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

该项目将为植物科学界提供研究工具和资源，以确定蛋白质在植物中的相互作用。该研究将发展双分子荧光互补（BMFC）方法来研究植物中蛋白质-蛋白质相互作用。BMFC涉及用互补的半YFP分子标记蛋白质，这两种分子本身都不发荧光。当半YFP分子与相互作用的蛋白质融合在一起时，荧光恢复。BMFC设计是一种新颖的植物“双杂交”系统。BMFC也可用于鉴定相互作用蛋白质的亚细胞位置。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。这些载体将采用Gateway技术，用于在各种载体之间高通量动员扩增的基因和cDNA文库。将几个拟南芥cDNA编码区克隆到BMFC载体中;这些基因代表细胞骨架、输入蛋白、染色质和F-box蛋白的大家族。该项目还将在这些载体中提供几个拟南芥cDNA表达文库，这将有助于蛋白质相互作用的全面筛选。作为概念证明，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VirE 2、VirD 2和VirF，以及拟南芥蛋白VIP 1。将基于先前的遗传和生物化学信息选择测试拟南芥蛋白。将使用高水平组成型启动子以及天然启动子来确定标记蛋白质表达的灵敏度极限。从该项目中获得的所有数据将公布在项目网站上：http://www.bio.purdue.edu/about/faculty/gelvin/gelvinweb/gelvin.htmlBroader该项目将开发第一个基础广泛的技术来研究植物中的蛋白质-蛋白质相互作用，并将另外鉴定其表达可能被操纵以增加农杆菌介导的柑橘属植物物种转化的频率的植物蛋白质。该项目还包含大量培训内容。除了培训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联计划，以确定来自代表性不足的少数群体的本科生，并通过与我们的研究小组的夏季和学年合作，向他们介绍科学研究的开展。将鼓励对这些学生进行多年培训，以巩固他们对从事科学职业的兴趣。当他们回到自己的家乡机构，学生将接触更多的学生在我们的实验室学到的技术。在该项目中接受培训的代表人数不足的少数民族学生将在其家乡的机构继续进行研究，从而扩大他们将接触科学研究的同龄人的人数。我们希望这些努力将鼓励本科生选择科学职业。

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.