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

维管束植物的基因组编码1000多种蛋白激酶。其长期目标是确定作物蛋白质组中所有的调节磷酸化位点。这些位点代表着植物生长的各个方面的潜在控制点，包括对生物和非生物胁迫的反应。重点是使用生物信息学来预测水稻蛋白质组中潜在重要的磷调节位点的子集，然后使用基于多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，以确定参与植物磷酸化信号转导的基序。对植物蛋白质组中所有含子序列的丝氨酸和苏氨酸进行聚类分析。设计多肽阵列平台，用于体外蛋白水解酶活性分析。确定可以扰乱体内信号通路的多肽(将测试50多个)。拟议研究的独特性和强度基于一个潜在的理论基础，即两个植物蛋白质组的计算比较可以被用作‘噪声过滤器’，以识别重要的序列基序，这些序列基序是单子叶植物和双子叶植物之间保守的信号通路的基础。这种过滤为实现‘多肽干扰物’提供了关键的基础，作为了解和操纵植物细胞磷酸化组动态的一种强大的新方法。所有基因的DNA构建将被存放在俄亥俄州立大学的ABRC，并将免费提供。这些基因编码了一种‘多肽干扰物’，或作为一种特定的激酶活性的报告。

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.