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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王愛玉(Ai-Yu Wang) | |
dc.contributor.author | Pei-Ju Lee | en |
dc.contributor.author | 李佩儒 | zh_TW |
dc.date.accessioned | 2021-06-08T01:02:06Z | - |
dc.date.copyright | 2015-01-27 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-10-14 | |
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Identification of genes differentially expressed during the growth of Bambusa oldhamii. Plant Physiology and Biochemistry 63: 217-26 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18375 | - |
dc.description.abstract | BoMSP41 (Monocot-specific protein-41 in Bambusa oldhamii) 為綠竹之未知功能基因,其基因表現在綠竹生長快速時期之節間部分會大幅提升。此基因轉譯出的蛋白質BoMSP41被預測為一個內生性不具穩定構型的蛋白質 (intrinsically disordered protein, IDP) 以及可能會被運送至成熟的葉綠體中。為了瞭解BoMSP41的特性,本研究用大腸桿菌中表現帶有 His-tag 的重組BoMSP41蛋白質並進行純化。重組BoMSP41被觀察到具有一些特殊的性質,包括:在SDS 膠體上的泳動率比預期的分子量應有的泳動率低、以膠體過濾層析預測的分子量高於預期的分子量以及具熱穩定性等,這些特性也是在IDPs中常被觀察到的特性。將純化過的重組BoMSP41利用圓二色光譜 circular dichroism (CD) 觀察其二級結構,結果顯示BoMSP41的二級結構中有8.6% alpha-helix、 37.0% beta-sheet、12.9% beta-turn 及 41.5% random coil。由上述結果顯示BoMSP41為具有disordered region,符合之前所預測的結果。以純化過後的重組BoMSP41蛋白質為抗原製備多株抗體,並對快速生長中的綠竹筍蛋白質粗抽液進行西方墨點法分析,結果顯示BoMSP41基因會在植物體內轉譯出BoMSP41蛋白質,而且可能不存在於葉綠體或細胞核分劃中。BoMSP41可能需要與其它蛋白質結合,以使其不具構形的區域轉變為具穩定的結構,以執行其功能。 | zh_TW |
dc.description.abstract | BoMSP41 (Monocot-specific protein-41 in Bambusa oldhamii), an unknown function gene, is highly up-regulated in the internode-containing region of rapidly elongating bamboo shoots. The protein encoded by this gene was suggested to be an intrinsically disordered protein (IDP) and might be translocated into chloroplasts. To understand the characteristics of BoMSP41, the His-tagged recombinant BoMSP41 proteins produced in E. coli were purified. The recombinant BoMSP41 had several unusual properties including a lower electrophoretic mobility on SDS-PAGE, overestimated molecular weight with gel filtration and high thermal stability, which are generally observed in many IDPs. Analysis of the purified recombinant BoMSP41 by circular dichroism (CD) spectroscopy suggested that there were 8.6% alpha-helix, 37.0% beta-sheet, 12.9% beta-turn and 41.5% random coil in BoMSP41. The results showed that BoMSP4 contained intrinsically disordered regions as previously predicted. The purified recombinant BoMSP41 proteins were used as antigen to raise polyclonal antibodies. Western analyses of proteins extracted from developing bamboo shoots with the polyclonal antibodies showed that the transcripts of BoMSP41 gene could be translated into proteins in planta and BoMSP41 might not be present in chloroplasts and nuclei. To perform its function, BoMSP41 might require binding partners to induce the transition of its disorder region to ordered conformation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:02:06Z (GMT). No. of bitstreams: 1 ntu-103-R01b22046-1.pdf: 3675685 bytes, checksum: 4703411dc9279f85cb726a813f98a9e0 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | Contents I
Abbreviation List IV Abstract VI 中文摘要 VIII Chapter 1. Introduction 1 1.1 BoMSP41, an unknown proteins found in bamboo 1 1.1.1 The sequence of BoMSP41 1 1.1.2 The subcellular localization of BoMSP41 2 1.2 Intrinsically disordered proteins (IDPs) 3 1.2.1 Recognition and characteristics of protein disorder 4 1.2.2 The functions of intrinsic disorder in proteins 5 1.3 The objectives of this research 7 Chapter 2. Materials and Methods 9 2.1 Expression and purification of recombinant BoMSP41 proteins in E. coli 9 2.1.1 Optimization of expression condition 9 2.1.2 Western blotting and immunodetection 10 2.1.3 Purification of recombinant BoMSP41 proteins 10 2.2 CD spectroscopy 14 2.3 Production of polyclonal antiserum against BoMSP41 14 2.4 ESI-MS-MS analysis 15 2.4.1 In gel digestion 15 2.4.2 Zip-tip purification 16 2.4.3 ESI-MS-MS analysis 17 2.5 Immunoprecipitation 17 2.6 Preparation of total proteins and subcellular fractions of bamboo shoots 18 2.6.1 Extraction of total protein from bamboo shoots 18 2.6.2 Preparation of subcellular fractions of bamboo shoots 18 Chapter 3. Results 20 3.1 Prediction of the secondary structure of BoMSP41 20 3.2 Characterization of the recombinant BoMSP41 proteins expressed in E. coli…. 20 3.2.1 Production of the recombinant BoMSP41-2 protein in E. coli 20 3.2.2 Electro-transfer efficiency of BoMSP41 using different transfer buffers. 21 3.2.3 Purification of the soluble recombinant BoMSP41-2 proteins from E. coli…… 22 3.2.4 Purification and refolding of recombinant BoMSP41-2 inclusion bodies.. 23 3.2.5 The secondary structure of rBoMSP41-2 analyzed by CD spectroscopy 25 3.3 Analysis of BoMSP41 subcellular localization from bamboo 25 Chapter 4. Discussions 27 Chapter 5. Perspectives 30 References 32 Tables 38 Figures 39 | |
dc.language.iso | en | |
dc.title | 重組綠竹 Monocot-Specific Protein-41 之純化與性質檢定 | zh_TW |
dc.title | Purification and characterization of the recombinant Bambusa oldhamii Monocot-Specific Protein-41 | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 楊健志(Chien-Chih Yang) | |
dc.contributor.oralexamcommittee | 陳佩燁(Rita P.-Y. Chen),宋賢一(Hsien-Yi Sung),張世宗(Shih-Chung Chang) | |
dc.subject.keyword | 綠竹,單子葉植物特有蛋白質,重組蛋白質,包涵體,純化,圓二色光譜,內生性不具穩定構型蛋白質,細胞內定位, | zh_TW |
dc.subject.keyword | bamboo,monocot-specific protein,recombinant protein,inclusion bodies,purification,circular dichroism,intrinsically disordered protein,subcellular localization, | en |
dc.relation.page | 56 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-10-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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