钙离子作为重要的第二信使，参与植物生长发育和逆境适应的众多生物学过程。除了细胞质有钙信号的发生外，植物细胞的其他细胞器也能产生不同的钙信号，尤其是细胞核钙信号受到了广泛关注。然而，核钙信号与胞质钙信号发生的相互关系并不清楚。此外，核钙信号与胞质钙信号调控转录的互作网络也未见报道。本论文利用大鼠小清蛋白（Parvalbumin，PV）结合钙离子的特性，将其分别连上核排除序列（Nuclear Export Sequence，NES）与核定位序列（Nuclear Localization Sequence，NLS），获得分别在胞质和核质特异表达PV的转基因拟南芥株系PV-NES与NLS-PV，以期特异地封闭相应亚细胞区域的钙离子浓度变化。利用这些转基因株系探索高渗和高盐与植物激素脱落酸（Abscisic Acid，ABA）和茉莉素（Jasmonates，JA）等外界刺激下胞质和核质钙信号发生之间的相互关系。随后，利用全基因组芯片技术分析了ABA和MeJA处理后的野生型拟南芥与PV-NES和NLS-PV株系中的差异表达基因，深入挖掘胞质钙信号与核质钙信号调控的ABA和JA响应基因，并通过蛋白互作网络与基因共表达网络解析胞质和核质钙介导的激素响应基因编码蛋白、钙信号解码蛋白（后简称钙解码蛋白）以及激素信号通路蛋白间的互作网络关系。主要研究结果如下： 首先，为探索胞质钙信号和核质钙信号的发生关系，分别构建了胞质和核质特异表达PV的转基因拟南芥株系PV-NES与NLS-PV。利用可特异检测胞质和核质钙信号变化的转基因拟南芥株系NES-YC3.6与NLS-YC3.6检测到高渗（250 mM Sorbitol）、高盐（125 mM NaCl）、ABA（100 μM）和MeJA（1 mM）处理都分别引起胞质和核质钙离子浓度的增加。通过将PV-NES植株与NES-YC3.6植株杂交，NLS-PV植株与NLS-YC3.6植株杂交并检测不同处理后胞质和核质钙离子浓度的变化，发现PV-NES阻断了不同处理引起的胞质钙离子浓度升高；同样，NLS-PV也阻断了不同处理引起的核质钙离子浓度升高。进一步将NLS-PV植株与NES-YC3.6植株杂交以阻断核质钙离子浓度变化，并检测高渗和高盐处理后胞质钙浓度的变化。结果表明，核钙变化被PV蛋白螯合后，不同处理导致的胞质钙动态变化与野生型中钙动态变化一致。同样的，通过将PV-NES植株与NLS-YC3.6植株杂交以封闭胞质钙来检测核钙的变化，发现封闭了胞质钙变化并不影响核钙的变化。以上结果表明，植物感受高渗和高盐胁迫后胞质和核质钙信号的发生是相互独立的。 其次，为探索胞质钙与核质钙信号参与的生物学功能，检测了PV-NES与NLS-PV幼苗在高渗、高盐、ABA和MeJA处理后植株生长发育的变化。结果如下：1）高盐处理下的PV-NES和NLS-PV植株的主根均长于野生型，而高渗处理的PV-NES和NLS-PV植株主根长度与野生型没有区别。另外，高渗处理后的PV-NES植株的侧根密度显著高于野生型和NLS-PV植株。2）PV-NES和NLS-PV植株中ABA抑制种子萌发的效果比野生型更强。3）高渗和高盐胁迫诱导的气孔关闭在PV-NES和NLS-PV植株中均被抑制，而ABA和MeJA诱导的气孔关闭只在PV-NES植株中被抑制。4）高渗和高盐处理后，部分胁迫诱导基因，如CML37、DREB2A、MYB2和RD29等，在野生型与PV-NES和NLS-PV植株中表达存在显著区别。以上结果表明，拟南芥胞质钙信号和核质钙信号调控不同的基因表达并参与植物生长发育的不同过程。 第三，为大规模挖掘受胞质钙和/或核质钙介导的基因表达，以野生型、PV-NES和NLS-PV株系为材料，利用全基因组芯片技术获得了受胞质钙调控的ABA响应基因210个（86上调，124下调），核质钙调控的ABA响应基因18个（14上调，4下调），既受胞质钙又受核质钙调控的ABA响应基因16个（14上调，2下调）。同时，也获得了受胞质钙调控的JA响应基因48个（31上调，17下调），核质钙调控的JA响应基因62个（43上调，19下调），既受胞质钙又受核质钙调控的JA响应基因34个（29上调，5下调）。进一步在这些钙调控基因的启动子上发现了多个转录因子结合的元件（Response elements），如bZIP、bHLH和TCP等转录因子结合元件等。本研究发现了多个未报道的受钙信号（尤其是受核钙信号）调控的响应元件。 第四，为解析钙信号通路与激素通路的交互联系，通过整合已经发表的拟南芥蛋白质互作数据，构建出蛋白互作网络图谱。利用该图谱分析了胞质和核质钙调控的激素响应基因编码蛋白、钙解码蛋白及激素通路蛋白之间的互作，挖掘三者直接或间接相互作用的众多节点。研究结果中的某些关键节点是已知的激素通路关键节点，但还有多个是新发现的网络富集关键节点，暗示着这些关键蛋白可能介导了钙信号与激素信号通路间的交互调控。进一步运用WGCNA算法对已有的拟南芥基因表达芯片数据库进行计算，构建了拟南芥的共表达网络。将蛋白互作网络中的节点蛋白放入共表达网络中进行分析，找到了植物潜在的受胞质钙和/或核质钙调控的激素响应关键调控因子。 综上，本项研究分别利用胞质或核质特异表达的PV蛋白构建了胞质或核质钙浓度增加受损的转基因拟南芥株系。利用这些株系发现，拟南芥根细胞中高盐和高渗胁迫诱导的胞质钙信号与核质钙信号的发生相互独立。进一步利用全基因组芯片技术，鉴定了胞质钙与核质钙介导的ABA和JA响应基因。并且，通过蛋白互作网络分析和共表达网络分析找出钙信号通路与激素通路的众多节点以及植物中响应激素的关键钙调控因子。本研究为钙信号通路的解析提供了重要线索和丰富资源，对相关蛋白功能的进一步解析将为探索钙信号转导通路与激素信号通路间交叉对话机制提供新的见解。

Calcium acts as a universal second messenger in both developmental processes and responses to environmental stresses. Previous researches have shown that a number of stimuli can induce [Ca2+] increases in different components of cells, especially in nucleoplasm which lead to intensive attention. However, the relationship between [Ca2+]cyt and [Ca2+]nuc is not clear. In addition, the gene expression networks deciphering cytosolic and/or nucleosolic calcium signaling remain obscure. In this study, the transgenic plants containing a fusion protein which is comprised of rat parvalbumin (PV) with either a nuclear export sequence (PV-NES) or a nuclear localization sequence (NLS-PV) were generated to selectively buffer [Ca2+]cyt and [Ca2+]nuc. Then, using these transgenic plants, we detected [Ca2+]cyt and [Ca2+]nuc response to osmotic stress, salt stress and phytohormone ABA and JA, respectively. Moreover, we identified [Ca2+]cyt- or / and [Ca2+]nuc-regulated ABA- and JA-responsible genes with the Arabidopsis Genome Oligo array. Using protein-protein interaction network and gene coexpression analysis, we have shown that the high intensity interactions among [Ca2+]cyt- / [Ca2+]nuc-regulated genes, calcium signaling decoding proteins and hormone signaling pathways proteins. The detail results are as follows: Firstly, using NES-YC3.6 and NLS-YC3.6 transgenic Arabidopsis, we found that 250 mM Sorbitol, 125 mM NaCl, 100 μM ABA or 1 mM MeJA seperately induced both [Ca2+]cyt and [Ca2+]nuc increase in Arabidopsis roots. Then, the osmotic stress-induced [Ca2+]cyt increase (OICIcyt), salt stress-induced [Ca2+]cyt increase (SICIcyt), ABA-induced [Ca2+]cyt increase and JA-induced [Ca2+]cyt increase were impaired in the PV-NES lines compared with the Arabidopsis wildtype (WT). Similarly, the osmotic stress-induced [Ca2+]nuc increase (OICInuc), salt stress-induced [Ca2+]nuc increase (SICInuc), ABA-induced [Ca2+]nuc increase and JA-induced [Ca2+]nuc increase were also disrupted in the NLS-PV lines. These results indicate that PV can effectively block the increase of [Ca2+] in response to various stimuli in Arabidopsis. However, the OICIcyt and SICIcyt in the NLS-PV plants were similar to those in the WT, and the OICInuc and SICInuc in the PV-NES plants were also same as those in the WT, suggesting that the cytosolic and nucleosolic calcium dynamics are mutually independent in Arabidopsis. Nextly, we found that osmotic stress- and salt stress-inhibited root growth was reduced dramatically in the PV-NES and NLS-PV lines compared with WT plant, and the osmotic stress-induced increase of the lateral root primordia was higher in the PV-NES plants than either in WT or NLS-PV plants. In addition, ABA-inhibited seed germination was more serious in both PV-NES and NLS-PV plants than WT plant. We also found that the osmotic stress-induced and salt stress-induced stomatal closure were disrupted in both PV-NES and NLS-PV plants, while ABA/MeJA-triggered stomatal closure were impaired only in PV-NES plants. Moreover, several stress-responsive genes, such as CML37, DREB2A, MYB2, RD29A, and RD29B, displayed diverse expression patterns in response to osmotic and salt stress in the PV-NES and NLS-PV lines compared with the WT. These results indicate that both of the cytosolic and nucleosolic calcium signaling play pivotol roles in regulating the growth and development and responses to environment stresses in plants. Thirdly, using whole-genome chip technology, we have identified calcium-regulated ABA-responsive genes: 210 [Ca2+]cyt-regulated genes (86 activated, 124 repressed),18 [Ca2+]nuc-regulated genes (14 activated, 4 repressed), and 16 [Ca2+]cyt- and [Ca2+]nuc-coregulated genes (14 activated, 2 repressed), and calcium-regulated JA-responsive genes: 48 [Ca2+]cyt-regulated genes (31 activated, 17 repressed)，62 [Ca2+]nuc-regulated genes (43 activated, 19 repressed), and 34 [Ca2+]cyt- and [Ca2+]nuc-coregulated genes (29 activated, 5 repressed). In addition, potential calcium-responsible element, such as bZIP, bHLH and TCP transcription factors binding DNA elements, were unveiled in these calcium-regulated genes by using MEME online program. Fourthly, we have constructed the Arabidopsis protein-protein interaction (PPI) network and found the high intensity interactions among [Ca2+]cyt- or / and [Ca2+]nuc-regulated ABA- and JA-responsible gene encoded proteins, calcium signaling decoding proteins and hormone signaling pathways proteins. In which, some of these interactions were previously reported, and others were newly unveiled through our network analysis. Moreover, we mapped the interaction proteins in PPI network in Arabidopsis co-expression networks, and found some potential [Ca2+]cyt- / [Ca2+]nuc-related or regulated components which may be crucial for plant stress tolerance. In summary, we constructed plant binary vectors containing rat PV with either NES or NLS sequence and generated the PV-NES and NLS-PV transgenic Arabidopsis. Through crossing PV-NES or NLS-PV with NES-YC3.6 or NLS-YC3.6 and measuring [Ca2+]cyt / [Ca2+]nuc elevation, we proved that cytosolic and nucleosolic calcium dynamics are mutually independent. In addition, we identified [Ca2+]cyt- and/or [Ca2+]nuc-regulated ABA- / JA-responsive genes, and found multiple crosspoints between calcium signaling and hormone signaling pathway by protein-protein interaction analysis and critical regulatory factors modulated by calcium in response to certain hormone stress. These results highlight the critical roles of cytosolic calcium and nucleosolic calcium in plant response to hormone with its modulation of transcription, also provide insight and rich resources for dissecting calcium signaling and hormone signaling.