Organellar metabolic networks in Stramenopiles (Heterokonts)

层生菌，包括硅藻和相关的无色卵菌，通过次生内共生进化。这一过程导致了代谢和遗传重组以及由于两个以前独立的真核细胞的内共生融合而导致的区室化的变化。目前对草莓属植物线粒体的生理生化研究还不多，特别是作为次生内吞作用宿主的真核细胞的起源还不清楚。在这个项目中，我们想研究硅藻细胞质、质体和线粒体之间的相互作用，以及它们的碳水化合物代谢。先前的工作已经证明，糖酵解的后半部分的每种酶的同工酶都靶向线粒体。为了研究这些平行的途径，我们想优化硅藻和卵菌的线粒体的制备，以测量各自的线粒体酶的活性。我们希望通过进行qPCR分析来确定单个基因的表达，来确定这两种（线粒体和胞质）途径是否在硅藻中起作用，以及细胞是否可以在两种途径之间转换，以响应内部或外部条件的变化。蛋白质水平的变化将通过蛋白质印迹法进行分析，活性的变化将通过酶分析进行分析。单个酶将在大肠杆菌中过表达，并进行酶学表征。通过遗传学的方法，三角褐指藻中的单个基因将被沉默使用诱导型启动子，然后对突变体进行生理学表征。

Stramenopiles, including diatoms and the related colourless oomycetes, evolved by secondary endosymbiosis. This process resulted in metabolic and genetic re-organization and changes in the compartmentation due to the endosymbiotic fusion of two formerly independent eukaryotic cells. Very little is known about the physiology and biochemistry of mitochondria of Stramenopiles, especially considering that the origin of the eukaryotic cell which served as host for secondary endocytobiosis is still unclear. In this project we want to study the interaction between cytosol, plastids and the mitochondria in diatoms with respect to their carbohydrate metabolism. Previous work has demonstrated that isoenzymes of each enzyme of the second half of the glycolysis are targeted to the mitochondria. To study these parallel pathways we would like to optimize the preparation of mitochondria from diatoms and oomycetes to measure activities of the respective mitochondrial enzymes. We want to find out whether both (mitochondrial and cytosolic) pathways are functional in diatoms and whether the cells can shift between both pathways, responding to changes of the internal or external conditions, by performing qPCR analysis to determine the expression of individual genes. The variation of the protein levels will be analysed by Western blots and the changes of activity by enzymatic analyses. Individual enzymes will be overexpressed in E.coli and characterized enzymatically. By genetic approaches individual genes in Phaeodactylum tricornutum will be silenced using inducible promoters followed by a physiological characterization of the mutants.