| 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. |
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