Peptide aptamers to investigate and disrupt protein function in plants

PI: Stanton B. Gelvin（普渡大学）CoPI: Vitaly Citovsky（纽约州立大学石溪分校）适体是随机氨基酸序列的小合成肽。当适体在细胞中表达时，它可以特异性结合靶蛋白并抑制功能。因此，它们可以用来定义蛋白质在各种细胞过程中的功能作用，以及定义这些蛋白质中的特定结构域，这些结构域对蛋白质介导这些作用很重要。此外，适体序列可以作为植物蛋白的功能鉴定“标记”，与靶蛋白自然相互作用。一个表达载体和数以百万计的随机适体库将开发用于瞬态转化实验。该载体将允许首先在酵母中表达和筛选适配体，然后在植物中表达和筛选适配体。这些构建将结合双分子荧光互补（BiFC）技术来检测适配体与蛋白质的相互作用，以及Gateway技术来促进适配体在各种载体之间的动员。作为概念的证明，标记对发育重要的植物蛋白和对农杆菌介导转化重要的农杆菌毒力蛋白将使用适体库进行探测。这项初步工作将测试随机适体是否可以用于识别酵母和植物这两种不同生物中相互作用的蛋白质。该项目将为植物科学界确定植物蛋白的功能和蛋白质-蛋白质相互作用提供研究工具和资源。数据将通过普渡大学的项目网站（可通过http://bio.purdue.edu/people/faculty/index.php?refID=128访问）按顺序发布，适配库将提供给ABRC分发。

PI: Stanton B. Gelvin (Purdue University)CoPI: Vitaly Citovsky (State University of New York, Stony Brook) Aptamers are small synthetic peptides of random amino acid sequence. When expressed in cells, aptamers can specifically bind to target proteins and inhibit function. They can thus be used to define the functional roles of proteins in various cellular processes, as well as defining specific domains within these proteins that are important for the proteins to mediate these roles. Furthermore, the aptamer sequences can be used as functional identification "marks" of plant proteins that naturally interact with the target proteins. An expression vector and libraries of millions of random aptamers will be developed for use in transient transformation experiments. The vector will allow expression and screening of aptamers first in yeast, and subsequently in plants. The constructions will incorporate bimolecular fluorescence complementation (BiFC) technology to detect aptamer-protein interactions, and Gateway technologies to facilitate mobilization of aptamers among various vectors. As proof of concept, tagged plant proteins important for development, and Agrobacterium virulence proteins important for Agrobacterium-mediated transformation will be probed with the aptamer library. This initial work will test whether random aptamers can be used to identify interacting proteins in two different organisms, yeast and plants. This project will provide research tools and resources for the plant science community to determine the functions of plant proteins, and protein-protein interactions in planta. Data will be released sequentially through a project website at Purdue University (accessible via http://bio.purdue.edu/people/faculty/index.php?refID=128) and the aptamer library will be provided to the ABRC for distribution.