Collaborative Research: Arabidopsis 2010: In Vivo Genomics: Visualizing G Protein Interactions in Arabidopsis

异源三聚体G蛋白以细胞特异性方式将受体识别的多种多样的信号（例如光、病原体、激素）偶联到下游效应物。在拟南芥中，假设G蛋白参与多个信号转导和发育过程可以使用该项目开发的工具进行评估。该基因组包括16个七螺旋膜蛋白（候选G蛋白偶联受体），一个典型的Ga亚基，一个Gb，和两个Gg亚基（异源三聚体复合物），和三个'extralarge' Ga相关蛋白（XLG）。将开发的资源/工具是：1）GUS构建体，其允许基因组的表达定位;和2）构建体，其允许使用青色荧光蛋白（CFP）标记的蛋白质和黄色荧光蛋白（YFP）标记的蛋白质之间的荧光能量转移（FRET），在真实的时间和空间维度中体内基因组成员之间的物理相互作用的可视化。GUS构建体将在第一年资助结束时可用，YFP和CFP翻译融合构建体将在第二年资助结束时可用。矢量图、表达水平和模式的图像、方案更新以及获取材料的说明将每周发布在以下URL上，这些URL将与TAIR链接：www.plantbiology.unc.edu/构建体将可通过网页订购。对于每个翻译融合，三个稳定的转基因品系将在验证后存放在储备中心。作为本项目目标的基因家族成员的功能将如下确定。将在植物中评估基因的表达模式。 将评估基因项目在根细胞增殖和保卫细胞信号传导中的作用，并鉴定调节植物G蛋白信号传导途径的新型小分子，如FRET“读数”所评估的。 该项目开发的工具将允许社区对假设的G蛋白参与任何信号转导或发育背景进行直接的体内测试。随着对植物细胞体内FRET分析用于监测G蛋白复合物和G蛋白偶联受体之间的相互作用的深入了解，该工具将可用于其他植物研究组，用于评估其他系统中的体内蛋白质：蛋白质相互作用。此外，YFP构建体和细胞系将与von Arnheim/约翰逊2010项目（田纳西大学）开发的BRET分析兼容。拟议研究的更广泛影响包括由两位主要研究者直接指导的博士前和博士后培训，以及PI组织的G蛋白研讨会期间的FRET测量实践教程。讲习班将特别在小型文理学院和少数民族院校进行宣传，本科生将有资格申请旅费赠款。

Heterotrimeric G proteins couple multiple and diverse signals (e.g. light, pathogens, hormones) recognized by receptors to downstream effectors in a cell-specific manner. In Arabidopsis, hypothesized G-protein involvement in multiple signal transduction and developmental processes can be assessed using the tools developed by this project. The gene set comprises 16 heptahelical membrane proteins (candidate G protein-coupled receptors), one canonical Ga subunit, one Gb, and two Gg subunits (of the heterotrimeric complex), and three 'extralarge' Ga-related proteins (XLGs). The resources/tools that will be developed are: 1) GUS constructs allowing localization of expression for the gene set and; 2) constructs allowing visualization of physical interactions between members of the gene set in vivo in real time and with spatial dimensions using fluorescence energy transfer (FRET) between cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged proteins. The GUS constructs will be available at the conclusion of the first year of funding, and the YFP and CFP translational fusion constructs will be available at the conclusion of the second year of funding. Vector maps, images of expression levels and patterns, protocol updates, and instructions for obtaining material will be posted weekly at the following URL, which will be TAIR-linked: www.plantbiology.unc.edu/ Constructs will be available for order via the webpage. For each translational fusion, three stable transgenic lines will be deposited in the stock center following validation.The significance of the proposed work in relation to the overall 2010 project objectives is twofold. Function of the members of the gene family that is the target of this project will be determined as follows. Expression patterns of the genes will be evaluated in plants. The role the gene projects have in root cell proliferation and guard cell signaling will be evaluated, and novel small molecules that modulate plant G protein signaling pathways, as assessed by the FRET 'readout', will be identified. Tools developed by the project will allow the community to perform direct in vivo tests of hypothesized G protein involvement in any signal transduction or developmental context. As insights are gained into use of plant cell in vivo FRET analysis for monitoring the interaction between G protein complexes and G protein-coupled receptors, this tool will be made available to other plant research groups for evaluation of in vivo protein:protein interactions in other systems. In addition, the YFP constructs and lines will be compatible with BRET analysis being developed by the von Arnheim/Johnson 2010 project (Univ. Tennessee).The broader impact of the proposed research includes pre- and postdoctoral training by direct mentorship of the two Principal investigators, and a hands-on tutorial on FRET measurements during a G protein workshop organized by the PIs. The workshop will be advertised in particular at small liberal arts colleges and minority institutions and undergraduate students will be eligible to apply for travel grants.