【作者】 刘辉；

【导师】 郁飞； 赵惠贤；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 作为重要的非生物胁迫因子之一,干旱严重地抑制了作物的生长、发育以及产量。小麦(Triticum aestivum L)是世界上最早栽培并广泛种植的禾本科植物,同时也是我国重要的粮食作物之一,易受干旱、高温等逆境胁迫的影响,引起籽粒品质和产量的下降,由干旱胁迫引起的小麦减产是其它非生物逆境胁迫所造成小麦减产的总和。因此,深入研究小麦干旱胁迫响应机制,培育抗旱小麦新品种,是当前小麦育种工作者的首要任务。六倍体普通小麦(Triticum aestivum L.)染色体组为AABBDD,基因组非常大,而且结构复杂,一定程度上制约了小麦抗旱相关基因分离鉴定。乌拉尔图小麦(Triticum urartu)和野生一粒小麦(Triticum boeoticum)均为二倍体(染色体组为AA),基因组相对简单;乌拉尔图小麦是普通小麦的祖先种之一,其基因组测序工作已经完成。与乌拉尔图小麦及普通小麦相比,野生一粒小麦具有很好的抗旱性,是改良普通小麦抗旱性的重要基因库。因此,以野生一粒小麦为材料,研究其响应干旱的分子机制将为揭示普通小麦抗旱分子机理提供参考。蛋白质是细胞功能的主要执行者;研究干旱条件下野生一粒小麦植株体内蛋白质组的变化将有助于揭示其抗旱的分子机制。为此,本研究以野生一粒小麦为供试材料,进行室内液体培养及干旱胁迫处理,干旱胁迫组是用含20%PEG-6000的Hoagland营养液处理三叶期小麦幼苗模拟干旱处理,对照组则在同样生长条件下正常供应Hoagland营养液。在干旱胁迫处理24 h和48 h分别采集叶片及根组织,分析其生理生化及蛋白质组变化。利用MALDI-TOF-TOF质谱技术鉴定叶片及根部的差异表达蛋白质,分析这些差异表达蛋白质的功能及参与生物学过程或代谢途径。本研究旨在分析野生一粒小麦叶片与根响应干旱胁迫的生理生化及蛋白质组的变化,为揭示野生一粒小麦对干旱的响应机制提供一些有价值的信息。本研究取得的主要研究结果如下:1.叶片与根中的ABA、可溶性糖和脯氨酸含量在干旱胁迫后上升,表明野生一粒小麦可通过增强信号传导和渗透调节能力来抵御干旱胁迫。干旱胁迫下野生一粒小麦叶片与根中的丙二醛(MDA)含量也上升,表明干旱导致小麦细胞膜的脂质过氧化,引起细胞损伤。同时,干旱胁迫引起叶片叶绿素a和叶绿素b含量下降,以及光合速率、蒸腾速率、胞间二氧化碳浓度的降低,表明干旱抑制了野生一粒小麦叶片的光合作用。2.比较蛋白质组学结果显示在干旱胁迫下野生一粒小麦叶片与根分别有115和102个差异(p≤0.01)表达倍数在1.5倍以上的蛋白质点。其中,17来自叶片和16个来自根的差异蛋白在干旱胁迫下诱导表达;而3个来自叶片和4个来自根的差异蛋白点是在干旱胁迫下消失了;这暗示着这些蛋白很可能在小麦响应干旱胁迫中有非常重要作用,值得深入研究。3.对上述差异蛋白点进行MALDI-TOF-TOF质谱鉴定,结果在叶片和根中分别成功地鉴定出98个和85个功能蛋白,分别代表了85和80个unique蛋白。比较根和叶的差异蛋白质发现仅有6个差异表达蛋白是根和叶中共有的,说明叶片与根对于干旱有不同的响应机制。4.对鉴定出的差异蛋白进行功能分类,结果表明这些叶片与根蛋白可分别分为14类和12类功能类别,包括光合作用、碳代谢、解毒和防御相关等。对鉴定出的差异蛋白进行亚细胞定位分析表明,叶片中的差异蛋白主要位于叶绿体(33.67%)、细胞质(29.59%)、线粒体(19.39%)及细胞核(14.29%)中。根中的差异蛋白则主要存在于线粒体(36.47%)和细胞质(49.41%)中。5.分别随机选择2-DE分析检测的5个根和叶差异表达蛋白质进行western blot分析,结果显示所选蛋白的的丰度变化模式与2-DE测得的变化模式一致,表明2-DE分析所得蛋白质差异表达结果是可靠的。6.对在干旱胁迫下根和叶片差异蛋白的功能及其涉及的代谢途径分析发现,在干旱胁迫下野生一粒小麦根组织信号感应及传导相关的蛋白显著上调表达;叶片与根中的抗氧化和防御相关的蛋白含量均极显著上升;干旱胁迫下根组织的糖酵解代谢受到抑制而磷酸戊糖途径增强;在干旱胁迫下叶片中光合作用和碳固定能力降低;而TCA循环增强,从而导致复杂的能量代谢变化,以建立一个新的内稳态系统;蛋白质代谢和氨基酸代谢在根中下调,而在叶片中上调。总之叶片与根对干旱胁迫的响应有一些共性,也有组织特异性。更多还原

【Abstract】 As a main abiotic stresses, drought stress seriously limit crop growth, development and production in majority of agriculture fields of the world. Wheat(Triticum aestivum L.) is one of the worldwidely cultivated and staple crops, which belongs to gramineous plants, its yield and quanlity are easily affected by climatic temperature change and water resources scarcity in the environment, the loss in wheat production caused by drought is equal to the sum of other non-biological adverse stress. As above, it is top priority task to systematically investigate the mechanism of wheat response to drought stress and cultivate the drought resistance of wheat varieties.The genome of the common wheat is AABBDD, such large and complecated genome significantly restrain the isolation and identification of genes related to drought response. Triticum urartu and Triticum boeoticum are both diploid wheat species(the genome is AA), the genome composition is much simpler compared to common wheat species. Triticum urartu is one of the ancestors of common wheats, and the genomic sequencing project for Triticum urartu were finished. Comparing with the Triticum urartu and common wheats, Triticum boeoticum is more drought-resistant, therefore it is an important gene pool for improving the drought resistance of common wheats. Hence, the investigation of the drought response mechanism underby Triticum boeoticum would greatly contribute to uncover the molecular base of drought response in common wheat. Proteins are the major executor of cellular function, the investigation of the proteome change in Triticum boeoticum under drought stress would greatly contributed to the investigation of the molecular mechanism for drought response.At present study, Triticum boeoticum are cultured and treated with drought stress in greenhouse conditions, which is induced by 20% PEG-6000 in Hoagland solution at the three leaves stage, while the control groups were cultured in Hoagland solution in the same growth conditions. The leaves and roots were collected at 24 and 48 h of drought treatment, the analysis of physiological, biochemical and proteomics changes in the leaves and roots were conducted. The MALDI-TOF-TOF analysis were applied to identify the differential expressed proteins(DEPs), the molcular function, biological and metabolic process of the DEPs involved in drought were also analyzed as well. The main purpose of this study is to analyze physiological and proteomic change in leaves and roots of Triticum boeoticum under drought stress, and to provide some valuable information for the uncovering of drought response mechanism in Triticum boeoticum. And the main results are shown as follows: 1. The content of ABA, soluble sugar and proline were increased under drought stress, as well as MDA content. Suggesting the signal transduction and capability of osmotic adjustment were enhanced, but the cell membrane was also damaged by lipid peroxidation. The content of chlorophyll a and b were decreased, the photosythsis rate, transpiration rate, intercellular CO2 content were also reduced, indicating the inhibition of photosynthetic process. 2. Comparative proteomics results indicated that 115 and 102 protein spots in leaves and roots respectively, exhibited at least 1.5-fold differences(p≤0.01). Of the differential expressed proteins(DEPs), 17 and 16 spots from leaves and roots respectively were induced under drought stress, while 3 leaves spots and 4 roots spots were totally suppressed. These proteins may act as the key proteins in responnse to drought stress, and be worth of subsequent in-depth study. 3. All the DEPs above were analyzed by MALDI-TOF-TOF. Totally, ninety-eight in the wild wheat leaves and 85 in roots were successfully identified. Of the 98 identities in leaves and 85 in roots, 85 and 80 unique proteins have been documented in the current database as putative functional proteins, respectively. After comparism of proteome between leaves and roots, only 6 unique proteins were common in leaves and roots, indicating the existence of different responsing mechanism between leaves and roots. 4. Molecular function classification results showed that all the successfully identified DEPs by MALDI-TOF-TOF could be classified into 14 and 12 function groups in leaves androots respectively, such as photosynthesis, carbon metabolism, detoxification and defense. We also analyzed the sub-cellular localization of these DEPs, and the results showed that most of the DEPs in leaves were localized in chloroplast(33.67%), cytoplasm(29.59%), mitochondria(19.39%) and nucleus(14.29%). For root proteins, most of the DEPs were localized in cytoplasm(36.47%) and mitochondria(49.41%). 5. To confirm reliability of the DEPs identified in 2-DE analysis, ten DEPs(Five from leaves and five from roots), were selected randomly to conducted Western bloting analysis with protein samples of the three time point(0, 24 and 48 h). The result indicated that the protein level of these DEPs were consistent with the 2-DE results. The Western bloting results seemed to agree well with the proteomic results. 6. Molecular function and Metabolic pathways analysis results showed that the signal transduction-involved proteins were greatly up-regulated in roots, indicating enhanced stress-responsive signaling pathways in the roots of the wild wheat seedlings exposure to short-term drought treatment; the ROS scavenge capability and pathogenesis defensive network were enhaced both in leaves and roots as well. The glycolytic pathway were inhibited, while the pentose phosphate pathway was enhanced in roots under drought stress. Photosynthesis and carbon fixation pathways were suppressed in the leaves of wild wheat plants exposure to short-term drought, while tricarboxylic acid cycle(TCA) pathway were enhanced. Consequently resulted in the complicated change of energy metabolism, and a new homeostasis was established. The amino acid and protein metabolism were suppressed in roots, while enhanced in leaves. Totally, there are some common character in leaves and roots in response to drought stress, as well as the existence of tissue specific mechanism.更多还原