Arabidopsis 2010: Bimolecular Fluorescence Complementation (BMFC) to Investigate Protein-protein Interactions in Planta

拟南芥 2010：双分子荧光互补 (BMFC) 研究植物中蛋白质-蛋白质相互作用

• 批准号: 0418709
• 负责人: Stanton Gelvin
• 金额: $ 100万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0418709&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Bimolecular; Fluorescence; Complementation

This project will provide research tools and resources for the plant science community to determine the in planta interactions of proteins. The research will develop Bimolecular Fluorescence Complementation (BMFC) methodology to investigate protein-protein interactions in planta. BMFC involves tagging proteins with complementary half-YFP molecules, neither of which fluoresces on its own. When half-YFP molecules are brought together as fusions with interacting proteins, fluorescence is restored. The BMFC design is a novel in planta "two hybrid" system. BMFC can also be utilized to identify the subcellular location of interacting proteins. The project will develop BMFC vectors to express half-YFP-tagged proteins for use both in transient transformation experiments, and for Agrobacterium-mediated transformation. The vectors will incorporate Gateway technology for high-throughput mobilization of amplified genes and cDNA libraries among the various vectors. Several Arabidopsis cDNA-coding regions will be cloned into the BMFC vectors; these genes are representative of large families of cytoskeleton, importin, chromatin, and F-box proteins. The project will also provide several Arabidopsis cDNA expression libraries in these vectors, which will be useful for comprehensive screens for protein interactions. As proof of concept, the project will test these tagged plant proteins for interaction with tagged Agrobacterium Virulence proteins in both transient and stable transformation assays. The tagged Virulence proteins include VirE2, VirD2, and VirF, as well as the Arabidopsis protein VIP1. The test Arabidopsis proteins will be selected based upon prior genetic and biochemical information. The limits of sensitivity of expression of the tagged proteins will be determined using both high-level constitutive as well as native promoters. All data derived from this project will be posted on the project web site: http://www.bio.purdue.edu/about/faculty/gelvin/gelvinweb/gelvin.htmlBroader Impacts: This project will develop the first broad-based technology to investigate protein-protein interactions in planta, and will additionally identify plant proteins whose expression may be manipulated to increase the frequency of Agrobacterium-mediated transformation of recalcitrant plant species. This project also contains a substantial training component. In addition to training postdoctoral research scientists and technicians, the project will include a vigorous outreach program with local colleges to identify undergraduate students from under-represented minority groups and introduce them, through summer and academic year collaborations with our research groups, to the conduct of scientific research. Multi-year training of these students to solidify their interest in pursuing a career in science will be encouraged. When they return to their home institutions, the students will expose additional students to the techniques learned in our laboratories. The under-represented minority students trained in the project during the summer will continue their research at their home institutions, thus broadening the number of their peers who will come into contact with scientific research. We expect that these efforts will encourage undergraduate students to select a career in science.

该项目将为植物科学界确定植物内蛋白质相互作用提供研究工具和资源。本研究将发展双分子荧光互补（BMFC）方法来研究植物中蛋白质与蛋白质的相互作用。BMFC包括用互补的半yfp分子标记蛋白质，这两种分子都不会自己发出荧光。当半yfp分子与相互作用的蛋白质融合在一起时，荧光就会恢复。BMFC设计是植物“双混合”系统中的一种新颖设计。BMFC还可用于鉴定相互作用蛋白的亚细胞位置。该项目将开发BMFC载体来表达半yfp标记的蛋白质，用于瞬时转化实验和农杆菌介导的转化。该载体将采用Gateway技术，在各种载体之间高通量地动员扩增基因和cDNA文库。拟南芥cdna编码区将被克隆到BMFC载体中；这些基因是细胞骨架、输入蛋白、染色质和F-box蛋白大家族的代表。该项目还将在这些载体上提供几个拟南芥cDNA表达文库，这将有助于蛋白质相互作用的综合筛选。作为概念验证，该项目将在瞬时和稳定转化试验中测试这些标记的植物蛋白与标记的农杆菌毒力蛋白的相互作用。标记的毒力蛋白包括VirE2、VirD2和VirF，以及拟南芥蛋白VIP1。测试拟南芥蛋白将根据先前的遗传和生化信息进行选择。标记蛋白表达的敏感性限制将使用高水平的组成和天然启动子来确定。从该项目获得的所有数据将发布在项目网站上：http://www.bio.purdue.edu/about/faculty/gelvin/gelvinweb/gelvin.htmlBroader影响：该项目将开发第一个基础广泛的技术来研究植物中蛋白质-蛋白质相互作用，并将额外识别可以通过操纵表达来增加农杆菌介导的抗性植物物种转化频率的植物蛋白。该项目还包含大量培训内容。除了培养博士后研究科学家和技术人员外，该项目还将包括与当地大学开展积极的外展计划，从代表性不足的少数群体中识别本科生，并通过与我们的研究小组的夏季和学年合作，向他们介绍进行科学研究。鼓励对这些学生进行多年的培训，以巩固他们从事科学事业的兴趣。当学生们回到自己的学校后，他们会让更多的学生接触到在我们实验室学到的技术。在夏季接受该项目培训的少数族裔学生将继续在其本国机构进行研究，从而扩大与科学研究接触的同龄人的数量。我们期望这些努力将鼓励本科生选择科学领域的职业。