【作者】 郝佩佩；

【导师】 刘康；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2011， 硕士

【摘要】 棉纤维是重要的纺织工业原料,在国民经济和人民生活中占有重要的地位。棉花纤维实质上是种皮毛,是从胚珠外表皮细胞分化而来的一种单细胞结构,是迄今为止发现的最长的植物细胞。作为研究细胞伸长的理想模式植物,科学家们一直致力于揭示棉纤维发育的分子机理。棉花分子生物学、功能基因组学的发展已大大促进了棉花纤维发育过程中基因的表达谱及其调控机制的研究。但是,在蛋白质翻译、蛋白质分解、运输和翻译后修饰(如磷酸化、糖基化、酰基化)等水平上的棉花蛋白质组学研究较少。本文着重于对棉花纤维初始发育时期的磷酸蛋白质组学研究,一方面是因为棉花原始纤维细胞的分化决定着种皮细胞是否能发育成为纤维；另一方面是因为可逆的磷酸化修饰是细胞信号转导的重要途径,在生物发育过程中起着关键的调节作用。因此,开展棉花纤维初始发育时期的磷酸蛋白质组学研究对于深入探究棉花纤维分化和突起的分子调控机制有着重要的意义。本文以陆地棉品种徐州142(WT)及其无绒无絮突变体(fl)初始发育时期的开花前3天(-3 DPA)、1天(-1 DPA)、开花当天(0 DPA)的胚珠为材料,通过比较磷酸化蛋白质组学的分析,鉴定了与棉花纤维初始发育相关的磷酸蛋白质,分析了蛋白质磷酸化修饰在棉花初始发育中所起的重要作用,并探讨了棉花纤维分化和突起的可能机理。研究结果如下：1.由于棉花富含酚类和多糖,而磷酸化蛋白质在提取的过程中又十分容易脱磷酸化,因此本研究首先评价了TCA-苯酚法和改良苯酚法两种蛋白提取方法在蛋白产量、蛋白纯度和电泳图谱等方面的差异,明确了改良苯酚法能最大限度的保证磷酸蛋白质的质量和产量,适用于提取棉花胚珠总蛋白应用于磷酸化蛋白质组学研究。同时发现,选用pH 4～7的18cm IPG胶条,以被动吸涨上样方式来进行IEF的双向电泳能很好地分离棉花胚珠磷酸蛋白质。2.采用MOAC(金属氧化物/氢氧化物亲和层析)先富集磷酸化蛋白质再对其进行2-DE分析,和先对总蛋白质进行2-DE分析再用Pro-Q Diamond磷酸化特异性染色染料进行染色这两种策略分析棉花磷酸化蛋白质,对比双向电泳图谱发现,直接对棉花总蛋白2-DE胶进行Pro-Q Diamond特异性染色所得到的磷酸蛋白质图谱蛋白质点较多,能对棉花胚珠中的磷酸蛋白质进行全景式的观察与分析。初步建立了双向电泳—特异性荧光染色—质谱鉴定的棉花磷酸化蛋白质组研究的技术路线。3.在此技术平台基础上,本文对WT和.fl纤维初始发育阶段的-3、-1、0 DPA胚珠的磷酸蛋白质进行了比较分析。经PDQuest软件分析,共检测到73个差异表达磷酸蛋白质,通过MALDI-TOF-TOF MS/MS分析,成功鉴定了63种磷酸蛋白质。这些蛋白质主要涉及氧化-还原动态平衡与调控；蛋白合成、加工、降解；糖代谢和呼吸代谢；细胞骨架等生物学过程。初步揭示了磷酸化作用在棉花纤维初始发育中起重要的调控作用,并为进一步研究其调控机制提供了线索和候选蛋白质。本研究是首次运用磷酸化蛋白质组学研究方法分析棉花纤维分化和起始发育的分子机理。更多还原

【Abstract】 Cotton fiber is an important textile industrial material, which plays a significant role in national economy and people’s lives. Cotton fiber is a tubular single cell derived from the outer epidermis of ovule, which is known as the longest single botanic cell in record. As an ideal mode plant for the investigation of cell elongation, scientists have been trying intensively to reveal the molecular mechanism of cotton fiber development. The progress made in cotton molecular biology and functional genomics has greatly promoted gene expression profiling and discovering regulation mechanisms underlying cotton fiber development. However, few proteomic studies have been conducted in cotton at protein translation, protein degradation, protein trafficking, and post translation modifications (eg. glycosylation, phosphorylation, acetylation) levels. The present work focuses on phosphoproteomic analysis during cotton fiber differentiation and initiation, given that on one hand, the differentiation of primordial fiber determnines the number of testa cells which can develop into fibers, on the other hand, the reversible protein phosphorylation is one of the most important ways in cell signal transduction playing key regulatory roles in biological development. Therefore, phosphoproteomic studies on early cotton fiber development will greatly deepen our knowledge about molecular mechanisms controlling cotton fiber differentiation and initiation.In this thesis, a comparative phosphoproteomic research were conducted between the 3 days before postanthesis (-3 DPA),1 day before postanthesis (-1 DPA) and 0 day before postanthesis (0 DPA) ovules of a fuzzless-linless mutant (fl) derived from the upland cotton variety of Xuzhou 142 (WT) and WT, phosphoproteins related to cotton fiber differentiation and initiation were identified, the significance of the protein phosphorylation modification in these stages, and the possible mechanisms of cotton fiber differentiation and initiation were discussed. The main results are as follows:1. Because cotton tissue is rich of phenol and polysaccharide compounds, and the phosphate residual on proteins are vulnerable in protein extraction, the yields, purities and 2-DE profiles of the proteins extracted by using TCA plus phenol extraction method and modified phenol extraction method were compared, and the modified phenol extraction method can extract high yield and high quality of protein from cotton ovules, therefore this method is applicable for phosphoproteomic research. In addition, we found that 2-DE using 18cm pH 4-7 IPG strips and the sample in-gel rehydration method for IEF allowed isolating phosphoproteoins in cotton ovules.2. Two strategies of MO AC (metal oxide/hydroxide affinity chromatography) enrichment of phosphoproteins followed by 2-DE analysis and separating total protein 2-DE and visualizing phosphoproteins with specific Pro-Q Diamond staining were evaluated in cotton phosphoprotein analysis. The 2-DE maps obtained by two strategies were compared and the results indicated that the strategy by total protein 2-DE combined with Pro-Q Diamond visualization can identify relatively more phosphoproteins on gels. A technology routine based on Two-dimensional electrophoresis/phosphoprotein specific Pro-Q Diamond staining/MS identification was established for cotton phosphoproteome analysis.3. Based on this technology platform, a comparative phosphoptein analysis was carried out between -3,-1,0 DPA ovules of WT and fl during fiber differentiation and initiation respectively.73 phosphoproteins were detected to be differentially expressed between WT and fl significantly by using PDQuest software, and 63 phosphoproteins were successfully identified by MALDI-TOF-TOF MS/MS analysis. These proteins are mainly involved in redox homeostasis, protein synthesis, protein processing and degradation, carbohydrate metabolism and respiratory metabolism, and cytoskeleton construction, etc. Our results initially reveal that phosphorylation plays important roles in the earl cotton fiber development, which provide clues and candidate proteins for further research on molecule regulating mechanisms. This is the first time to our knowledge to use phosphoproteomics to analyze the molecular mechanism of cotton fiber differentiation and initiation development.更多还原