FACS-based in planta BiFC screening

基于 FACS 的植物 BiFC 筛选

• 批准号: 38601546
• 负责人: Professor Dr. Klaus Harter
• 金额: --
• 依托单位: Abteilung für Pflanzenphysiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/38601546?language=en
• 关键词: FACS; based; planta; BiFC; screening

For the first round of the SPP1212, our proposed research was divided into two parts. In the first part we proposed the exploration of the functional role of two-component signalling systems (TCS) in Arabidopsis-microbe interactions. This part was not recommended for funding. In the second part we had proposed to establish a novel Bimolecular Fluorescence Complementation (BiFC)-based in vivo two-hybrid system for the identification and characterisation of protein-protein interactions in living plant cells by using fluorescence activated cell sorting (FACS). The establishment of the in vivo BiFC based interaction screen was more challenging than expected. However we have managed to develop a working protocol. Our approach is of great interest to most groups in and also outside of the SPP1212 that are working on in planta protein-protein interaction and protein complex formation. This proposal is targeted towards further improving the FACS-based in planta BiFC screening approach and offering the technology and expertise to the members of the SPP1212 in a dedicated fashion. Furthermore, we also offer research support, by providing dedicated cytometric and FACS expertise for other applications besides the BiFC screen to the SPP1212 community such as promoter transactivation, sorting of cells and nuclei for “-omics” work (e.g. transcriptomics, proteomics, ChIP-chip) and monitoring cell cycle (e.g. DNA content).

对于第一轮的SPP1212，我们提出的研究分为两部分。在第一部分中，我们提出了两组分信号系统(TCS)在拟南芥-微生物相互作用中的作用的探索。这一部分不被建议用于资助。在第二部分中，我们提出建立一种新的基于双分子荧光互补(BIFC)的活体双杂交系统，用于利用荧光激活细胞分选(FACS)来鉴定和表征活植物细胞中蛋白质-蛋白质的相互作用。基于体内BIFC的交互屏幕的建立比预期的更具挑战性。然而，我们已经设法制定了一项工作协议。我们的方法对SPP1212内外致力于植物蛋白质相互作用和蛋白质复合体形成的大多数小组都非常感兴趣。这项建议旨在进一步改进基于PANTA BIFC的FACS筛查方法，并以专门的方式向SPP1212的成员提供技术和专业知识。此外，我们还提供研究支持，为SPP1212社区的BIFC筛查之外的其他应用提供专门的细胞学和FACS专业知识，如启动子反式激活、细胞和细胞核的“组学”工作(例如转录组学、蛋白质组学、芯片)和监测细胞周期(例如DNA含量)。