【作者】 周玉亮；

【导师】 黄上志；

【作者基本信息】 中山大学 ， 植物学， 2011， 博士

【摘要】 种子活力是影响种子质量的一个重要性状,对于植物的繁殖和作物的产量有着重要的影响。但是,种子在贮藏的过程中,其活力和生活力逐渐下降,对于萌发过程中的环境因子变得越来越敏感,最终导致种子丧失萌发能力。尽管在种子活力领域已经开展了大量的研究工作,但是迄今为止对于影响种子活力的分子和遗传机制仍然不是十分清楚。莲(Nelumbo nucifera Gaertn.)是一种原始的被子植物,在中国已有大约5000年的栽培历史。经科学鉴定,莲种子保持着大约1300年的寿命记录,是目前为止发现的最长寿的种子之一。此外,莲种子也能耐受极端高温和土壤中的伽马射线辐射。据此,我们认为莲种子为鉴定与种子寿命和活力相关的基因提供了极好的植物基因资源。金属硫蛋白(Metallothioneins,MTs)是一类能够结合金属离子的低分子量、富含半胱氨酸的蛋白质,其主要参与维持重金属离子的稳态和活性氧的清除反应。在植物中,小分子热激蛋白(small heat shock proteins,sHSPs)极其丰富和多样,并且与多种非生物胁迫逆境有关。尽管植物MTs和sHSPs已经被广泛的研究,但是关于他们在种子中的功能却鲜有报道。在本文中,我们利用基因表达和植物转基因等方法,重点研究了莲MTs和sHSPs在植物种子中的功能。通过利用莲种子和转基因拟南芥种子,我们揭示了NnMT2a、NnMT3和NnHSP17.5在逆境条件下种子萌发活力中的重要作用,取得了以下的主要研究结果:1.我们分离了三个莲MT基因NnMT2a、NnMT2b和NnMT3以及一个细胞质II类的莲sHSP基因NnHSP17.5。2.实时定量PCR的结果表明,NnMT2a、NnMT2b和NnMT3转录本在莲种子发育和萌发过程中大量存在,并且在萌发的莲种子胚轴中能够被高盐和氧化胁迫大量上调。此外,正常的生长条件下NnHSP17.5在种子中特异表达,NnHSP17.5在萌发的莲种子胚轴中的表达能够被热激和氧化胁迫显著上调。3. SDS-PAGE电泳结果显示重组蛋白Trx-6His-NnMT2a、Trx-6His-NnMT3和6His-NnHSP17.5在大肠杆菌中高效表达。利用亲和层析的方法,我们成功的纯化了这些融合蛋白,并且将它们注射大白兔,制备了特异性的多克隆抗体。4.将植物瞬时表达载体导入到拟南芥悬浮细胞制备的原生质体后12-16 h,通过激光共聚焦显微镜对融合蛋白NnMT2a-YFP、NnMT3-YFP和NnHSP17.5-YFP在原生质体中的定位进行了观察,结果显示所有的融合蛋白都定位于细胞质和核质中。5.为了研究NnMT2a、NnMT3和NnHSP17.5在体内的功能,我们通过构建植物表达载体将这些基因分别置于CaMV35S启动子的调控下,通过遗传转化获得了转基因的拟南芥。半定量RT-PCR结果表明,NnMT2a和NnMT3在转基因植株的干种子和发育的果荚中都能检测到表达,NnHSP17.5也能在转基因株系的干种子中检测到表达。在蛋白水平上,我们通过Western blot证实这些蛋白在转基因干种子有积累,但是对照的野生型拟南芥种子中检测不到这些蛋白的存在。6.人工加速老化(accelerated aging,AA)通过模仿自然界的老化进程达到将种子快速老化的目的,从而用以评价种子的活力水平。本实验采用人工老化的方法研究了NnMT2a、NnMT3和NnHSP17.5在种子中的功能。结果显示,过表达NnMT2a、NnMT3或NnHSP17.5的转基因拟南芥种子与野生型种子相比表现出更强的抵抗人工老化的能力,表明这些基因在种子活力中发挥着重要的作用。另外,人工老化后转基因种子中的超氧化物歧化酶活性显著高于野生型种子。7. TTC活力染色可以用于种子生活力的检测。在人工老化后,野生型种子不能被TTC染上色的死种子的数量要远大于过表达NnMT2a和NnMT3的转基因种子。8.逆境条件下的萌发实验显示,过表达NnMT2a和NnMT3的转基因种子耐受高盐胁迫和氧化胁迫的能力显著高于野生型种子。另外,NnHSP17.5转基因拟南芥幼苗的耐热性明显增强。总之,这些基因在种子活力方面的生物学功能具有重要的理论价值和应用前景,如果将其转入到水稻、小麦、玉米和大豆等重要的农作物中,对于种质资源的保存和作物的产量或将产生积极的影响。更多还原

【Abstract】 Seed vigor is an important seed quality for the success of plant propagation and food production. However, seeds gradually lose vigor and viability during storage, become more sensitive to stresses during germination and eventually lose germination ability. Although seed viability and vigor has been the subject of a number of studies, the underlying molecular mechanisms remain poorly understood.As a primitive angiosperm, sacred louts (Nelumbo nucifera Gaertn.) has been a special crop in China for nearly 5000 years. Sacred lotus seeds are characterized with 1300-year astonishing seed viability, and resistance to extreme high temperature and soilγ-radiation. Owing to these characteristics, sacred lotus seeds could therefore be an excellent plant genetic resource for identification of genes controlling seed viability and vigor.Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses. Although plant MTs and sHSPs have been intensively studied, their roles in seeds remain to be clearly established. In this study, we focus on the functions of NnMT2a, NnMT2b, NnMT3 and NnHSP17.5 in seeds by gene expression analysis and transgenic approach. We have used sacred lotus seeds and Arabidopsis seeds overexpressing NnMT2a, NnMT3 and NnHSP17.5 to study their unique roles in seed germination vigor under sub-optimal conditions. The main results are as following:1. We report the isolation and characterization of three MT genes, NnMT2a, NnMT2b and NnMT3, and a cytosolic class II sHSP gene NnHSP17.5 from sacred lotus seeds.2. The results of real-time RT-PCR showed that the transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity and oxidative stresses. On the other hand, NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses.3. SDS-PAGE analysis revealed that the recombinant Trx-6His-NnMT2a, Trx-6His-NnMT3 and 6His-NnHSP17.5 were highly expressed in E. coli cells, and the fusion proteins were purified by nickel-resin affinity chromatography. After purification, the recombinant proteins were injected into rabbits to produce specific antibodies.4. Transient expression of NnMT2a-YFP, NnMT3-YFP and NnHSP17.5-YFP in protoplasts prepared from Arabidopsis suspension cultured cells was evaluated by confocal microscopy after electroporation 12-16 h. The results showed that all the tested YFP fusion proteins were localized in cytoplasm and nucleoplasm.5. In order to investigate the in vivo functions of NnMT2a, NnMT3 and NnHSP17.5, we generated transgenic Arabidopsis plants overexpressing these genes under the control of the cauliflower mosaic virus 35S promoter. RT-PCR analysis demonstrated that both NnMT2a and NnMT3 were overexpressed in dry mature seeds and green developing siliques of transgenic plants, while NnHSP17.5 was overexpressed in the dry mature seeds of transgenic plants. Similarly, immunoblot analysis of the protein extracts using specific antibodies confirmed the presence of NnMT2a, NnMT3 and NnHSP17.5 in the transgenic seeds but not in the wild-type seeds.6. The in vivo functions of NnMT2a, NnMT3 and NnHSP17.5 in seeds were evaluated by subjecting dry mature seeds of transgenic and wild-type lines to accelerated aging (AA) treatments, which was used to mimic natural aging to assess seed vigor. Transgenic Arabidopsis seeds overexpressing NnMT2a, NnMT3 or NnHSP17.5 displayed improved resistance to AA treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment.7. Vital stain with tetrazolium salt can be used to demonstrate the differences in seed viability. After AA treatment, the wild-type line displayed a higher number of dead seeds than those of the transgenic lines overexpressing NnMT2a and NnMT3, as indicated by the numbers of unstained seeds.8. We also showed that NnMT2a and NnMT3 conferred improved germination ability to high salinity and oxidative stress on transgenic Arabidopsis seeds. In addition, improved basal thermotolerance was observed in the transgenic seedlings overexpressing NnHSP17.5.In summary, the biological roles of NnMT2a, NnMT3 and NnHSP17.5 in seed vigor are of significantly theoretical and practical values, which may be applicable to agricultural crops such as rice, wheat, maize and soybean for the success of germplasm conservation and crop yield.更多还原