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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志 | zh_TW |
| dc.contributor.advisor | Chien-Chih Yang | en |
| dc.contributor.author | 陳逸甄 | zh_TW |
| dc.contributor.author | I-Chen Chen | en |
| dc.date.accessioned | 2025-02-27T16:31:23Z | - |
| dc.date.available | 2025-02-28 | - |
| dc.date.copyright | 2025-02-27 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-02-13 | - |
| dc.identifier.citation | Binder, B. M. (2020). Ethylene signaling in plants. Journal of Biological Chemistry, 295(22), 7710-7725.
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K. (2016). Abiotic stress signaling and responses in plants. Cell, 167(2), 313-324. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97172 | - |
| dc.description.abstract | Heptahelical proteins 1 (HHP1) 是一個阿拉伯芥的穿膜蛋白，主要參與ABA和滲透壓信號的調控。HHP1的N端片段 (第1至第96個胺基酸) 被推測包含固有無序區域 (Intrinsically disordered regions, IDRs)。我們先前的研究發現，HHP1的N端可能與冷逆境調節因子ICE1 產生交互作用。由於ICE1是阿拉伯芥對抗冷逆境的關鍵調節因子，我們推測 HHP1 的 N 端可能在冷訊號及滲透壓訊號傳遞中扮演一定的角色。為了研究HHP1 N 端片段的生物物理及生化特性，我們使用E coli以及pET16b質體建構了重組蛋白生產系統，並表現重組蛋白10His_nHHP1 (13.96kDa)。最佳的表現條件是在25°C下，以0.2 mM IPTG誘導E. coli Rosetta (DE3)，並經由LC-MS/MS確認為目標蛋白。利用圓二色光譜 (Circular Dichroism, CD) 進行二級結構分析，我們發現10His_nHHP1在加熱至95°C後，原本呈現的random coils結構部分轉變為α-helices結構。根據這一觀察，我們初步推測，可能是樣品中的雜蛋白干擾了nHHP1的實際構像，導致其光譜特徵顯示為random coils。因此，我們計劃在純化前進行熱處理，藉此降解雜蛋白以驗證此假設。然而，經過熱處理後的10His_nHHP1，並未顯現出有序結構的特徵。我們推測，這可能與環境因素改變有關，不僅僅是因為溫度，還可能包括鹽濃度和 pH 的變化。基於這些結果，我們推論nHHP1的結構轉變可能需要其他因子的協助。總而言之，我們成功建立了 nHHP1 的重組蛋白生產系統，並初步揭示了其在各種環境條件下的結構動態特性。 | zh_TW |
| dc.description.abstract | Heptahelical Proteins 1 (HHP1) is a transmembrane protein in Arabidopsis thaliana, primarily involved in regulating ABA and osmotic stress signaling. The N-terminal of HHP1 (1 A.A to 96 A.A) is predicted to be intrinsically disordered regions. Our previous research found that the N-terminal of HHP1 may interact with the cold stress regulator ICE1. Given that ICE1 is a key factor in Arabidopsis cold stress response, we hypothesize that the N-terminal of HHP1 may play a role in cold and osmotic stress pathways. In order to study the biophysical and biochemical properties of this fragment, we established a recombinant protein production system using E. coli and pET16b plasmid to express 10His_nHHP1 (13.96 kDa). Optimal expression was achieved in E. coli Rosetta (DE3) with 0.2 mM IPTG at 25°C, and soluble protein was confirmed by LC-MS/MS. CD spectroscopy revealed that nHHP1 undergoes a structural transition from random coils to partially α-helices upon heating to 95°C. This observation led us to hypothesize that other proteins might influence the actual conformation of nHHP1, resulting in spectral characteristics resembling random coils. To test this hypothesis, we implemented heat treatment prior to purification to degrade other proteins. However, following heat treatment, 10His_nHHP1 did not exhibit features of ordered structure. These findings suggest that the observed effects may be influenced not only by temperature but also by other environmental factors, such as variations in salt concentration and pH. In summary, we successfully established a recombinant protein production system for nHHP1 and gained preliminary insights into its structural dynamics under diverse environmental conditions. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-02-27T16:31:23Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-02-27T16:31:23Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
謝辭 ii Abstract iii 摘要 iv 縮寫 v 目次 vii 第一章 緒論 1 1.1 阿拉伯芥 Heptahelical proteins 1 (HHP1) 1 1.1.1 HHP 家族 (HHP family) 2 1.1.2 HHP1已知的生理功能 2 1.1.3 HHP1可能作為冷信號以及滲透壓路徑的反應交叉點 3 1.1.4 HHP1已知結構域及其功能 5 1.1.5 穿膜蛋白的裂解與後續基因調控的關聯 6 1.2 固有無序蛋白質／固有無序區域 (IDPs/IDRs) 7 1.2.1 IDPs/IDRs 序列與結構特徵 8 1.2.2 IDP/IDRs 有諸多生物功能 8 1.2.3 IDP/IDRs 的特殊物理特性使其在蛋白質—蛋白質交互作用中扮演重要樞紐 9 1.2.4 IDPs純化之挑戰 10 1.3 研究動機與目標 11 第二章 材料與方法 14 2.1 實驗材料 14 2.2 菌株 14 2.2.1 大腸桿菌培養基 15 2.2.2 載體 15 2.3 實驗方法 15 2.3.1 生物資訊學分析 15 2.3.1.1 序列資料 15 2.3.1.2 蛋白質結構預測分析 15 2.3.2 質體建構 16 2.3.3 蛋白質表現與純化 16 2.3.3.1 勝任細胞製備 16 2.3.3.2 大腸桿菌轉型 16 2.3.3.3 菌液培養及重組可溶蛋白質表現測試 17 2.3.3.4 重組可溶蛋白質破菌及離心 17 2.3.3.5 親和管柱層析 17 2.3.3.6 超微薄膜過濾法 18 2.3.3.7 煮沸法 (Boiling method) 18 2.3.3.8 分子篩色譜法 18 2.3.4 蛋白質定量與定性分析 19 2.3.4.1 蛋白質變性電泳 19 2.3.4.2 Coomassie Brilliant Blue R250 staining 20 2.3.4.3 西方墨點法 (Western Blot) 21 2.3.5 蛋白質濃度測定 21 2.3.5.1 Bradford assay 21 2.3.5.2 紫外-可見光光譜 (Ultraviolet–visible spectroscopy，UV-Vis) 22 2.3.6 結構與化學分析 22 2.3.6.1 藉由遠紫外線圓二色光譜分析蛋白質的二級結構 22 第三章 結果 23 3.1 HHP1的生物資訊學分析 23 3.1.1 HHP1之胺基酸組成分析 23 3.1.2 HHP1片段之N端片段含有IDR 24 3.2 重組蛋白HHP1表現與純化 27 3.2.1 10His_nHHP1之序列 27 3.2.2 使用勝任細胞Rosetta (DE3) 及低溫誘導下表現效果最佳 27 3.2.3 使用HisTrap純化重組蛋白10His_nHHP1 ，純化條件已成功建立 28 3.2.4 使用分子篩色譜法再次純化重組蛋白10His_nHHP1，使其純度更高 29 3.2.5 透過LC-MS/MS 鑑定10His_nHHP1之身份 30 3.3 nHHP1之光譜分析 30 3.3.1 以圓二色光譜分析10His_nHHP1的二級結構，並推測為random coils 30 3.3.2 經由變溫處理分析，10His_nHHP1的二級結構會在95度時部分轉變為alpha-helix 31 3.4 煮沸法使用時機在純化過程中對 10His_nHHP1 特性的影響 32 3.4.1 高溫處理可提升10His_nHHP1之純度及RNA殘留問題 32 3.4.2 優化煮沸法之使用時機以提高蛋白純度並去除 RNA 33 3.4.3 再次確認加熱對 nHHP1 構型變化的影響 34 3.4.4 純化方法、溫度與鹽濃度對 nHHP1 特性與構型的影響 34 第四章 討論 35 4.1 nHHP1表現與純化 35 4.2 IDPs/IDRs於SDS-PAGE 上之分子量異常遷移 36 4.3 nHHP1構型改變 37 4.4 未來展望 38 參考文獻 41 圖次 46 圖ㄧ、HHP1 全長與N端之胺基酸組成分析 47 圖二、HHP1之生物資訊學研究 49 圖三、重組蛋白10His_nHHP1之序列 50 圖四、重組蛋白10His_nHHP1之粗萃取小量表現測試 51 圖五、重組蛋白10His_nHHP1破菌後之粗萃取小量表現及可溶性測試 53 圖六、重組蛋白10His_nHHP1之大量表現與蛋白質純化分析 55 圖七、重組蛋白10His_nHHP1之身份鑑定。 56 圖八、10His_nHHP1之紫外-可見光光譜分析 57 圖九、10His_nHHP1之圓二色光譜分析 58 圖十、溫度對10His_nHHP1構象和二級結構的影響。 60 圖十一、經由煮沸法處理後之10His_nHHP1蛋白質純化分析 (1) 62 圖十二、經由煮沸法處理後之10His_nHHP1蛋白質1之紫外-可見光光譜分析 63 圖十三、經由煮沸法處理後之10His_nHHP1蛋白質純化分析 (2) 65 圖十四、經由煮沸法處理後之10His_nHHP1蛋白質圓二色光譜分析 67 附錄 69 問答集 72 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 植物逆境 | zh_TW |
| dc.subject | 構型變化 | zh_TW |
| dc.subject | 固有無序蛋白 | zh_TW |
| dc.subject | 蛋白質折疊 | zh_TW |
| dc.subject | protein folding | en |
| dc.subject | IDPs/IDRs | en |
| dc.subject | conformational changes | en |
| dc.subject | plant stress | en |
| dc.title | 阿拉伯芥穿膜蛋白HHP1之N端結構解析 | zh_TW |
| dc.title | Structural characterization studies on the N-terminal of HHP1 from Arabidopsis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 李昆達;陳佩燁 | zh_TW |
| dc.contributor.oralexamcommittee | Kung-Ta Lee;Pei-Yeh Chen | en |
| dc.subject.keyword | 固有無序蛋白,構型變化,植物逆境,蛋白質折疊, | zh_TW |
| dc.subject.keyword | IDPs/IDRs,conformational changes,plant stress,protein folding, | en |
| dc.relation.page | 75 | - |
| dc.identifier.doi | 10.6342/NTU202500640 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-02-13 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| dc.date.embargo-lift | 2030-02-11 | - |
| 顯示於系所單位： | 生化科技學系 | |
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