臺灣博碩士論文加值系統

古生菌的感覺型視紫質 (Sensory Rhodopsin, SR) 可藉由感受不同可見光波長之光源，並藉由其對應之傳導元 (Transducer, Htr) 傳遞訊息，以調控嗜鹽古生菌之趨、避光反應。目前發現之SRs共有三種型態 : SRI, SRII及SRM；其感受之可見光波長分別分佈於紅藍綠光波段。基因體的序列中，嗜鹽古生菌Natronomonas pharaonis 僅具有調控吸收 ~498 nm 而驅動避光的NpSRII、 Halobacterium salinarum 擁有SRII及吸收~590 nm 來調控趨光的SRI 。本實驗室先前發表基因體的 Haloarcula marismortui，為一三色感光系統的嗜鹽古生菌，除了SRI, SRII以外，尚有一個功能未定的 SRM。SRI 及 SRII 之傳導元藉由類同細菌中化學趨性的 two-component system ，將光訊號轉化為化學訊息傳遞至鞭毛，進而調控菌體之泳動 ; 然而， SRM的傳導元 HtrM 結構上缺乏了許多構件，因此我們推測 SRM-HtrM應由其他機制調控嗜鹽古生菌之光趨性。本篇研究，先建立 SRM 及SRM-HtrM 在二色感光系統 H. salinarum菌株中的表現及確立其功能，再以兩種本篇研究發展之光趨性研究方法 (ELISA Reader 之量測及顯微鏡之觀察)，量化 Ｈ. salinarum 及其轉形株的光趨反應。初步結果顯示， SRM-HtrM減少了 H. salinarum在綠光及藍光下的避光反應; 並且，在 HtrM 缺乏的轉形株中，該現象並沒有被觀察到。因此，我們認為 HtrM 的存在，對於 SRM-HtrM 複合蛋白質在 H. salinarum中調控的趨光反應是重要的。未來，可以嘗試將 SRM-HtrM 複合蛋白質嵌入磷脂中，分析其與化學趨性相關蛋白質之間的交互作用，以解出其分子機制。

A group of photoreceptors, sensory rhodopsin (SR), regulates phototaxis in haloarchaea through absorbing diverse range of visible light and relaying the signals to the cell by their cognate transducer (Htr). To date, there are three types of SRs identified: SRI, SRII and SRM, and they response to red, blue and green light, respectively. Among the annotated archaeal genomes, Natromonas pharaonis solely own the NpSRII, which response blue light and mediates photorepellent; Halobacterium salinarum holds HsSRII and HsSRI, and HsSRI is known to absorb ~590 nm of light to mediate photoattractant. However, in Haloarcula marismortui, there exists three SRs, namely SRI, SRII and functionally unknown SRM. Previous studies showed that SRI and SRII transduce photo signal to flagellum through transducer and chemotaxis proteins, similar to two-component system of chemotaxis in bacteria. However, it is speculated that the SRM-HtrM complex regulates the phototaxis responses through new pathway as SRM-HtrM lacks many structural components seen in other transducers. In this study, we transplanted SRM and SRM-HtrM into H. salinarum cells and compare the phototaxis responses of H. salinarum and its transformants under different wavelengths of light through two new measurements developed in this study. It is found that SRM-HtrM decreased the photorepellent response of H. salinarum in green and blue light; but not in SRM transformant. The importance of HtrM in SRM-HtrM signaling was concluded. In future, the molecular mechanism of SRM-HtrM can be examined through measurements of the interaction between lipid-reconstituted SRM-HtrM with related chemotaxis proteins.

目錄
目錄………………………………………………..…….…..………..……..………. i
圖目錄…………………………………………………..…….……..….…………… vi
表目錄…………………………………………………………………...…………... viii
摘要…………………………………………………………..………...……………. ix
Abstract…………………………………………………………….…..…………… x
第一章 緒論……………………………………………………..……………... 1
第一節 嗜鹽古生菌之介紹…………………………………..…..………...... 1
第二節 微生物視紫質…………………………………….….….….....…….. 2
第三節 古生菌感覺型視紫質………………….....……………..…….……. 7
I-3.1 紫外光/可見光譜……………………….…….....……………...….. 7
I-3.2 光週期……………………………………….....…….………….….. 8
I-3.3 質子傳輸………………………………….....…….…………...…… 9
I-3.4 重要胺基酸……………………………….....…….…...…………… 11
I-3.5 演化探討………………………………………......…..….………… 13
第四節 傳導元 (Transducer) 的結構與特性………….....….….……..…… 14
第五節 嗜鹽古生菌的光趨性…………………………...……….….….…… 16
I-5.1 SRs於嗜鹽古生菌中的光趨性…………….....…...…....….……… 16
I-5.2 嗜鹽古生菌的光趨性…………………………......….…….….…… 16
I-5.3 SRs調控光趨性之機制……………….....……….…..….….……... 18
第六節 研究動機及策略……………….....…….……….……..…………... 20


第二章 材料與方法……………………………………….....……...………...……. 22
第一節 生物試劑……………………………..…….....…………...........…… 22
II-1.1 菌種………………………………….....…….….………………… 22
II-1.2 質體…………………………………....…….…………………….. 23
II-1.3 蛋白質藥品……………………….....…..…...…...…………..…… 23
第二節 化學藥品……………………………….....…..…….……….………. 23
第三節 儀器設備………………………………….....…..….…..….………... 25
II-3.1 核酸電泳…………………………………….…….......…..…….… 25
II-3.2 蛋白質電泳及轉印………………………….....………….…….… 25
II-3.3 離心機…………………………………….…………..….....…...… 25
II-3.4 光學設備………….....…..………………………….…..…….…… 26
II-3.5 恆溫培養箱………………….....…..……………………...….…… 27
II-3.6 酸鹼度計……………………………….....…..……….…..….…… 27
II-3.7 其他………………………………………….…….....…..…...…… 27
第四節 實驗方法…………….....…..……………………………….…..…… 28
II-4.1 生物資訊學分析…………….....…..……………………………… 28
II-4.1.1 基因資料庫……………………….....…..………….…..…… 28
II-4.1.2 序列親緣比對………………………….…….....…..…..…… 28
II-4.1.3 蛋白質結構分析……………………….……….....………… 28
II-4.2 DNA建構及轉形…….....…..………………………………..….….. 29
II-4.2.1 小量核酸萃取………….....…..………………….…….…… 29
II-4.2.2 聚合酶鏈鎖反應 (PCR) ………….....…..…….…..……….. 29
II-4.2.3 DNA膠體純化………………………….....…..…….……… 30
II-4.2.4 限制酶截切……………………………….....…..……...…… 30
II-4.2.5 DNA黏合………………………………….....….…....….…. 30
II-4.2.6 大腸桿菌轉形…………………………….....…..…….…..… 30
II-4.2.7 嗜鹽古生菌H. salinarum轉形………….....…..….…..….… 31
II-4.2.8 轉形株鑑定.....…..……………………………………….….. 32
II-4.3重組蛋白質之表現及純化….…..………………………….……… 32
II-4.3.1 重組視紫質表現………….....…..………………….……..… 32
II-4.3.2 重組可溶蛋白質表現………………….....…..……….…..… 33
II-4.3.3 重組視紫質純化……………………………….........…....…. 33
II-4.3.4 重組可溶蛋白質純化 (Hexa-His-tagged) .....…..…….….… 34
II-4.3.5 重組可溶蛋白質純化 (GST-tagged)…….....…..…….…….. 34
II-4.3.6 H. salinarum細胞膜之純化……...….……………………… 35
II-4.4 蛋白質定量及定性….......…………..…………………….…….… 35
II-4.4.1 蛋白質定量…………….....…………………………….…… 35
II-4.4.2 蛋白質變性電泳………………..….....…..………….……… 36
II-4.4.3 蛋白質原態電泳…………………..………….....…....……… 37
II-4.4.4 蛋白質轉印……….....…..…………..……………….……… 38
II-4.4.5 免疫呈色…………………….....…..…..…………….……… 38
II-4.5 感光蛋白質光學分析…………………………....…..…….……… 38
II-4.5.1 吸收光譜測定……………………………..……….....…..…. 38
II-4.5.2 視紫質光週期量測…………….....…..…..………….……… 39
II-4.5.3 光驅動離子幫浦活性測定………………….......…..….…… 39
II-4.6 嗜鹽古生菌生理分析……..………………........…..……….…….. 39
II-4.6.1 泳動菌株挑選……………………….......….....…………....…… 39
II-4.6.2 顯微鏡觀察…………………….....…..……...…..……………… 40
II-4.6.3 光趨性分析……….…..………………………….……………… 41
II-4.6.4 光照生長曲線…….......………………….……………………… 41

第三章 實驗結果………………….....…..…………………………………….….... 43
第一節 蛋白質性質….....…..…………………………………...…….….…… 43
III-1.1 序列比對………….....…..…………………………….….….…… 43
III-1.2 結構模擬…………………….....…..……...……….…..….……… 44
III-1.3 pI 及 pKa…………………………….....…..…….….....….…… 46
III-1.4 光誘發質子傳輸………………………….……….....…........…… 49
III-1.5 光週期……......……………………………………………...……. 50
第二節 微生物光趨性研究方法….....…..…………………………….…....… 51
III-2.1 顯微鏡觀察…………………….....…..….…………….…...…..… 51
III-2.2 ELISA Reader測定……………..………….....……….....………. 56
第三節 SRM 及 SRM-HtrM 之移植……………………....……...……...…. 60
III-3.1 DNA 確認…………..……………………………….……..…….. 60
III-3.2 蛋白質表現確認………....…..……………………..……….……. 60
III-3.3 SRM功能性測試……………….....…..………..…………...…… 62
第四節 嗜鹽古生菌之生理探討……………………….....…..……...…..…… 64
III-4.1 細胞型態……………………………………….….....….....….….. 64
III-4.2 生長曲線………………….....…..…………………………...…… 65
III-4.3 光趨性研究………………………….....…..…………...………… 67
第四章 結論與探討………………………………….....…..……….…….….…….. 70
第一節 微生物之光趨性研究………….....…..……………………....……… 70
第二節 光趨性在生理上之意義………….....…..………………..….….…… 71
第三節 H. salinarum的SRM-HtrM功能獲得型轉殖株………...........…..... 72
第四節 嗜鹽古生菌的光趨性機制…………………….......………........…… 73

第五章 未來展望……………………………….....…..………………..….……….. 74
第一節 感覺型視紫質的轉介…………....…..……………...…….………… 74
第二節 光趨性機制之研究方向……………………………....…...………… 74

第六章 參考文獻………………………………………………………….....…...… 76
附圖………………………………………………………………..…….....….......….83











圖目錄

圖1 : 微生物視紫質之共通特性……………………….....…..…….……..........… 2
圖2 : 嗜鹽古生菌中視紫質的分佈……….....…..………………...……….……... 4
圖3 : 嗜鹽古生菌中感覺型視紫質與其傳導元………………......………...….… 6
圖4 : H. salinarum感覺型視紫質的質子傳輸現象………......…..……...….…… 10
圖5 : 感覺型視紫質重要胺基酸比對……………….….....…..………………..… 12
圖6 : 傳導元之結構及比較…………….....…..…………….………………..…… 14
圖7 : 菌體泳動分析……………………….....…..……………….…….….……… 17
圖8 : NpSRII傳遞避光反應的訊息路徑…………………………...……..……… 19
圖9 : 實驗流程表…………………………………..……….....…….…………..… 21
圖10 : 感覺型視紫質之間的蛋白質多序列比對…………….……....………….… 45
圖11 : HmCheR及HmHtrM之模擬結構 ……………….………………...……… 46
圖12 : SRM及其複合蛋白質之pKa測定………………….……………..…….… 47
圖13 : 原態等電聚焦電泳……………………….……………………….....….….. 48
圖14 : SRM及SRM-HtrM的氫離子傳輸能力……………….………….…..…… 49
圖15 : 顯微鏡之架設…………………………….……………………….….……... 52
圖16 : LED燈源之波譜………………………….…………….………………...… 53
圖17 : 樣品薄片…………………………….……………………….…...….……… 53
圖18 : 顯微鏡刺激光源的照射範圍……………….……………………….……… 54
圖19 : 光源刺激時間………………………….…………………………………… 55
圖20 : 光趨性影像分析………………………………………………………..…… 55
圖21 : 光趨性分析裝置架設………………….………………………….………… 56
圖22 : 照射光源的波譜……………….…………………………….……………… 57
圖23 : 偵測光源範圍……………….…………………………..……..….………… 58
圖24 : 光源限制片…………………………….…………..…………………...…… 58
圖25 : C. reinhardtii於光趨性分析裝置之分析……….…………….…..…...….… 58
圖26 : C. reinhardtii在白光下的巨觀避光現象………………….………....…..… 59
圖27 : H. salinarum 轉形株的菌落PCR…………………………………..….…… 61
圖28 : H. salinarum 轉形株之Western Blot……………….……….…..…...…..… 61
圖29 : H. salinarum 野生株及轉形株細胞膜的可見光吸收波譜…………...…… 62
圖30 : SRM 及 SRM-HtrM 於不同條件之光週期分析……………….....……… 63
圖31 : H. salinarum 野生株及轉形株型態…………………………….…..……… 64
圖32 : 嗜鹽古生菌於不同光照下之生長曲線………….…………….….…...…… 66
圖33 : 嗜鹽古生菌在顯微鏡之光趨性分析……….…………………….……....… 68
圖34 : 嗜鹽古生菌在 ELISA Reader 之光趨性測定………….……….…….…… 69
圖35 : E. coli BL21表現HmCheR …………………….………...…………..…..… 75
圖36 : E. coli BL21小量表現GST嵌合蛋白質…………….…...………….…..… 75









附圖目錄

附圖1 : 光照培養箱之設計……………………………………….….…..….….… 83
附圖2 : H. salinarum 的趨氧現象……………………………….………..……… 83
附圖3 : 嗜鹽古生菌於不同光照下之菌體狀況…………….………….………… 84



表目錄

表1 : 各類視紫質在四種嗜鹽古生菌中的分佈及吸收峰…………....…..…… 8
表2 : 各蛋白質長度及帶電胺基酸的占比…………………….…......……...… 44
表3 : HmSRs在不同pH值下的光週期速率………………..………………… 50
表4 : 嗜鹽古生菌於不同光照環境下之世代時間………….…….....………… 65
表5 : 單隻嗜鹽古生菌對RGB光源刺激之反應…………….……...………… 67

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