Functional characterisation of the two-component signalling system of Arabidopsis

该提案的目的是研究拟南芥中双组分信号系统（TCS）功能的具体方面。在高等真核生物中，TCS是植物所特有的。拟南芥含有8种杂交传感器激酶（AHKs）、5种组氨酸磷酸转移蛋白（AHPs）和22个编码两种不同类型应答调节因子（A型和B型ARR基因）的基因。TCS成员的大部分功能，特别是它们参与的信号通路以及它们如何通过与拟南芥其他蛋白质的相互作用和基因激活实现信息传递尚不清楚。我们将以两种方式促进对TCS的更好理解。我们克隆并表达了大部分TCS基因，利用双杂交筛选和亲和共纯化结合质谱技术，鉴定了TCS蛋白与拟南芥蛋白质组其他蛋白之间的蛋白-蛋白相互作用。通过全基因组表达分析，我们已经确定了一组细胞分裂素的即时早期反应基因，这些基因通过TCS发出信号。除了一般的表型分析外，这些基因还可以作为内源性报告基因来表征单个和多个细胞分裂素受体敲除突变体以及表达功能获得和显性阴性TCS转录因子的细胞系，从而识别单个TCS途径。对TCS这些具体方面的详细分析是对参与阿富汗政府网络或2010年项目的其他团体的方案的补充。

The objective of the proposal is to study specific aspects of the function of the two-component signalling system (TCS) in Arabidopsis. The TCS is, among the higher eukaryotes, unique to plants. Arabidopsis harbours eight hybrid sensor kinases (AHKs), five histidine phosphotransfer proteins (AHPs) and 22 genes encoding two different types of response regulators (type A and type B ARR genes). Most of the functions of members of the TCS, in particular in which signalling pathway they participate and how they realize information transfer via interactions with other proteins of Arabidopsis and through gene activation are unknown. We will contribute to an improved understanding of the TCS in two ways. We have cloned and expressed most of the TCS genes in order to identify proteinprotein interactions between proteins of the TCS and other proteins of the Arabidopsis proteome by means of twohybrid screens and affinity co-purification coupled with mass spectrometry. By genome-wide expression analysis we have identified a set of immediate early response genes to cytokinin, which signals through the TCS. These will serve, beside a general phenotypical analysis, as endogenous reporter genes to characterize single and multiple cytokinin receptor knockout mutants and lines expressing gain-of-function and dominant-negative TCS transcription factors in order to identify individual TCS pathways. The detailed analysis of these specific aspects of the TCS is complementary to programmes of other groups participating in AFGN or the 2010 project.