Vivek Padmanabhan Jayanthi, Tuteja Narendra, Soniya Eppurathu Vasudevan
Plant Molecular Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
PLoS One. 2013 Oct 23;8(10):e76392. doi: 10.1371/journal.pone.0076392. eCollection 2013.
In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) - PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.
在植物中,已观察到在高盐、干旱、极端温度和机械干扰等胁迫条件下,细胞质中钙浓度会发生瞬时变化。钙依赖性蛋白激酶(CDPKs)在将这些钙信号传递至下游效应中发挥重要作用。在本研究中,利用cDNA末端快速扩增(RLM-RACE)-PCR技术从生姜的胁迫cDNA中分离出一个胁迫响应性CDPK基因ZoCDPK1,并对其在胁迫耐受性中的作用进行了表征。在对推导蛋白的分析过程中发现的一个重要方面是,在连接域中存在核定位序列与类钙调蛋白结构域的EF-手环中的共有序列之间存在罕见的耦合。ZoCDPK1在根茎中大量表达,并被高盐胁迫、干旱和茉莉酸处理迅速诱导,但不受低温胁迫或脱落酸处理的诱导。使用共聚焦激光扫描显微镜在转基因烟草表皮细胞中研究了ZoCDPK1-GFP融合蛋白的亚细胞定位。生姜CDPK1基因在烟草中的过表达赋予了对盐度和干旱胁迫的耐受性,这体现在种子发芽率高、相对含水量更高、胁迫响应基因的表达、叶片叶绿素含量更高、光合效率提高以及其他光合参数上。此外,与正常条件下的野生型植物相比,遭受盐度/干旱胁迫的转基因烟草在胁迫条件下的生长量多出50%。T3转基因植物能够在持续的盐度或干旱胁迫下生长至成熟,提前开花并结出可育种子,且产量不受影响。ZoCDPK1上调了烟草植株中胁迫相关基因RD21A和ERD1的表达水平。这些结果表明,ZoCDPK1在生姜对盐度和干旱胁迫响应所涉及的信号通路的正向调节中发挥作用,并且可能以不依赖DRE/CRT的方式起作用。