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完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor楊啓伸(Chii-Shen Yang)
dc.contributor.authorYu-Fong Linen
dc.contributor.author林育鋒zh_TW
dc.date.accessioned2021-06-16T05:08:56Z-
dc.date.available2023-08-20
dc.date.copyright2020-09-17
dc.date.issued2020
dc.date.submitted2020-08-14
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55817-
dc.description.abstract嗜鹽古細菌利用感覺型視紫質 (SR) 調控光趨性反應。本研究室於2010年發表了嗜鹽方形古菌 (Haloarcula marismortui) 為一株同時具有六種菌式視紫質的嗜鹽古細菌,其中包括三種光驅動離子幫浦和三種SR,除了被廣泛研究的SRI和SRII外,首次發現了SRM以及其配對傳導元HtrM。SRM-HtrM獨特之處在於SRM本身特徵吸收峰波長為503 nm,介於另外兩種感覺型視紫質之間;而HtrM相較於SRI和SRII的傳導元,同樣具有兩個穿膜螺旋,但在胞內側僅具一個HAMP-domain而非兩個,並且缺乏能與下游Che蛋白質作用的methyl-accepting domain。針對SRM-HtrM的特異性,2019年本研究室陳政良同學將SRM-HtrM移植到一株只有SRI和SRII兩種感覺型視紫質的Halobacterium salinarum中,發現活化SRM-HtrM同時會抑制菌體原先對藍光的迴避行為及對紅光的趨向行為,但其中的分子機制尚不清楚。本研究以生物資訊學分析SRM-HtrM發現:(一)HtrM和HtrII的第二個穿膜區進入胞內側的區域有一保守且與功能相關之轉角結構;(二)HtrM的C端有一個可能與Che蛋白質進行交互作用的區域。針對以上兩個功能相關區域進行突變得到突變型SRM-HtrM重組融合蛋白,並完成其特性測試。而為了往後測試HtrM與Che蛋白質間是否有交互作用,從Hm全基因序列中擷取CheW1基因並成功純化出帶有GST-tag之重組蛋白質。另一方面,截至目前為止,SR-Htr的功能性測試皆需要在嗜鹽古細菌中測試菌體光趨性,而嗜鹽古細菌的轉形相當耗時且不易成功。本研究以SRM和SRM-HtrM作為實驗對象,並結合全細胞光電流和蛋白質光電流測試發展出簡易且能快速測試SR與Htr之間是否有適當交互作用之功能性測試。zh_TW
dc.description.abstractHaloarchaea utilize sensory rhodopsin (SR) to regulate phototaxis responses. Among the haloarchaea, Haloarcula marismortui contains six microbial rhodopsins- three sensory rhodopsin (SR) and three light-driven ion-pump. In addition to SRI and SRII, in 2010, our lab discovered a new kind of SR, SRM, which has a maximum absorbance wavelength of 503 nm, between that of SRI and SRII. When comparing the cognate transducers, HtrM, to HtrI and HtrII, cytoplasmic domain of HtrM possesses only one HAMP-domain rather than two, therefore lacks the methyl-accepting domain to interact with Che proteins. These unique properties raise the questions on both the biological function and possible molecular mechanism of SRM-HtrM. In our past publication, we acknowledge that the blue-light photo-repellent and the red-light photo-attractant responses were both attenuated by light-activation of SRM-HtrM in SRM-HtrM knock-in Halobacterium salinarum cells, however, its molecular mechanism remains unknown. In this study, the bioinformatics analysis results of HtrM show that: (1) HtrM owns a functional-related turn structure behind the second transmembrane helix; (2) the C-terminal of HtrM remains a conserved region that could interact with Che proteins. For further investigation on SRM-HtrM interaction with Che protein, a construction of the HmCheW1 was made. Mutagenesis at those functional-related regions on HtrM and the characterization of those SRM-HtrM mutants were conducted. Until present, the functional assay of SR-Htr can only be done by testing phototaxis responses in haloarchaea cells. Our study combines the whole-cell and protein-based photocurrent assay to develop a more efficient and accurate functional assay for SR-Htr.en
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dc.description.tableofcontents目錄 I
圖目錄 IV
附圖目錄 V
表目錄 V
誌謝 VI
摘要 VII
Abstract VIII
第一章 緒論 1
第一節 嗜鹽古細菌與菌氏視紫質 2
1-1-1 紫外光/可見光譜最大吸收波長 3
1-1-2 光週期之時間長度 3
第二節 菌氏視紫質功能性分類 4
1-2-1 光驅動離子運輸蛋白 4
1-2-2 光趨性受器 5
第三節 Two-component system 10
1-3-1 微生物化學趨性 12
1-3-2 古細菌光趨性 14
第四節 死海鹽古菌六個視紫質系統 17
第五節 研究動機及策略 19
第二章 材料與方法 21
第一節 生物試劑 21
2-1-1 菌種 21
2-1-2 質體 21
第二節 藥品 21
第三節 儀器設備 23
2-3-1 核酸電泳 23
2-3-2 蛋白質電泳 23
2-3-3 離心機 23
2-3-4 光學設備 24
2-3-5 酸鹼度計 25
2-3-6 其他 25
第四節 實驗方法 25
2-4-1 蛋白質生物資訊學分析 25
2-4-2 DNA建構及轉形 26
2-4-3 重組蛋白質表現與純化 29
2-4-4 蛋白質定量及定性 31
2-4-5 視紫質性質分析 32
第三章 實驗結果 35
第一節 生物資訊學分析 35
3-1-1 蛋白質親緣關係比對 35
3-1-2 傳導元二級結構分析 41
3-1-3 建構三級結構模型 42
第二節 蛋白質突變與建構 45
3-2-1 HtrM突變設計 45
3-2-2 建構HmCheW1重組質體 46
第三節 HtrM mutants性質分析 48
3-3-1 紫外光/可見光譜 48
3-3-2 光週期 49
第四節 光電流測試 50
3-4-1 全細胞光電流量測 51
3-4-2 蛋白質光電流量測 52
第四章 結論與討論 53
第一節 HtrM HAMP-like domain 53
第二節 HtrM與Che蛋白質作用區域 55
第三節 HtrM突變之影響 57
第四節 感覺型視紫質與其傳導元功能性測試 58
第五節 SRM-HtrM光電流測試 59
第五章 未來展望 60
第六章 參考文獻 61
附圖 73
dc.language.isozh-TW
dc.subjectChe蛋白質zh_TW
dc.subject嗜鹽方形古菌zh_TW
dc.subject感覺型視紫質Mzh_TW
dc.subject光電流zh_TW
dc.subjectsensory rhodopsin Men
dc.subjectHaloarcula marismortuien
dc.subjectphotocurrenten
dc.subjectChe proteinen
dc.title感覺型視紫質SRM與其傳導元HtrM影響嗜鹽方形古菌光趨性之分子機制探討zh_TW
dc.titleA Study on the Possible Molecular Mechanism of SRM-HtrM Attenuates Phototaxis Responses in Haloarcula marismortuien
dc.typeThesis
dc.date.schoolyear108-2
dc.description.degree碩士
dc.contributor.oralexamcommittee梁博煌(Po-Huang Liang),李昆達(Kung-Ta Lee),徐駿森(Chun-Hua Hsu),吳亘承(Hsuan-Chen Wu)
dc.subject.keyword嗜鹽方形古菌,感覺型視紫質M,Che蛋白質,光電流,zh_TW
dc.subject.keywordHaloarcula marismortui,sensory rhodopsin M,Che protein,photocurrent,en
dc.relation.page74
dc.identifier.doi10.6342/NTU202001997
dc.rights.note有償授權
dc.date.accepted2020-08-17
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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