RNAi transfection.The results suggest that allantoicase is interfered,which is required for the development of Dictyostelium discoideum.更多还原