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研究生(外文):Jui-Ying Tsao
論文名稱(外文):Secretome analysis of Deinococcus ficus at different culture conditions
指導教授(外文):Chao-Hsiung Lin
外文關鍵詞:Deinococcus ficussecretomeproteomics
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菩提奇異球菌 (Deinococcus ficus) 是由中興大學楊秋忠教授從台灣菩提樹根部周圍土壤分離出的一種革蘭氏陽性新菌種。生理及生化學的測試顯示此菌具有抗輻射、抗UV、耐鹼至pH 10並能夠分解多種有機化合物。為了能夠了解菩提奇異球菌的功能及探討其可能在極端環境下消化有機物質之應用,本研究建立了研究方法以分析菩提奇異球菌的分泌蛋白體,主要為使用一維或二維電泳分離配合後續之質譜儀分析來儘可能鑑定出最多之蛋白質。在二維電泳膠片上約可觀察到630個蛋白質點,其中208點可以鑑定出166個不同蛋白質;另一方面,一維電泳可以鑑定出99個蛋白質。在所有鑑定到的244個蛋白質,其中199個蛋白質可以比對到其他物種功能,其中包含了serine protease, aminopeptidase或amylase等類型的酵素可能在有機物質分解上扮演重要的角色;剩餘之conserved hypothetical及hypothetical proteins則進一步以Phyre軟體來預測結構蛋白質以評估他們可能的功能。另外,其中有47個鑑定到的蛋白質是符合之前實驗室以有無signal peptide或細胞中分佈位置的標準所推測之分泌蛋白質。另外,本研究也嘗試探討不同多寡營養成分培養基對菩提奇異球菌培養條件的影響,例如M9 和 RPMI-1640培養基。本篇研究結果顯示菩提奇異球菌在營養複雜及相對簡單的培養基中的確會改變所產生之分泌蛋白質體,從這些資料希望能連結菩提奇異球菌在不同之有機碳源培養條件下的分泌蛋白質體差異與其分解能力。
Deinococcus ficus is a newly-identified Gram-positive microorganism isolated from the rhizosphere of the sacred tree Ficus religiosa L. in Taiwan by Professor Chiu-Chung Young at NCHU. Physiological and biochemical examinations showed that this species has the distinctive characteristics, i.e. the resistance to UV and gamma radiation as well as the ability to grow at pH 10 and digest diverse organic compounds. In order to understand the functional role of Deinococcus ficus and to illustrate its application potential in digesting organic substances at extreme alkaline condition, this study have established the methodology to investigate the secreted proteome of D. ficus. The one-dimensional (1D) or two-dimensional (2D) gel electrophoresis followed by mass spectrometry, using peptide mass fingerprinting or peptide sequencing, were concurrently used to maximally identify the secreted proteins. In a typical 2D gel, approximate 630 spots were visualized and 208 of them have been identified to a total of 166 distinct proteins. On the other hand, 99 proteins could be identified from 1D gel electrophoresis. Among the total identified 244 proteins, 199 of them could be further assigned with function in other species, including types of serine protease, aminopeptidase, and nucleotidase which may participate in the degradation of extracellular macromolecules. The remaining conserved hypothetical and hypothetical proteins were further analyzed using Phyre structural prediction software to expand their functional evaluation. Interestingly, only 47 of these secreted proteins matched to previous prediction of secretory proteins, which has been carried out in lab based on the presence of signal peptides and cellular location prediction. Moreover, the culture conditions of D. ficus using rich or less nutrition medium, the M9 and RPMI-1640 medium were also studied in this research. The result has showed that the constitution of secreted proteins was accordingly changed upon the selection of culture medium. With the current data, future intention is to establish the linkage between secretome changes and the digestive function of D. ficus under conditions using different organic compounds as carbon sources.
Contents i
Table contents iii
Figure contents iv
Abbreviations v
中文摘要 1
Abstract 2
Introduction 4
Materials and methods 7
Materials 7
Instruments 8
Methods 9
Bacterial strain and growth conditions 9
Secreted protein sample preparation 10
One- and two-dimensional electrophoresis analysis 11
Silver-staining, image acquisition and data analysis 12
In-gel digestion 12
MALDI-TOF MS and LC-MS/MS analysis and database search 13
Results 15
1. Identification of secreted proteins of D. ficus grown in yeast extract and tryptone medium by 1D and 2D electrophoresis followed with LC-MS/MS and MALDI-TOF MS analyses 15
2. Establishment of D. ficus culture using chemical-defined RPMI-1640 medium 18
3. Proteomic analysis of secreted proteins from D. ficus cultured in RPMI-1640 medium 19
Discussion 20
Functional analysis of the 244 identified proteins in D. ficus secretome 20
KEGG pathway analysis of the 225 identified proteins with homologs in D. radiodurans 23
Structure-based functional prediction of eight function-known proteins 24
Secreted protein prediction and Non-classical secreted proteins 25
References 29
Tables 36
Figures 72
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