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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
| dc.contributor.author | Tsun-Hsuan Yi | en |
| dc.contributor.author | 尹存瑄 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:20:12Z | - |
| dc.date.available | 2015-08-12 | |
| dc.date.copyright | 2010-08-12 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-10 | |
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(2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4, 873-886. 32. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., and Mann, M. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635-648. 33. Macek, B., Gnad, F., Soufi, B., Kumar, C., Olsen, J. V., Mijakovic, I., and Mann, M. (2008) Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol Cell Proteomics 7, 299-307. 34. Macek, B., Mijakovic, I., Olsen, J. V., Gnad, F., Kumar, C., Jensen, P. R., and Mann, M. (2007) The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol Cell Proteomics 6, 697-707. 35. Hatch, M. D., and Slack, C. R. (1968) A new enzyme for the interconversion of pyruvate and phosphopyruvate and its role in the C4 dicarboxylic acid pathway of photosynthesis. 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(2006) Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax: key pieces in the puzzle of archaeal carbohydrate metabolism. Mol Microbiol 60, 287-298. 42. Burnell, J. N. (2010) Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase--Pi-dependent pyrophosphorylase from bacteria. BMC Biochem 11, 1. 43. Furbank, R. T., and Hatch, M. D. (1987) Mechanism of C(4) Photosynthesis: The Size and Composition of the Inorganic Carbon Pool in Bundle Sheath Cells. Plant Physiol 85, 958-964. 44. Chastain, C. J., Lee, M. E., Moorman, M. A., Shameekumar, P., and Chollet, R. (1997) Site-directed mutagenesis of maize recombinant C4-pyruvate,orthophosphate dikinase at the phosphorylatable target threonine residue. FEBS Lett 413, 169-173. 45. Burnell, J. N., and Hatch, M. D. (1986) Activation and inactivation of an enzyme catalyzed by a single, bifunctional protein: a new example and why. Arch Biochem Biophys 245, 297-304. 46. Han, C. L., Chien, C. W., Chen, W. C., Chen, Y. R., Wu, C. P., Li, H., and Chen, Y. J. (2008) A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease. Mol Cell Proteomics 7, 1983-1997. 47. Ravichandran, A., Sugiyama, N., Tomita, M., Swarup, S., and Ishihama, Y. (2009) Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species. Proteomics 9, 2764-2775. 48. Soufi, B., Gnad, F., Jensen, P. R., Petranovic, D., Mann, M., Mijakovic, I., and Macek, B. (2008) The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins. Proteomics 8, 3486-3493. 49. G. Salahas, Y. M. a. N. A. G. (1990) Assaying for pyruvate, orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme Photosynthesis Research 24, 183-188. 50. Chastain, C. J., Botschner, M., Harrington, G. E., Thompson, B. J., Mills, S. E., Sarath, G., and Chollet, R. (2000) Further analysis of maize C(4) pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues. Arch Biochem Biophys 375, 165-170. 51. Pawson, T., and Scott, J. D. (2005) Protein phosphorylation in signaling--50 years and counting. Trends Biochem Sci 30, 286-290. 52. Stock, A. M., Robinson, V. L., and Goudreau, P. N. (2000) Two-component signal transduction. Annu Rev Biochem 69, 183-215. 53. Cozzone, A. J., Grangeasse, C., Doublet, P., and Duclos, B. (2004) Protein phosphorylation on tyrosine in bacteria. Arch Microbiol 181, 171-181. 54. Backert, S., and Selbach, M. (2005) Tyrosine-phosphorylated bacterial effector proteins: the enemies within. Trends Microbiol 13, 476-484. 55. Gosse, J. L., Engel, B. J., Rey, F. E., Harwood, C. S., Scriven, L. E., and Flickinger, M. C. (2007) Hydrogen production by photoreactive nanoporous latex coatings of nongrowing Rhodopseudomonas palustris CGA009. Biotechnol Prog 23, 124-130. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47815 | - |
| dc.description.abstract | 紫色非硫光合菌Rhodopseudomonas palustris (R.palustris) 廣泛分布於土壤和河水中，分類階層屬於alpha-proteobacteria。R. palustris能利用太陽光當作能量來源，也可吸收大氣中的二氧化碳轉變成本身的重量，除此之外，它還能代謝並回收植物的木質組織。由於R. palustris和碳的利用與回收有密切的關係，因此被DOE Carbon Management Program選為基因體定序的物種。R. palustris能夠生長在光合異營、光合自營、化學異營以及化學自營等不同的代謝環境，並且可能利用磷酸化的方式來調控不同代謝路徑的轉變。在本研究中，我們在化學異營以及光合異營的條件下，利用shotgun的方式分析R. palustris的磷酸化蛋白質體表現。我們在化學異營的條件下鑑定到80條磷酸化胜肽，其可對應到73個蛋白質；而在光合異營的條件下則有74條磷酸化胜肽，可對應到68個蛋白質。另外，針對磷酸化胜肽定量的結果中發現，pyruvate phosphate dikinase (PPDK, RPA1051) 當中的胜肽“GGMpTSHAAVVAR”，在光合異營下蘇胺酸的磷酸化程度比化學異營條件下高出兩倍以上。目前已知PPDK在C4及CAM植物中是受磷酸化調控活性，並且在固碳上扮演重要的角色，但磷酸化的PPDK在原核生物的研究當中目前仍是未知。在我們的光合異營及化學異營內生性PPDK酵素活性測試實驗中發現，PPDK在光合異營條件下的活性較高；另外，在我們點突變磷酸化位置的實驗中也顯示，蘇胺酸的磷酸化位置對PPDK的活性是重要的。因此，從我們的結果推測，PPDK受到光刺激後，使蘇胺酸磷酸化並且同時具有活性，而磷酸化的PPDK也可能調控了R. palustris在光合異營與化學異營間的轉換機制。 | zh_TW |
| dc.description.abstract | Rhodopseudomonas palustris (R. palustris) is a purple nonsulfur anoxygenic phototrophic bacterium that belongs to the alpha-proteobacteria. It is a common soil and water bacterium that makes its living by converting sunlight to cellular energy and by absorbing atmospheric carbon dioxide and converting to biomass. This microbe can also degrade and recycle components of the woody tissues of plants. Because of its intimate involvement in carbon management and recycling, R. palustris was selected by the DOE Carbon Management Program for genome sequencing. R. palustris exhibits the ability to grow under photoheterotrophic, photoautotrophic, chemoheterotrophic and chemoautotrophic conditions, and can switch between the four different modes of metabolism for survival by protein phosphorylation. Here, we analyzed the phosphoproteome of R. palustris in photoheterotrophic condition with a shotgun approach and identified 80 phosphopeptides from 73 phosphoproteins and 74 phosphopeptides from 68 phosphoproteins at chemohetrotrophic and photoheterotrophic condition, respectively. Our results revealed that the threonine phosphorylated peptide “GGMpTSHAAVVAR” from pyruvate phosphate dikinase (PPDK, RPA1051) in photoheterotrophic condition is elevated more than 2 folds than in chemoheterotrophic condition. PPDK performs a light-dependent activity and plays an important role in carbon fixation in C4 and CAM plants. However, the function of phosphorylated PPDK is still unknown in prokaryotic cell. Here, we showed that PPDK enzyme activity is higher in photoheterotrophic than in chemoheterotrophic. In our in vitro point mutation experiment revealed that threonine phosphorylation site played an important role in regulating PPDK activity. We suggested that light could stimulate threonine phosphorylation of PPDK and might enhance its activity which could regulate the switch mechanism of R. palustris in photoheterotrophic and chemoheterotrophic conditions. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:20:12Z (GMT). No. of bitstreams: 1 ntu-99-R97b43002-1.pdf: 8997195 bytes, checksum: b34ae4e9331ac69d45718784b29bbffb (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 誌 謝......................................................I
中文摘要................................................. II 英文摘要................................................ III 縮寫表 ....................................................V 第1章簡介................................................. 1 1.1 Rhodopseudomonas palustris ........................... 1 1.2 磷酸化蛋白質體學(Phosphoproteome) .................... 2 1.3 丙酮酸磷酸雙激酶(PPDK) ............................... 4 1.3.1 序列分析............................................ 4 1.3.2 磷酸化活性調控...................................... 5 1.4 實驗動機與目的........................................ 5 第2章材料與方法 .......................................... 7 2.1 培養Rhodopsudomonas palustis方法...................... 7 2.2 穿透式電子顯微鏡觀察R. palustris外觀.................. 7 2.3 西方轉漬(Western blot) ............................... 7 2.4 磷酸化蛋白質體分析.................................... 8 2.4.1 定性分析............................................ 8 2.4.1.1 準備光合異營及化學異營條件的樣品.................. 8 2.4.1.2 破菌.............................................. 8 2.4.1.3 丙酮沉澱(Acetone precipitation)................... 9 2.4.1.4 蛋白質濃度定量(Protein assay) .................... 9 2.4.1.5 In-solution digestion ............................ 9 2.4.1.6 樣品酸化.......................................... 9 2.4.1.7 SCX陽離子交換樹脂................................ 10 2.4.1.8 TiO2純化步驟..................................... 10 清洗平衡 TiO2 ........................................... 10 TiO2純化磷酸化胜肽鏈..................................... 10 2.4.1.9 C18 desalting cartridge去除鹽類及雜質.............11 活化C18 desalting cartridge ............................. 11 注入樣品 ................................................ 11 2.4.1.10 LC-MS/MS 分析................................... 11 2.4.1.11 原始檔資料分析.................................. 11 2.4.1.12 生物資訊軟體分析................................ 12 2.4.2 定量分析........................................... 12 2.4.2.1 凝膠............................................. 13 2.4.2.2 洗膠............................................. 13 2.4.2.3 In-gel digestion ................................ 13 2.4.2.4 胜肽萃取......................................... 13 2.4.2.5 TiO2純化步驟..................................... 13 2.4.2.6 LC-MS/MS 分析.................................... 13 2.4.2.7 生物資訊軟體分析................................. 13 2.5 點突變............................................... 14 2.5.1 建構RPA1051基因質體................................ 14 2.5.2 膠體電泳回收(Gel-extraction) ...................... 14 2.5.3 樣品末端加A........................................ 15 2.5.4 TA cloning ........................................ 15 2.5.5 轉型作用(Transform) ............................... 16 2.5.6 菌落PCR篩選(Colony pcr screen) .................... 16 2.5.7 小量質體萃取( Plasmid extraction) ................. 17 2.5.8 T487V以及H489N的點突變............................. 17 DpnI digest原始的質體.................................... 18 2.5.9 DNA限制酵素反應(Double digestion) ................. 18 2.5.10 接合作用(Ligation) ............................... 19 2.5.11 轉型作用(Transform) .............................. 20 2.6 誘導表現PPDK (Induction) ............................ 20 2.7 蛋白質純化(Protein purification)..................... 21 2.8 PPDK 酵素活性測試(Enzyme activity assay)............. 21 第3章結果................................................ 23 3.1 光合異營與化學異營不同生長條件比較................... 23 3.1.1 外觀與生長......................................... 23 3.1.2 蛋白質變化......................................... 23 3.2 磷酸化蛋白質體學分析................................. 24 3.2.1 定性實驗........................................... 24 鑑定結果................................................. 24 Blast2Go分析磷酸化蛋白質功能............................. 26 3.2.2 定量實驗........................................... 27 鑑定結果................................................. 27 IdealQ- label free定量軟體分析........................... 27 3.3 菌體內PPDK活性測試................................... 29 3.4 點突變T487V、T487D與H489N in vitro PPDK活性測試...... 30 第4章討論................................................ 32 第5章參考資料 ........................................... 37 第6章圖.................................................. 44 第7章表.................................................. 70 附錄 .................................................... 86 | |
| dc.language.iso | zh-TW | |
| dc.subject | 化學異營 | zh_TW |
| dc.subject | 紫色非硫光合菌 | zh_TW |
| dc.subject | 磷酸化蛋白質體學 | zh_TW |
| dc.subject | 磷酸化胜肽 | zh_TW |
| dc.subject | 光合異營 | zh_TW |
| dc.subject | chemoheterotrophic | en |
| dc.subject | photoheterotrophic | en |
| dc.subject | phosphopeptide | en |
| dc.subject | phosphoproteome | en |
| dc.subject | Rhodopseudomonas palustris | en |
| dc.title | 紫色非硫光合菌之磷酸化蛋白質體學研究 | zh_TW |
| dc.title | Phosphoproteome of Rhodopseudomonas palustris | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃宣誠,吳世雄,陳水田,陳仁治 | |
| dc.subject.keyword | 紫色非硫光合菌,磷酸化蛋白質體學,磷酸化胜肽,光合異營,化學異營, | zh_TW |
| dc.subject.keyword | Rhodopseudomonas palustris,phosphoproteome,phosphopeptide,photoheterotrophic,chemoheterotrophic, | en |
| dc.relation.page | 107 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-10 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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