Prediction and Validation of Phospho-Regulatory Sites in Crop Plant Proteomes

维管植物的基因组编码超过1000种蛋白激酶。长期目标是确定作物植物蛋白质组中所有的调控磷酸化位点。这些位点代表了植物生长各个方面的潜在控制点，包括对生物和非生物胁迫的反应。重点是利用生物信息学来预测水稻蛋白质组中磷酸化调控的潜在重要位点子集，然后使用基于肽的实验策略来开始验证这些预测。这四个具体目标是：1。改进生物信息学工具以识别植物中磷酸化信号的基序。对植物蛋白质组中所有含Ser和Thr的亚序列进行聚类分析。设计肽阵列平台，在体外分析激酶的活性。鉴定可以破坏体内信号通路的肽（将测试超过50种）。该研究的独特性和优势在于其基本原理，即两种植物蛋白质组的计算比较可以用作“噪声过滤器”，以识别对单子叶和双子叶之间保守的信号通路至关重要的序列基序。这种过滤为实现“肽干扰物”作为理解和操纵植物细胞磷酸化体动力学的强大新方法提供了关键基础。所有编码“肽干扰物”或作为特定激酶活性报告者的基因的DNA构建体将存放在俄亥俄州立大学的ABRC，并将免费提供。

The genomes of vascular plants encode more than 1000 protein kinases. The long-term goal is todefine all the regulatory phosphorylation sites in crop plant proteomes. Such sites represent potential control points for all aspects of plant growth, including responses to biotic and abiotic stress. The focus is to use bioinformatics to predict a subset of potentially important sites of phospho-regulation in the rice proteome, and then use a peptide-based experimental strategy to start validating those predictions. The four Specific Aims are:1. Improve bioinformatics tools to identify motifs involved in phospho-signaling in plants.2. Conduct a clustering analysis of all Ser and Thr containing sub-sequences in plant proteomes.3. Design peptide array platform to profile kinase activities in vitro.4. Identify peptides that can disrupt in vivo signaling pathways (more than 50 will be tested).The uniqueness and strength of the proposed research rests upon the underlying rationale that acomputational comparison of two plant proteomes can be used as a 'noise filter' to identifyimportant sequence motifs that are fundamental to signaling pathways conserved between monocots and dicots. This filtering provides the critical foundation for implementing 'peptide-disruptors' as a powerful new approach to understanding and manipulating the dynamics of the plant cell phosphorylome. DNA constructs for all genes found to encode a 'peptide disruptor' or function as a reporter for a particular kinase activity will be deposited at the ABRC at Ohio State University, and will be freely available.