钙离子作为细胞内普遍存在的第二信使，几乎参与植物生长发育、激素应答、各种生物/非生物胁迫信号传导的所有调控过程。钙信号通路被认为是植物中主要的信号转导途径。我们采用生物化学及分子生物学手段结合高通量的表达谱芯片技术与质谱鉴定技术，围绕钙信号通路进行了两方面的探索性研究：首先，我们利用小清蛋白基因获得了分别特异螯合植物细胞胞质和核质钙离子的PV-NES和NLS-PV两种转基因拟南芥植株。ABA、JA和ACC处理后的幼苗经表达谱芯片检测后，分别获得受胞质和核质钙信号调控的应答基因。然后，以蛋白相互作用网络数据库为基础，构建植物激素、钙感受器与胞质和核质钙信号应答基因间的相互作用网络。其次，我们以水稻悬浮细胞为材料，通过生物化学的方法，分离细胞的膜组分蛋白，用Heparin-agarose亲和纯化及同位素结合实验确定与IP3有特异结合的蛋白组分。采用高通量的质谱鉴定技术，获得了IP3结合蛋白数据库，为进一步寻找植物IP3受体奠定了基础。获得的主要研究结果如下：1、利用小清蛋白螯合钙离子的特性，构建了两种可以特异螯合胞质钙离子和核质钙离子的过表达PV-NES和NLS-PV的转基因拟南芥植株。使用ABA、JA和ACC三种激素分别处理PV-NES和NLS-PV幼苗，结合表达谱芯片技术分析受胞质钙信号和核质钙信号调控的目标基因。结果表明，我们获得了与胞质钙信号相关的差异表达基因（DEG_PV-NES）552条以及与核质钙信号相关的差异基因（DEG_NLS-PV）342条。2、对现有的拟南芥蛋白相互作用数据库进行整合，获得了14,656个蛋白之间的47,984条相互作用数据库，并构建出蛋白相互作用的网络。将552条DEG_PV-NES和342条DEG_NLS-PV基因编码蛋白、已知的134个钙感受器以及666个涵盖三种激素信号通路相关蛋白投射到蛋白相互作用网络中，分析三种激素信号通路与胞质钙信号和核质钙信号间相互作用。聚类分析表明不同激素的基因表达pattern不同。通过蛋白网络分析获得了具有差异表达特性的3个钙结合蛋白和12个编码蛋白能够直接与钙离子结合蛋白有相互作用。构建了6个相互作用的信号通路网络，分析得到了在不同激素刺激条件下特异受胞质钙离子和核质钙离子影响的基因以及和它们有直接相互作用的钙离子结合蛋白。3、用水稻悬浮细胞为材料，分离纯化膜组分蛋白。用同位素结合实验方法确定与IP3特异结合的膜蛋白组分。采用高通量质谱鉴定技术，对IP3结合蛋白进行了鉴定。获得了297条蛋白作为水稻IP3受体候选蛋白数据库。进一步对得297条蛋白进行GO分析、亚细胞定位分析和跨膜结构域的分析。GO分析结果包括cellular_component（25 terms）、biological_process （33 terms）和 molecular_function（20 terms）。亚细胞定位结果共得到137条膜蛋白，主要定位在质膜、液泡膜、内质网膜、高尔基体膜上等，其中有15条蛋白共定位在液泡膜和内质网膜上。这为植物IP3受体介导的信号转导途径提供了一定的蛋白信息资源。综上，本研究利用拟南芥14,656个蛋白间的47,984条相互作用数据库，分析了不同激素条件下，分别受胞质和核质钙信号调控的蛋白相互作用网络。并第一次用亲和纯化结合质谱鉴定方法，构建了第一个水稻IP3结合蛋白数据库。本研究将为植物钙信号转导的研究提供全新的信息资源，为进一步解析钙信号转导分子机制鉴定基础。

The calcium ion, as an ubiquitous intracellular second messenger, is involved in various progresses, such as growth and development, phytohormone signal transduction as well as the adaptation to biotic and abiotic environmental conditions in plants. Therefore, calcium signaling is considered as the core signal transduction pathway in plants. Here, we have used the biochemical and molecular biological techniques combined with MS and bioinformatics ananlysis to investigate the role mechanism of calcium sensing and sigaling transduction in the plant cell. First, we obtained the PV-NES and PV-NLS transgenic plant lines in which the cytosolic and nuclear Ca2+ have been specifically blocked, respectively. We collected the seedlings which were respectively treated with ABA, JA and ACC for 7 days. RNA samples were extracted from these samples and then hybridized with Affymetrix Arabidopsis ATH1 genome arrays to investigate the whole genome-wide DEG (differentially expressed genes) in PV-NES vs. WT and PV-NLS vs. WT, respectively. GO and Cluster analysis were conducted to study the cellular activities and biological processes in which the DEGs are involoved. Next, we have constructed a protein & protein interaction network database by combing the three published database and analysized the crosstalk between cytosolic or nuclear Ca2+ signal and phytohorme signalling pathways. Finally, we isolated the microsomal fraction from rice suspension cultured cells followed by enrichment of putative IP3-binding proteins by heparin-agarose affinity purification. Subsequently, the fractions in which the putitive IP3 binding proteins exist were confimed by isotope labled-IP3 ([3H]IP3) binding assay and the proteins were isolated and identified by mass spectrometry analysis. That will provide insight into the IP3 signalling pathways in plants. The major results are showed as the follows:1) Based on the specific calcium binding ability of parvalbumin (PV) we have constructed the vectors containing PV-NLS and PV-NES and generated the transgenic lines in which PV-NES and PV-NLS can specifically chelate the cytosolic and nuclear Ca2+, respectively, resulting in the elimination of Ca2+ siganals in plants. Then, we have performed microarray analysis and obtained 342 differentially expressed genes from NLS-PV transgenic plant and 552 differentially expressed genes from PV-NES transgenic plant which were treated with ABA, JA and ACC respectively.2) We integrated three public database into the protein-protein interaction database containing 47984 pairs of 14656 proteins. We respectively analysised the 342 DEG_NLS-PVs (differentially expressed genes from NLS-PV transgenic plants VS WT) and 552 DEG_PV-NESs with 134 calcium-regulated proteins and 666 proteins involved in the hormones signal pathway. We found that 3 DEGs which encoded calcium-binding proteins and 12 other DEGs which directly interacted with calcium-binding protein. The cluster study showed the various distribution patterns of different hormone. Finally, three hormones signaling subnetwork were constructed on the condition of suppression of nuclear Ca2+ signals, and another three hormones signaling subnetwork were constructed on the condition of suppression of cytosolic Ca2+ signals.3) We isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP3-binding proteins by heparin-agarose affinity purification. The IP3 binding activities of these protein fractions were determined by [3H]IP3 binding assay. SDS-PAGE and MS analysis were then performed to characterise these proteins. Totally, we have identified 297 proteins from the eluates of heparin-agarose column chromatography. Then we carried out the GO and TMs analysis of the identified proteins. The GO categories included cellular component, molecular function and biological process which represented 25, 20 and 33 terms, respectively were refered. We also investigated the subcellular localizations of the identified proteins. The results indicated that we identified 137 membrane proteins which were mainly located on the membrane of plasma membrane, vacuole, ER and Golgi apparatus in our database. 15 of them were calssfied on both vacuole and ER membrane. Our results will provide insight into the IP3 signalling pathways in plants.In conclusion, we have constructed the protein & protein interaction network which contain 47,984 protein-pairs interaction of 14,656 proteins in Arabidopsis. Then, six signaling networks integrating ABA, JA and ethylene signaling mechanisms which were mediated by cytoplasmic and nuclear Ca2+ signal were demonstrated. Finally, we have established the database of IP3 binding protein in rice suspension cells using high-throughput affinity purification and mass spectrometry identification methods. These results provides new information resources for functional study of cytoplasmic and nuclear Ca2+ signaling pathway in plant cell.