【作者】 楚璞；

【导师】 黄上志；

【作者基本信息】 中山大学 ， 植物学， 2011， 博士

【摘要】 高温是影响植物生长和繁殖的最重要的环境压力之一。由于其固着生长的特性,植物更容易受到温度变动的影响。为应对高温对其产生的威胁,植物进化出了一系列的适应机制。膜联蛋白是一类多功能的蛋白,该蛋白家族的特点在于它们能结合钙离子和带负电荷的脂类。越来越多的证据表明植物膜联蛋白可能具有过氧化物酶的活性,暗示其与植物逆境应答相关,然而,植物膜联蛋白在高温逆境中的作用还未见报道。目前关于植物高温耐性的分子生物学研究已有不少,但极少是以种子为材料进行的,尽管植物种子是具有重要价值的基因材料。曾有研究指出莲种子具有耐受极端高温的能力,为寻找种子适应机制中的新基因/蛋白提供了宝贵的材料。本研究采用比较蛋白质组学的方法以寻找高温胁迫信号通路中的新蛋白,由此在莲种子中鉴定到膜联蛋白,将其命名为NnANN1,并研究了该蛋白在高温逆境以及种子活力中的功能。本研究的主要结果如下:1.为研究莲种子的基础耐热性,用高温处理成熟莲种子,结果约50%种子在90°C处理24h后仍能萌发。为鉴定莲种子中的高温响应蛋白,从未处理的对照种子和90°C处理24h的种子的胚中提取蛋白经双向电泳分离用于比较蛋白质组学研究。10个高温处理后含量上调2倍以上的蛋白得到鉴定,其中大部分为能量代谢相关或逆境相关蛋白。在这些热响应蛋白中,选取被鉴定为膜联蛋白的蛋白点2作进一步研究。2.实时定量PCR(qRT-PCR)和蛋白质印记(western blot)的结果显示,与未经处理的莲种子相比,经高温处理的种子中NnANN1的表达在mRNA水平和蛋白水平都有大幅提高,证实与上述蛋白质组学的结果一致,NnANN1是热响应蛋白。亚细胞定位分析发现NnANN1表现出典型的细胞质定位模式,qRT-PCR分析发现NnANN1主要在种子发育和萌发过程中大量表达,暗示该蛋白可能在种子发育和萌发中发挥作用。3.为证实NnANN1在体内的保护作用,在大肠杆菌细胞和植物种子中进行了功能研究。结果表明,与对照相比,在高温逆境下,NnANN1转化的大肠杆菌细胞和拟南芥的种子耐热性提高。此外,加速老化实验表明NnANN1的表达提高了转基因拟南芥种子的活力。相反,与野生型种子相比,拟南芥同源基因AtANN1、AtANN2的T-DNA插入突变株的种子对于高温逆境和加速老化处理表现得更为敏感。NnANN1转基因种子中观察到过氧化物酶活性提高,并伴随活性氧(ROS)释放水平的降低,这可能帮助阐释NnANN1在体内的保护功能。综上所述,本研究实验结果表明NnANN1在耐热性和种子活力中发挥着重要功能,支持NnANN1在清除ROS中的作用,为研究植物高温应答的分子机制,提高作物耐热性和种子活力开拓了新的可能性。更多还原

【Abstract】 High temperature is one of the major environmental factors that negatively impact plant growth and productivity. As sessile organisms, plants are more vulnerable to temperature fluctuations. To cope with these challenges, plants have evolved a variety of adaptive mechanisms. Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids. There is increasing evidence showed that plant annexins may acting as peroxidases and implicating their importance in plant stress responses. However, the involvement of plant annexins in high temperature stress response has not yet been described.Although molecular aspects of high temperature tolerance in plants have been investigated, few studies have been conducted in seeds in spite of their importance as a source of valuable genetic materials. Sacred lotus (Nelumbo nucifera Gaertn.) seed appeared to have the ability to withstand extremely high temperatures. Therefore, it could provide an excellent system for discovering new components involved in seed’s adaptive mechanisms. In this study, comparative proteomic analysis was used to identify novel components in heat stress signaling and leading to the identification of a sacred lotus annexin NnANN1. The functions of NnANN1 in high temperature stress and in seed vigor were also assessed. The main results obtained in this study were as follows:1. To demonstrate basal thermotolerance of sacred lotus seeds, mature sacred lotus seeds were treated at high temperatures for 24 h. Approximately, 50% of sacred lotus seeds remained alive and geminated after subjected to 90°C treatment for 24 h. Proteins were extracted from embryo axes of sacred lotus seeds that were treated at 90°C for 24 h, and untreated embryos were used as control. To identify proteins responsive to high temperature in sacred lotus seeds, both treated and untreated protein samples were resolved by 2D gel electrophoresis for comparative proteomic analysis. Ten protein spots displaying more than two fold up-regulation in response to heat stress were identified and most of the proteins are energy metabolism-related or stress-related proteins. Among the heat-responsive proteins, protein spot 2 which was identified as an annexin was chosen for further study.2. The results of quantitative Real-time PCR (qRT-PCR) and western blot showed that compared to untreated seeds, the expression of NnANN1 increased considerably in both mRNA and protein level in high-temperature treated seeds, demonstrated that NnANN1 is heat responsive, concurring with the observations obtained from our proteomic analysis. Subcellular localization analysis of NnANN1 showed that NnANN1 exhibited a typical cytosolic localization, and qRT-PCR analysis revealed that NnANN1 is expressed preferentially during seed development and germination, suggesting some fundamental function of this protein in seed development and germination.3. In an attempt to prove the in vivo protective function for NnANN1, we studied its functions in both E.coli cells and plant seeds. Under heat stress, the NnANN1 transformed E. coli cells and Arabidopsis seeds showed increased tolerance to high temperature compared with control. Furthermore, as revealed by accelerated aging (AA), the expression of NnANN1 improved seed vigor in transgenic Arabidopsis. In contrast, seeds from T-DNA insertion mutants of the Arabidopsis paralogs AtANN1 and AtANN2 were more sensitive to heat stress and AA treatment. NnANN1 transgenic seeds showed enhanced peroxidase activities, accompanied with reduced ROS release level, which may help to explain its protective function in vivo. The results support the role of NnANN1 in ROS detoxification and indicate that NnANN1 plays an important role in thermotolerance and seed vigor. The identification of NnANN1 as a high temperature responsive protein with demonstrable effects on tolerance to high-temperature stress and AA treatment may present new possibilities for exploring molecular mechanisms of high-temperature response in plants and improving thermotolerance and seed vigor in crop plants.更多还原