The role of CDPKs in calcium sensing and ABA signaling

描述（由申请人提供）： 钙依赖性蛋白激酶（CDPKs）是一类含有激酶结构域和钙离子结合结构域的蛋白激酶家族。拟南芥基因组中编码有34个cdpk，但尚未发现cdpk功能缺失突变体的表型。申办方实验室的初步结果表明，两种保卫细胞表达的CDPKs（CDPK-GC）的双突变体在ABA诱导的气孔关闭中表现出缺陷。这一cdpk-gc双突变体在阿坝信号转导中的表型为分析CDPKs在Ca 2+感应和阿坝信号转导中的作用提供了遗传工具。[Ca2+]cyt振荡对于气孔的长期关闭是必不可少的。通过钙离子成像和气孔运动分析确定CDPK-GCs在阿坝信号通路中与[Ca ~（2+）]cyt振荡的相对位置。CDPK-GC的亚细胞定位将通过共聚焦荧光显微镜和免疫金电子显微镜来确定。CDPK-GC的底物和相互作用蛋白将通过酵母双杂交测定和串联亲和纯化/质谱法分离。长期目标是使用标记的CDPK底物来检验CDPK作为[Ca 2 +]cyt振荡解码器的假设。了解胞内[Ca 2 +]振荡的解码机制将有助于包括记忆研究在内的广泛的Ca 2+信号转导领域的研究。

DESCRIPTION (provided by applicant): Calcium-dependent protein kinases (CDPKs) constitute a specific protein kinase family containing both a kinase domain and a Ca2+ binding domain. There are 34 CDPKs encoded in Arabidopsis genome, but no phenotypes have been published for any loss-of-function cdpk mutants. The preliminary results of the sponsor's lab suggest that double mutants in two guard cell expressed CDPKs (CDPK-GCs) exhibit defects in ABA-induced stomatal closure. The phenotype of this cdpk-gc double mutant in ABA signaling provides a genetic tool to analyze the role of CDPKs in Ca2+ sensing and ABA signaling. [Ca2+]cyt oscillations are essential for long-term stomatal closure. The relative location of CDPK-GCs to [Ca2+]cyt oscillations in ABA signaling pathway will be determined by calcium imaging and stomatal movement analyses. The subcellular localization of CDPK-GCs will be determined by confocal fluorescent microscopy and immunogold electron microscopy. Substrates and interacting proteins of CDPK-GCs will be isolated by yeast two-hybrid assay and tandem affinity purification/mass spectrometry. The long-term goal is to use tagged CDPK substrates to test the hypothesis that CDPKs function as decoders of [Ca2+]cyt oscillations. Understanding the decoding mechanism of [Ca2+]cyt oscillation will contribute to the broad field of Ca2+ signaling including memory study in human.