Zeng Houqing, Zhang Yaxian, Zhang Xiajun, Pi Erxu, Zhu Yiyong
College of Life and Environmental Sciences, Hangzhou Normal UniversityHangzhou, China.
College of Resources and Environmental Sciences, Nanjing Agricultural UniversityNanjing, China.
Front Plant Sci. 2017 May 24;8:877. doi: 10.3389/fpls.2017.00877. eCollection 2017.
Calcium ion (Ca) is a universal second messenger that plays a critical role in plant responses to diverse physiological and environmental stimuli. The stimulus-specific signals are perceived and decoded by a series of Ca binding proteins serving as Ca sensors. The majority of Ca sensors possess the EF-hand motif, a helix-loop-helix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPK-related protein kinases, 2 Ca- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related -elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca-binding ability of these EF-hand proteins, four CMLs (CML1, CML13, CML39, and CML95) were randomly selected for SDS-PAGE mobility-shift assay in the presence and absence of Ca. Results showed that all of them have the ability to bind Ca. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses.
钙离子（Ca）是一种通用的第二信使，在植物对各种生理和环境刺激的反应中起着关键作用。特定刺激信号由一系列作为钙传感器的钙结合蛋白感知并解码。大多数钙传感器具有EF手基序，这是一种形成转角-环结构的螺旋-环-螺旋结构。尽管模式植物如拟南芥中的EF手蛋白已得到充分描述，但大豆中含EF手蛋白的鉴定、分类及其生理功能尚未有系统报道。在本研究中，在大豆基因组中总共鉴定出至少262个可能编码含有一到六个EF手基序蛋白的基因。这些基因包括6个钙调蛋白（CaMs）、144个类钙调蛋白（CMLs）、15个钙调神经磷酸酶B样蛋白、50个钙依赖蛋白激酶（CDPKs）、13个CDPK相关蛋白激酶、2个钙和钙调蛋白依赖蛋白激酶、17个呼吸爆发氧化酶同源物以及15个未分类的EF手蛋白。这些基因中的大多数（87.8%）在其-1500 bp启动子区域含有至少一种激素信号和/或应激反应相关元件。通过探索已发表的微阵列和Illumina转录组测序数据进行的表达分析表明，这些EF手基因的表达在大豆的不同器官中广泛检测到，并且几乎一半的总EF手基因对各种环境或营养胁迫有反应。定量RT-PCR用于确认它们对几种胁迫处理的反应性。为了确认这些EF手蛋白的钙结合能力，在有钙和无钙的情况下，随机选择四个CMLs（CML1、CML13、CML39和CML95）进行SDS-PAGE迁移率变动分析。结果表明它们都具有结合钙的能力。本研究首次对大豆中编码EF手蛋白的基因进行了全面分析。大豆EF手基因在不同组织以及各种环境和营养胁迫下的分类、系统发育关系和表达谱信息，将有助于鉴定在钙信号介导的生理过程（包括生长发育、植物-微生物相互作用以及对生物和非生物胁迫的反应）中具有潜在作用的候选基因。
