阮雪芬Juan, Hsueh-Fen臺灣大學:分子與細胞生物學研究所陳定遠Chen, Ting-YuanTing-YuanChen2010-06-022018-07-062010-06-022018-07-062008U0001-2207200800482100http://ntur.lib.ntu.edu.tw//handle/246246/184685Rhodopseudomonas palustris (R. palustris) (沼澤紅假單胞菌)為紫色非硫光合菌的一種，可生活於各種不同的代謝環境；在自然環境中，廣泛分布於土壤與河水中。R. palustris可利用日光或是自然環境中的各種有機與無機物做為能量的來源，並利用大氣中的二氧化碳或是綠色植物衍生物做為碳源。除此之外特別的是，R. palustris尚可在行固氮作用時，產生氫氣做為其副產物。R. palustris也可以藉由調整自身的基因表現，使其能夠在各種不同的光源、碳源、氮源及不同電子來源的環境生存。在本研究中，我們使用了蛋白質二維電泳合併質譜儀研究 R. palustris不同代謝情況的蛋白質體表現；以及利用微陣列晶片研究其基因表現。我們的結果發現，在沼澤紅假單胞菌的四種不同代謝環境：光合自營、光合異營、化學自營、化學異營中，有38 個蛋白質產生變化。在化學自營和光合自營中比較，有 1357 個基因表現差異、光合自營和化學自營有 1629 個基因表現差異、化學異營和化學異營有 2512 個基因表現差異、光合異營和化學異營中有 168 個基因表現差異。有些蛋白質，像是60kDa chaperonin2、ABC transporters 相關蛋白質、PEPCK、catalase和phosphoglycerate dehydrogenase在三種以上的代謝條件均有表現，ABC transporter 並和自營與異營的調控相關。將這些結果利用蛋白質-蛋白質交互作用網路預測，發現60kDa chaperonin 2、transcription terminator rho、acyl-CoA dehydrogenase、S-adenosylmethionine synthetase、alcohol dehydrogenase扮演了重要的角色，甚至60kDa chaperonin 2 被預測和許多 ABC transporter 的蛋白質有交互作用的關係。根據這些變化，我們可以進一步了解光合細菌在不同代謝環境下的調控機制。Rhodopseudomonas palustris (R. palustris), a purple nonsulfur bacteria, is among the most metabolically versatile bacteria and ubiquitous in soil and water. The microbe could use sunlight, organic or inorganic compound for cellular energy, and use atmosphere carbon dioxide, or green plant-derived compound for cell material. It also could produce hydrogen gas by biological nitrogen fixation. The microbe could adjust and reweave itself in response to changes in light, carbon, nitrogen and electron sources. The ability of R. palustris to use carbon dioxide depends on two metabolic states: photoautotrophic and chemoautotrophic state. In this study, we used microarray for transcriptomic study, and two-dimensional polyacrylamide gel electrophoresis and mass spectrometry for proteomic study, respectively. Our results showed that 1357, 2512, 1629, 168 genes were differentially expressed over two-fold in photoautotrophic verus chemoautotrophic conditions, chemoautotrophic versus chemoheterotrophic conditions, photoautotrophic versus photoheterotrophic conditions, and chemoheterotrophic versus photoheterotrophic conditions, respectively. We identified 38 proteins differentially expressed over two-fold between two of the four major metabolic states. Some proteins, such as 60kDa chaperonin 2, ABC transporter related proteins, phosphoenolpyruvate carboxykinase, catalase, and phosphoglycerate dehydrogenase, could differentially expressed in three of these four major metabolic states. Interestingly, ABC transporter pathway was related to switch of autotroph and heterotroph. With computational prediction of plausible protein-protein interactions among these 38 proteins, we found 60kDa chaperonin 2, transcription terminator rho, acyl-CoA dehydrogenase, S-adenosylmethionine synthetase, alcohol dehydrogenase were the hub proteins in the predicted interaction network. These findings help us understand the regulatory mechanism of R. palustris.口試委員審定書 I謝 II文摘要 III文摘要 IV錄 VI目錄 VIII目錄 IX一章 前言 1.1 光合作用與光合細菌 1.2 代謝多樣性 1.3 紫色細菌 (Purple bacteria) 2.3.1 紫色含硫光合細菌 (Purple sulfur bacteria) 3.3.2 紫色非硫光合細菌 (Purple non-sulfur photosynthetic bacteria) 3.3.3 沼澤假單胞菌 (Rhodopseudomonas palustris) 4.4 蛋白質體學簡介 6.4.1 蛋白質體學研究 6.4.2 二維凝膠電泳法(two-dimensional gel electrophoresis ) 7.4.3 質譜分析方法簡介 7.4.4 蛋白質交互作用 (Protein-Protein Interaction) 8.5 微陣列晶片分析 9二章 實驗流程 11.1 實驗目的 11.2 實驗流程 11三章 實驗方法 12.1 藥品與儀器 12.1.1 研究材料 12.1.2 藥品 12.1.3 儀器 14.2 養菌基本操作 15.2.1 PM 培養基配方 16.2.2 Luria-Bertani 培養基 20.3 蛋白質體學分析 21.3.1 蛋白質定量 21.3.2 蛋白質二維電泳 22.3.3 影像分析 27.3.4 In-gel digestion 29.3.5 MALDI-TOF MS及MASCOT 29.4 微陣列分析 31.4.1 總RNA萃取與純化 31.4.2 將RNA轉成cDNA並標定螢光 32.4.3 微陣列晶片實驗 34.4.4 GeneSpring 軟體分析 36.5 穿透式電子顯微鏡操作 37四章 結果與討論 38.1 細菌形態結果 38.2 蛋白質結果 39.2.1 光合自營和光合異營之比較 40.2.2 光合自營與化學自營之比較 40.2.3 光合異營與化學異營之比較 41.2.4 化學自營與化學異營之比較 41.2.5 蛋白質交互作用網路之預測 42.2.6 重要蛋白質探討 43.3 微陣列結果 43六章 未來展望 48考資料 49錄 88application/pdf5866059 bytesapplication/pdfen-USRhodopseudomonas palustris光合細菌蛋白質二維電泳微陣列晶片蛋白質體學系統生物學purple non-sulfur bacteriatwo-dimensional electrophoresismicroarrayproteomicssystems biologyhttp://ntur.lib.ntu.edu.tw/bitstream/246246/184685/1/ntu-97-R95b43035-1.pdf