gp150蛋白对盘基网柄菌多细胞发育基因表达的影响
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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
The social amoeba Dictyostelium discoideum is a lower eukaryote protozoan. The amoeboid cells grow as separate, independent cells feeding on bacteria by phagocytosis and multipling by equal mitotic division. Exhaustion of the food source triggers a development programme, in which more than 100,000 cells aggregate by chemotaxis to form a multicellular structure. Morphogenesis and cell differentiation then culminate in the production of spores, enabling the organism to survive unfavourable condictions. A wide range of fundamental cellular processes are common to D. discoideum and metazoans. These processes include cytokinesis> motility、 phagocytosis、 chemotaxis、 signal transduction and aspects of development such as cell sorting、 pattern formation and cell-type determination. Many of these cellular behaviors and biochemical mechanisms are either absent or less accessible in other model organisms, making D. discoideum unique model organism for the study of the cellular and molecular mechanisms of these processes.After food source is depleted, the wild-type strain KAX-3 undergoes chemotaxis, cell-aggregating stage、 mound-formation stage、 slug stage and forms normal fruiting-body in the end. However, the mutant-type strain AK127, as a result of absence of gpl50, undergoes normal chemotaxis, but is arrested at the loose aggregate stage. Aggregates of AK127 cells eventually dissociate into single cells. In addition, cell-type differentiation is blocked in these mutant cells. It is likely that gpl50 plays a regulatory role in the transition from the initial multicellular stage to the tight mound stage and in cell-type specification. gpl50 is a membrane glycoprotein which has been implicated in cell-cell adhesion and may function as a key molecule transferring signal between cells, further regulating intracellular enzyme-catalyzed reactions and activing expression of genes needing for development.To study the influence of the cell adhesion molecule gpl50 on the expression of genes needing for D. 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. Total RNAs from KAX-3 cells and AK127 cells, developed for 12h、 14h and 16h, were isolated. After the reverse transcription and PCR reaction, distinct differential fragments could be seen on 1.5% agarose gel. The five characteristic differential bands were recovered and cloned. Northern analysis recognized the three differential cDNA fragments, named d12、 d14a and d16a, respectively specific for the 12h、 14h and 16h of development. Sequence querying and comparing with nucleic acid and protein in NCBI data-base revealed dl2b is 131 bp in length, having low homology with other known genes and no high homology proteins found. dl4a is 602 bp in length, having low homology with other known genes. We found its translated amino acid sequence and confirmed it IgC2 by SMART. dl6a is 496 bp in length, having relatively high homology with D. discoideum mitochondrial ribosomal protein S4 (Dd- mtRPS4). Dd-mtRPS4 plays a pole in the transition of D. discoideum cells to differentiation, and mitochondrial also regulates D. discoideum development, participating in prespore cell differentiation during slug stage.Conclusion: dl2b may be a novel gene in D. discoideum development. The protein of dl4a may be IgC2, related to gpl30. dl6a may be a ORF of mitochondrial DNA and be involved in prespore cells differentiation.Next, we need to acquire cDNA whole sequence , design carrier to express protein and deeply identify their biological functions during D. discoideum development by experiment.
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
The social amoeba Dictyostelium discoideum is a lower eukaryote protozoan. The amoeboid cells grow as separate, independent cells feeding on bacteria by phagocytosis and multipling by equal mitotic division. Exhaustion of the food source triggers a development programme, in which more than 100,000 cells aggregate by chemotaxis to form a multicellular structure. Morphogenesis and cell differentiation then culminate in the production of spores, enabling the organism to survive unfavourable condictions. A wide range of fundamental cellular processes are common to D. discoideum and metazoans. These processes include cytokinesis> motility、 phagocytosis、 chemotaxis、 signal transduction and aspects of development such as cell sorting、 pattern formation and cell-type determination. Many of these cellular behaviors and biochemical mechanisms are either absent or less accessible in other model organisms, making D. discoideum unique model organism for the study of the cellular and molecular mechanisms of these processes.After food source is depleted, the wild-type strain KAX-3 undergoes chemotaxis, cell-aggregating stage、 mound-formation stage、 slug stage and forms normal fruiting-body in the end. However, the mutant-type strain AK127, as a result of absence of gpl50, undergoes normal chemotaxis, but is arrested at the loose aggregate stage. Aggregates of AK127 cells eventually dissociate into single cells. In addition, cell-type differentiation is blocked in these mutant cells. It is likely that gpl50 plays a regulatory role in the transition from the initial multicellular stage to the tight mound stage and in cell-type specification. gpl50 is a membrane glycoprotein which has been implicated in cell-cell adhesion and may function as a key molecule transferring signal between cells, further regulating intracellular enzyme-catalyzed reactions and activing expression of genes needing for development.To study the influence of the cell adhesion molecule gpl50 on the expression of genes needing for D. 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. Total RNAs from KAX-3 cells and AK127 cells, developed for 12h、 14h and 16h, were isolated. After the reverse transcription and PCR reaction, distinct differential fragments could be seen on 1.5% agarose gel. The five characteristic differential bands were recovered and cloned. Northern analysis recognized the three differential cDNA fragments, named d12、 d14a and d16a, respectively specific for the 12h、 14h and 16h of development. Sequence querying and comparing with nucleic acid and protein in NCBI data-base revealed dl2b is 131 bp in length, having low homology with other known genes and no high homology proteins found. dl4a is 602 bp in length, having low homology with other known genes. We found its translated amino acid sequence and confirmed it IgC2 by SMART. dl6a is 496 bp in length, having relatively high homology with D. discoideum mitochondrial ribosomal protein S4 (Dd- mtRPS4). Dd-mtRPS4 plays a pole in the transition of D. discoideum cells to differentiation, and mitochondrial also regulates D. discoideum development, participating in prespore cell differentiation during slug stage.Conclusion: dl2b may be a novel gene in D. discoideum development. The protein of dl4a may be IgC2, related to gpl30. dl6a may be a ORF of mitochondrial DNA and be involved in prespore cells differentiation.Next, we need to acquire cDNA whole sequence , design carrier to express protein and deeply identify their biological functions during D. discoideum development by experiment.
引文
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