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

POWRE：用于研究 CDPK-底物相互作用的细菌双杂交系统。

• 批准号: 9973770
• 负责人: Alice Harmon
• 金额: --
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-15 至 2000-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9973770&HistoricalAwards=false
• 关键词: POWRE; Bacterial; Two; Hybrid; System

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.

这是POWRE奖。该项目的科学重点将是建立一个细菌双杂交系统，用于分析植物钙依赖蛋白激酶(CDPKs)及其底物之间的相互作用。PI将在杰弗里·哈珀博士的实验室里度过该项目的前六个月，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，并将提供提高克隆技能和简化实验室程序的机会。细菌双杂交系统将有助于识别大型拟南芥CDPK家族的哪些成员与给定蛋白质相互作用并将其磷酸化。在这个项目中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，有助于揭示这一多功能蛋白激酶大家族的细节，这些蛋白激酶的生理作用尚未很好地确定。在项目的后六个月，PI将准备磷酸化的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仪分析这些样品的磷酸化部位。