一条全新的蛋白激酶与转录因子信号通路调控ABA与干旱应答的分子机制研究

Calcium ions (Ca2+) are one type of imporant sencond messengers mediating abiotic stress responses including dought and salinity. Abscisic acid(ABA) is a famous phytohormone regulating signal transduction of abiotic stresses. Mining calcium-associated protein kinase genes and understanding their roles in drought and ABA signaling, is expected to be helpful to improve the tolerance or resistance of crop plants to abiotic stresses. In our previous work, we identified a drought and ABA-induced calcium-associated protein kinase gene from both Canola (Brassica napus L.) and Arabidopsis, with the T-DNA insertion mutant sensitive to both ABA and drougth treatments. Therefore, this mutant was named as ksa1(kinase sensitive to ABA 1). Moreover,yeast two-hybrid (Y2H) screeing of cDNA library using KSA1 as a bait demonstrated that it interacted with two core transcription factors (TFs), ABF2 and ABF4(ABA responsive element binding factor 2 and 4), which are two known TFs mediating ABA resoponse during seed germination, drought response, and post-germination seedling growth. Besides, bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaf cells indicated that KSA1 indeed formed a complex with both ABF2 and ABF4 in nuclei, suggesting that a novel pathway of protein kinase mediated phosphorylation of ABF TFs possibly modulates ABA and drought responses. In this proposal, we plan to analyze the phenotypes of relevant single, double and triple mutants, promoter activity and related cis-elements, test the phenotype of KSA1-overexpressing plants, as well as phosphorylation assy and target gene identification, using a combination of techniques involving plant physiology, biochemistry and molecular genetics. Through this proposal, we aim to dissect the molecular mechanism of signal transduction of a novel KSA1-ABF pathway modulating plant responses to ABA and drought and, provide candidate gene and lay theoretic foundation for molecular breeding and genetic improvement of crop plants.

钙离子是参与干旱与盐害等逆境应答的重要第二信使，脱落酸(ABA)是调控逆境信号转导的重要激素。发掘钙离子相关蛋白激酶基因，并解析它们在干旱与ABA应答中的功能，将为提高作物耐逆性奠定基础。我们在前期研究中发现一个新颖的钙相关蛋白激酶基因，受干旱与ABA处理诱导，并且突变体对ABA与干旱处理均高度敏感，命名为ksa1。进一步通过酵母双杂交筛选发现它分别能与调控ABA应答的核心转录因子ABF2、ABF4互作，又通过BiFC验证了互作的真实性，暗示激酶磷酸化转录因子调控下游靶基因的转录与抗逆性。本立项拟利用植物生理学、生物化学与分子遗传学方法，围绕这个蛋白激酶-转录因子信号通路，从启动子分析、蛋白表达与磷酸化修饰后的活性分析、过表达以及抗逆性鉴定等层面，着重解析它们调控干旱与ABA的信号转导分子机制。预计将阐明一条全新的调控干旱与ABA应答的信号通路及作用机制，为抗旱育种提供候选基因并奠定基础。

钙依赖的蛋白激酶（calcium-dependent protein kinase, CDPK）能够结合钙离子并发挥磷酸化底物蛋白的作用，从而调控植物对多种非生物和生物逆境的响应。在本研究中，我们发现拟南芥的一个新的CPKa既参与了调控脱落酸ABA应答过程，又参与了植物体内活性氧ROS的累积过程。qRT-PCR表明CPKa的转录本在多个器官组织中有表达且在多种非生物逆境下变化不大。 启动子-GUS活性分析显示CPKa在顶端分生组织和保卫细胞中有特异性表达。CPKa是一个具有膜定位特性的蛋白，其在细胞质膜、内质网膜和液泡膜都有定位；其组成型活化形式CPKaca也是一个具有膜定位特性的蛋白，而其点突变形式CPKacaG2A主要定位在细胞核中，而非膜上。.随后，发现CPKa的两个T-DNA突变体cpka-1和cpka-2在种子萌发水平对ABA处理不敏感，而其过表达株系比WT对ABA并没有明显的表型。通过双分子荧光互补（BiFC）发现CPKa能与ABA信号通路中的多个转录因子互作。进一步发现双突变体cpka-1abi5-1对ABA的耐受性强于abi5-1。其次，CPKa的突变体对干旱胁迫耐受，而abi5-1对干旱胁迫敏感，cpka-1abi5-1在干旱逆境下的表型和野生型无明显区别。.进而，发现瞬时表达CPKaca后，能够引起烟草叶片以超敏反应为标志的细胞坏死症状。如：相对电导率、DNA片段化、丙二醛（MDA）以及过氧化氢（H2O2）含量在CPKaca瞬时表达部位明显比对照GFP的表达部位高。而CPKaca激酶区域第202位点的点突变形式CPKacaD202A的瞬时表达却没能诱发烟草叶片细胞坏死的产生，说明CPKaca的功能发挥依赖其激酶活性。qRT-PCR表明CPKaca的瞬时表达能够诱导ROS相关以及防御相关的基因表达，并通过双荧光素报告系统（Dual-LUC）验证了部分结果。BiFC鉴定了CPKa能和AtRBOHD互作。诱导CPKaca的表达，发现能够明显地抑制种子的萌发和根系生长，而CPKacaD202A的诱导表达并没有导致上述的表型。在rbohd突变体背景下，诱导表达CPKaca仍然明显地抑制种子萌发和根系生长以及叶片黄化。我们的这一研究不仅鉴定了一个全新的具有双重功能的CPK基因，而且对理解该CPK的作用机理并探索可能的应用打下了基础。

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