Functional Genomics of Protein Phosphorylation in Arabidopsis Chloroplasts

拟南芥叶绿体蛋白质磷酸化的功能基因组学

• 批准号: 5388608
• 负责人: Professor Dr. Dario Leister
• 金额: --
• 依托单位: Lehrstuhl für Molekularbiologie der Pflanzen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5388608?language=en
• 关键词: Functional; Genomics; Protein; Phosphorylation; Arabidopsis

Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cell architecture. Chloroplast or mitochondrial localization is predicted or has been found for 152 protein kinases (OPKs, organellar protein kinases) in Arabidopsis thaliana. The goal of this project is to define systematically targets and physiological role of phosphorylation mediated by OPKs by a combined approach, including (i) intracellular localization of the 50 OPKs with disputable organelle targeting by transient expression of RFP-fusions, (ii) mRNA expression profiling of OPKs for a number of organelle-relevant conditions, (iii) identification of knock-out mutants for 75 OPKs and their characterization with respect to key organellar functions in a gauntlet-type approach, (iv) identification of substrates of OPKs by differential proteomics (2-D gel analyses of 32P-labelled organellar proteins combined with mass spectrometry for wild-type [WT] versus OPK knock-outs). (v) For selected OPKs, transgenic plants (overexpressors and/or gain-offunction mutants) will be generated, and interactors will be identified by systematic two-hybrid screens.

蛋白质磷酸化是调节代谢、基因表达和细胞结构的主要模式。拟南芥中152种蛋白激酶（OPKs，细胞器蛋白激酶）的叶绿体或线粒体定位已经被预测或发现。该项目的目标是通过综合方法系统地定义由opk介导的磷酸化的靶标和生理作用，包括(i)通过rfp融合的瞬时表达对有争议的细胞器靶向的50种opk进行细胞内定位，（ii）在许多细胞器相关条件下对opk的mRNA表达谱进行分析，（iii）鉴定75种OPK的敲除突变体，并在gaunlet型方法中对其关键细胞器功能进行表征；（iv）通过差异蛋白质组学鉴定OPK的底物（对32p标记的细胞器蛋白进行二维凝胶分析，结合质谱分析野生型[WT]与OPK敲除型）。(v)对于选定的OPKs，将产生转基因植物（过度表达和/或获得功能突变体），并将通过系统的双杂交筛选确定相互作用。