【作者】 张红梅；

【导师】 张林生；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 干旱是植物主要的非生物胁迫因子之一，它严重影响植物的生长、发育和产量。小麦是世界重要的粮食作物之一。利用差异蛋白质组学的技术来研究小麦对干旱胁迫的响应机理,发现耐旱基因和干旱响应蛋白,将不仅有助于整体揭示小麦干旱应答的遗传基础,而且为抗旱机理的阐明具有重要的意义。小麦幼苗长至二叶一心期时，分别进行20%PEG处理,测定处理0h、24h、48h、72h时的小麦根系生物量、株高、冠层生物量、根冠比、叶片质膜透性、脯氨酸含量以及SOD酶活力的变化情况。结果表明，干旱胁迫导致小麦幼苗叶片卷曲，须根增多；根系生物量增加极慢，严重抑制了株高的生长，导致高生长基本停止；根冠比呈现先增加后下降的趋势。相对电导率上升，随着干旱处理的延长其电导率急剧升高；游离脯氨酸含量先是缓慢增加，后急剧增加；SOD酶活性表现出先升高后降低的变化趋势，但均高于对照。表明20%PEG模拟的干旱已造成了生长的抑制、质膜受到伤害，但小麦能在如此极端条件下继续生长，表明植物已启动了干旱响应机制防止死亡。采用双向电泳的技术分析了小麦幼苗在干旱胁迫48h时的蛋白质图谱,在2D凝胶上检测到27个蛋白丰度超过2倍变化的差异蛋白，通过MALDE-TOF/TOF方法鉴定了这些差异蛋白，成功对其中23个进行了鉴定，包括19个上调表达的蛋白点及4个下调表达的蛋白点。采用生物信息学对其功能进行分类，可分为8类,即参与ROS的清除、光合作用和碳的固定、信号传导、蛋白质的代谢、氨基酸代谢、细胞骨架、细胞防御及膜脂修饰的蛋白。下调表达的蛋白主要与光合作用和碳的固定以及细胞骨架相关，而上调表达的蛋白主要参与了ROS的清除和蛋白质的代谢。LEA蛋白是一类与植物胁迫耐受性关系密切的蛋白质。根据LEA蛋白的理化特性，利用煮沸处理和3%TCA处理后，获得小麦叶片中热稳定的酸溶蛋白组分，并进行了Western Blot的分析验证。从双向电泳凝胶上选取28个差异蛋白质点，进行MALDI-TOF/TOF分析,其中26个蛋白点被成功地鉴定为12个蛋白质。9个蛋白属于LEA蛋白，包括LEA1组的Em蛋白；LEA2组的Cor39、21-kDa dehydrin、16-kDa dehydrin、wheat cold induced16；LEA3组的ABA inducible protein和group3late embryogenesisabundant protein；LEA5组的LEA D-34和late embryogenesis abundant protein。此研究加深了对小麦LEA蛋白分离的全面理解。利用生物信息学的方法，预测了LEA蛋白的序列、二级结构、亚细胞定位和生物学功能，明确了它们在数据库中的预测注释，为更深入的研究LEA蛋白的功能提供了非常重要的信息。9个LEA蛋白多为亲水性的天然非结构蛋白，二级结构中多含高比例的无规则卷曲结构；不同的LEA蛋白定位特点不尽相同，但多分布于细胞核、细胞质及线粒体中；不同的LEA蛋白生物功能也不尽相同，主要集中于酶的保护，Ca2+结合，核酸保护，细胞骨架等方面。LEA蛋白的生物信息学分析为其深入研究提供了指导性作用。本研究以2个抗旱性不同的小麦品系为材料，在小麦生长的4个生育期进行了不同程度的自然干旱，研究了小麦LEA2组蛋白-脱水素的表达与发育阶段、小麦品系和水分胁迫程度之间的关系。结果表明：在小麦不受干旱时，无脱水素表达；受到干旱胁迫后，脱水素的表达量会随着干旱程度的加剧而增加，并且随着复水的进行而减少；小麦脱水素的表达与小麦的发育阶段相关，不同的发育阶段表达的脱水蛋白不尽相同；小麦脱水素的表达与小麦的品系也相关，抗性不同的小麦品系虽然有相同的脱水蛋白表达，但也有品系特异性脱水素的表达。9个LEA蛋白中的Cor39(45-kDa)可能为在两个小麦品系和四个发育时期内发挥基础且重要作用的脱水蛋白，这为该蛋白的具体功能研究奠定了研究基础。更多还原

【Abstract】 Drought is one of the major abiotic factors that strong influe nces plant growth,development and productivity. Wheat (Triticum aestivum L.) is one of the most importantfood crops in the world. Differential proteomics is an ideal means that has be en applied tofind new genes associated with drought resistance and drought responsive proteins. This willcontribute not only to the overall reveal of the genetic basis of drought response but also toclarifying the mechanism of drought resistance.Wheat seedlings with two leaves were treated with20%PEG. At the predetermined timepoint (0h、24h、48h and72h), the changes of root biomasses、seedling height、crownbiomasses、root/shoot ration、leaf plasma-membrane permeability、proline content and SODactivity were measured. The results showed that drought stress resulted in curly leaves andincreased number of fiberous roots. Under drought conditions, the increasement of rootbiomasses is very slowly and the seedling height was primarily suspended. The crownbiomasses showed a reduced trend. The ratio of root and crown was increased at first, then itwas decreased. The relative conductivity showed an increased trend, and with the stresscontinued, this value increased rapidly. The proline content was gradually increased at first,then it was increased sharply. The activity of SOD showed an increased trend at first, then adecreased one. However, all the values of SOD activity were higher than it from controlsamples at the same time point. It indicated, the drought stress simulated by20%PEGinhibited the growth inhibition and damaged the membrane. However, the survival of wheatplants suggested that they ha ve started a positive response mechanism to avoid death. Thephysiological response to drought stress proved the molecular mechanism in wheat plants.Using two-demensional electrophoresis (2-DE) method, we analyzed the proteinsprofiles of wheat seedlings which were drought treated for48h.27differentially expressedprotein spots with higher than2-fold spot thickness were found in gel image. These proteinspots were identified with MALDI-TOF/TOF and23spots were identified successfully.Compared to control, the abundance of19proteins increased and of4decreased. According tobioinformatics, these proteins were grouped into8categories, which mainly related to ROSscavenging and detoxifying enzymes、 photosynthesis and carbon fixation、 protein metabolism、 signal transduction、 amino acid metabolism、 cytoskeleton、 defense andmembrane lipid modification. The downregulated proteins are mainly related tophotosynthesis、carbo n fixa tion and cytoskeleton. The upregulated proteins are principallyinvolved in ROS scavenging and protein metabolism.LEA proteins are a group of proteins which are closely related with stress tolerance inplants.According to physicochemical properties of LEA proteins, we separated the heatstableand acid-soluble factions by boiling and3%TCA. And each fraction after each treatment wasverified by western blot analysis. We separated the heatstable acid soluble proteins by2-DE.After compared with the control samples,28differental protein spots were selected tosubjected MALDI-TOF/TOF analysis.12proteins represented by26spots were identifiedsusccesfully. Among them,9proteins are LEA proteins and three other proteins. LEA1groupincluded Em protein; LEA2group included Cor39、21-kDa dehydrin、16-kDa dehydrin andwheat cold induced16; LEA3group included ABA inducible protein and group3lateembryogenesis abunda nt protein; LEA5group included LEA D-34and late embryogenesisabundant protein. This research made our understanding to the sepration of LEA proteinsmore deeply.Using bioinformatics, we analyzed the sequences、seconde ry stucture、subcellularlocation and biological function of LEA proteins. The relevant pr edictive annotation indatabase provided very important theory foundation for futher research on these LEA proteins.All LEA proteins identified were mostly hydrophilic and natively unfolded, and theirsecondary structure contained a high propo rtion of rando m coil structure. Different LEAproteins localization features varied, while most of them were distributed in the nucleus,cytoplasm and mitochondria. The biological functions of the LEA proteins were different, andmainly focused on the protection of enzyme, binding with Ca2+, nucleic acid protection andcytoskeleton. Bioinformatics analysis of LEA proteins provides guidance for deeply study.Two cultivars of winter wheat (Triticum aestivum L.) differing in their drought tolerancewere subjected to a progressive soil water stress and recovery at four developmental stages.The relationship between the expression of dehydrins（LEA2） and developmental stages、genotypes and the level of water stress was studied. Some results were obtained. No dehydrinexpressed in the leaves from the well-watered plants. With water loss, the amount of thedehydrins gradually increased and the amount decreased after rehydration. The expression ofdehydrins is dependent on the developmental stage. Not all the same dehydrins wereexpressed at different de velop mental stages. The expression of dehydrins is depe nde nt on thegenotype. Although there were same de hydrin expressed in different genop tyes, there wereanother de hydrins expressed in a ceratin wheat genotype. Combined with LEA proteomics in our resuarch, Cor39(45-kDa), which were sperated with boiling and TCA treatment, mayplay a fundamental and impor tant role in two wheat genotypes and four develop mental stages.This research laid the foundation for the specific function of45-kDa dehydrin.更多还原