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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊啟伸 | |
dc.contributor.author | Yu-Wei Cheng | en |
dc.contributor.author | 鄭有為 | zh_TW |
dc.date.accessioned | 2021-06-17T07:00:03Z | - |
dc.date.available | 2019-08-19 | |
dc.date.copyright | 2019-08-19 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-02 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72506 | - |
dc.description.abstract | 嗜鹽古細菌通常在有高鹽濃度及營養不充分的居住環境下被發現,他們演化出各種視紫蛋白質,協助將光能轉變為生物所需的能量,或是感受外界環境的訊息,使他們能夠在原生環境下生存;主要有離子型和幫浦型兩種。在所有光驅動離子型幫浦視紫蛋白質中,氫視紫質 (BR) 是氫離子向胞外幫浦,並藉此建立氫離子濃度梯度,推動F1Fo ATP合成酶產生ATP。氯視紫質 (HR) 目前已知為氯離子向胞內幫浦,維持細胞的滲透壓和膜電位。而感受型視紫蛋白質緊密地與其專一的傳導元(transducer) 連接,進行光趨性的反應。目前已知有三種感受型視紫蛋白質,一為感受型視紫質第一型 (SRI),會幫助正趨光反應;二為感受型視紫質第二型(SRII),會對藍光產生反應;三為感受型視紫質M (SRM),對藍綠光敏感,會抑制前兩種感受型視紫質傳遞訊號。截至目前為止,Halobacterium salinarum 以及 Haloarcula marismortui 為嗜鹽古細菌中主要的研究模式品種,其各至少包含上述四種視紫質 (BR、HR、SRI、SRII)。先前X光結晶繞射的結構解析揭開了BR、HR、SRII的原子結構以及作用機制;而SRI則因為其結構在低鹽的不穩定性而尚未有原子結構被解出。因此我們將重點放在一個能耐受低鹽的HtSRI,是來自台灣南部本土新品種嗜鹽古細菌,Haloarcula taiwanesis,藉由研究其耐受低鹽的機制,期望對解SRI結構上有所助益。在本篇論文中,我們發現其BC loop為貢獻此特性的重要區位。
關鍵字:感受型視紫蛋白質第一型;低鹽耐受性 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:00:03Z (GMT). No. of bitstreams: 1 ntu-108-R06b22003-1.pdf: 6728781 bytes, checksum: 93ff2b5fc2d34d2162afa6e30396fb34 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 目錄……………………………………………………………………………………..2
圖目錄…………………………………………………………………………………..5 表目錄…………………………………………………………………………………..6 摘要……………………………………………………………………………………..7 Abstract………………………………………………………………………………….8 第一章 緒論 9 第一節 古細菌的分類地位與嗜鹽古細菌之介紹 9 第二節 微生物視紫蛋白質 (microbial rhodopsin) 10 第三節 古細菌感受型視紫質 (Sensory rhodopsin, SR) 13 1.3.1 紫外光/可見光譜 (UV/Vis Spectrum) 13 1.3.2 光週期 (Photocycle) 15 1.3.3 質子運輸 16 1.3.4 重要胺基酸 17 1.3.5 胞內環及胞外環對於視紫質的影響 18 第四節 感受型視紫質趨光性調控機制 19 1.4.1 傳導元的結構與特性 19 1.4.2 嗜鹽古細菌的光趨性 22 1.4.3 感受型視紫質調控光趨性之詳細機制 23 第五節 研究動機及流程 24 第二章 實驗材料與方法 25 第一節 生物試劑 25 2.1.1 菌種 25 2.1.2 質體 25 2.1.3 蛋白質藥品 25 2.1.4 化學藥品 26 第二節 儀器設備 28 2.2.1 核酸電泳 28 2.2.2 蛋白質電泳及轉印 28 2.2.3 離心機 28 2.2.4 光學設備 29 2.2.5 恆溫培養箱 30 2.2.6 酸鹼度計 30 2.2.7 其他 30 第三節 實驗方法 31 2.3.1 生物資訊學分析 31 2.3.2 DNA建構及轉形 31 a. 小量核酸萃取 31 b. HtSRI突變株之質體建構 31 c. 聚合酶鏈鎖反應 (PCR) 33 d. DNA膠體純化 33 e. 限制酶截切 33 f. DNA黏合 33 g. 大腸桿菌轉形 34 h. 大腸桿菌轉形 34 i. 轉形株鑑定 34 2.3.3 重組蛋白質之表現及純化 35 a. 重組視紫質表現 35 b. 重組視紫質純化 35 2.3.4 蛋白質定性測量 36 a. 蛋白質吸收光譜測定 36 b. 視紫質光週期量測 36 c. 光照生長曲線 36 2.3.5 蛋白質耐受性測試 37 a. 強烈光照激發測試 37 b. 溫度耐受性測試 37 c. 酸鹼度測試 37 d. SDS解構測試 37 第三章 結果與討論 38 第一節 生物資訊系統分析 38 3.1.1 TMHMM 穿膜區預測及序列比對 38 3.1.2 PyMol 分析 40 第二節 菌體之光照生長曲線 42 第三節 感受型視紫蛋白質之穩定性測試 43 3.3.1 不同鹽濃度下之最高吸收光譜 44 3.3.2 光週期 47 3.3.3 感受型視紫蛋白質之強光盲化恢復能力測試 49 3.3.4 感受型視紫蛋白質之界面活性劑耐受性測試 51 3.3.5 感受型視紫蛋白質之溫度耐受性測試 52 3.3.6 酸鹼度測試 53 第四章 結論 54 第五章 未來展望 55 第六章 參考文獻 56 第七章 附錄 60 7.1.1 NMR 簡介 60 7.1.2 Material and methods 61 7.1.3 Results 62 圖目錄 圖一、發光基團retinal變化 10 圖二、四種類型之微生物視紫質 11 圖三、感受型視紫質第一型及第二型之作用機制 12 圖四、視紫質於光譜上之分布關係圖 14 圖五、四種微生物視紫質之光週期變化 15 圖六、SRs 胺基酸序列比對 17 圖七、NpSRII結構(PDB: 1JGJ) 18 圖八、傳導元之結構及比較 19 圖九、H. taiwanensis之transducer I 蛋白質序列分析 21 圖十、NpSRII 傳遞避光反應的訊息路徑 23 圖十一、光週期實驗器材設置簡圖 29 圖十二、利用TMHMM預測HtSRI的穿膜區 38 圖十三、利用TMHMM預測HsSRI的穿膜區 39 圖十四、SRs 胺基酸序列比對 39 圖十五、HtSRI模擬模型以及表面電荷分布示意圖 41 圖十六、HsSRI模擬模型以及表面電荷分布示意圖 41 圖十七、H. taiwanensis與H. salinarum 在不同鹽濃度下的生長曲線 42 圖十八、HsSRI 野生株、HtSRI野生株及突變株在不同鹽濃度下之吸收光譜 45 圖十九、HsSRI 野生株、HtSRI野生株及突變株在不同鹽濃度下之最大吸收峰值比較 46 圖二十一、HsSRI 野生株、HtSRI野生株及其突變株之光週期tau value 48 圖二十六、HtSRI野生株及突變株共三種蛋白質之bleaching test(無靜置於黑暗回復) 50 圖二十七、HtSRI野生株及突變株共三種蛋白質之bleaching test(有靜置於黑暗回復) 50 圖二十八、HtSRI野生株及突變株共三種蛋白質之SDS耐受性測試 51 圖二十九、HtSRI野生株及突變株共三種蛋白質之溫度耐受性測試 52 圖三十、HtSRI野生株及突變株共三種蛋白質之pH titration 53 圖三十一、pET-21b(空質體)轉型至E. coli C43 63 圖三十二、HtSRI轉型至E. coli C43 63 圖三十三、HwMR轉型至E. coli C43 64 圖三十四、HmSRM轉型至E. coli C43 64 表目錄 表 1、各種視紫質在4 M NaCl, 50 mM MES, 0.02% DDM, pH 5.8下的最大吸收波長 13 | |
dc.language.iso | zh-TW | |
dc.title | 探討Haloarcula taiwanensis感受型視紫質第一型之穩定性的關鍵區域 | zh_TW |
dc.title | Probing the key region conferring stability of Sensory Rhodopsin I From Haloarcula taiwanensis | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李昆達,吳韋訥,吳?承,梁博煌 | |
dc.subject.keyword | 感受型視紫蛋白質第一型,低鹽耐受性, | zh_TW |
dc.subject.keyword | Haloarcula taiwanensis,sensory rhodopsin I,low salt concentration, | en |
dc.relation.page | 64 | |
dc.identifier.doi | 10.6342/NTU201902291 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-05 | |
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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