POWRE: A Bacterial Two-Hybrid System for Studying CDPK-Substrate Interaction.

这是power奖。该项目的科学重点将是建立细菌双杂交系统，用于分析植物钙依赖性蛋白激酶（CDPKs）与其底物之间的相互作用。项目负责人将在杰弗里·哈珀博士的实验室里度过项目的前六个月，在那里将建立基本系统。这项工作将需要广泛使用重组DNA技术，并将提供改进克隆技术和简化实验室程序的机会。细菌双杂交系统将有助于鉴定大拟南芥CDPK家族的哪些成员与给定蛋白相互作用并使其磷酸化。本项目产生的材料可能在未来发展成为筛选CDPKs底物的cDNA文库的系统。这项工作将提供基础知识，将有助于揭示这一大家族的多功能蛋白激酶，其生理作用尚未很好地定义。在项目的后六个月，PI将制备磷酸化CDPKs和底物的样品，并在Michael Sussman博士的实验室中使用电喷雾质谱法分析磷酸化位点。

This is a POWRE award. The scientific focus of this project will be the establishment of a bacterial two-hybrid system for analysis of the interaction between plant calcium-dependent protein kinases (CDPKs) and their substrates. The PI will spend the first six months of the project in Dr. Jeffery Harper's lab where the basic system will be set up. This work will require extensive use of recombinant DNA techniques and will provide the opportunity to improve cloning skills and streamline laboratory procedures. The bacterial two-hybrid system will be useful for identifying which members of the large Arabidopsis CDPK family interact with and phosphorylate a given protein. Materials generated in this project may be developed in the future into a system for screening cDNA libraries for substrates of CDPKs. This work will contribute basic knowledge that will help reveal details of this large family of multifunctional protein kinases whose physiological roles are not well defined. In the second six months of the project, the PI will prepare samples of phosphorylated CDPKs and substrates and analyze these for the sites of phosphorylation in the laboratory of Dr. Michael Sussman using electrospray mass spectrometry.