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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
dc.contributor.author | Pei-Chun Chen | en |
dc.contributor.author | 陳佩君 | zh_TW |
dc.date.accessioned | 2021-06-16T02:47:41Z | - |
dc.date.available | 2016-07-20 | |
dc.date.copyright | 2015-07-20 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54269 | - |
dc.description.abstract | 科學界對古生菌的感光蛋白質研究,已有四十年以上之歷史,但其研究對象尚未包含台灣之菌種。本實驗室在2010年發表死海唯一倖存的嗜鹽古生菌,不僅有目前同類古生菌中,單一菌種最多的六個感光蛋白質,其中 HmBRII 性質和已知同類蛋白質具有差異。因此,不同地區感光蛋白質的性質,是值得研究和比較的。本研究要從台灣的嗜鹽古生菌著手,瞭解本地之感光蛋白質之性質。為達到此目的,有三個目標要完成,一是發現台灣本土菌種,二是選殖其感光蛋白質並加以大量表達,三是對其生化及生物物理性質做研究。首先,本實驗室已於井仔腳鹽田分離出一株暫命名為Ht之嗜鹽古細菌,初步利用16S rRNA 定序結果得知,Ht為一株屬於 Haloarcula 屬的嗜鹽古細菌,並與Haloarcula vallismortis 和 Haloarcula marismortui 具有高度16S rRNA序列相似度。因就選殖出來之感光蛋白質數量和生長酸鹼值之不同,知其非 H. marismortui。先前也已利用 FTIR,發現 Ht 與 H. vallismortis 具有不同的細胞膜成分,因此,將Ht視為一株台灣的特殊菌株。在本研究中,進一步利用不同鹽濃度下之生長曲線,發現 Ht 與 H. vallismortis 具有不同的鹽耐受性,因此,我們提出Ht為一新種,並暫名為 Haloarcula taiwanensis。第二目標上,我們選殖並比較其四個感光蛋白質,並進行感光蛋白質之大量表達。就感知環境因子及對外界環境反應對生物而言,感光是十分重要的生理功能,本研究先進一步針對H. taiwanensis之光趨性 (phototaxis response) 進行分析,觀察菌體被不同波長的光照射下之泳動現象。針對第三目標,先在分子層面,利用H. marismortui 之六個視紫蛋白質引子,以 H. taiwanensis 全基因體為模板,進行聚合酶連鎖反應,得到微生物感光型視紫蛋白質的擴增產物,包括 sensory rhodpsin I (SRI) 和 sensory rhodopsin II (SRII) 與其觸發器蛋白質。同時,建構其感光型蛋白質,利用大腸桿菌進行異源表現,進行其蛋白質的功能性分析。其中,實驗發現感光型視紫蛋白質I (Sensory rhodopsin I, SRI) 十分穩定,和現有 SRI 皆不同,目前發現嗜鹽古細菌中的 SRI 都十分不穩定,不利於蛋白質養晶過程,這也是目前為止,尚未解析出SRI蛋白質結構之原因。另外,在本研究中發現HtSRI-HtrI fusion protein 可以偵測650 nm的微弱光源,行使光週期,且其最大吸收峰為602 nm。此現象尚未在其他菌種之感光型視紫蛋白質發現。有別於其他嗜鹽古細菌,再度提高H. taiwanensis為一新種之可能性外,並對 HtSRI 進行養晶條件的篩選,如能解出結構,可望將過去四個主要的嗜鹽古細菌中的微生物視紫蛋白質結構研究中,唯一未能解出的結構補齊,啟發同類蛋白質研究之新可能性。鑑於所有證據都支持此一臺灣井仔腳古生菌為新種,本實驗對此菌做全基因定序,並進行基因註解 (gene annotation),已確定所有菌視紫蛋白質之存在及胺基酸順序,並初步得知H. taiwanensis之基因圖譜。 | zh_TW |
dc.description.abstract | Studies in proteins from local species offer valuable information in biological diversity. The main aim of this study was to investigate whether microbial rhodopsins in haloarchaea from Taiwan exhibit a region-dependent adaptation. To achieve this goal, we first tried to isolate local haloarchaea and proved them to be a new species, then microbial rhodopsins from it were cloned for bioinformatic, biochemical and biophysical studies before we attempted to compare their differences with other known microbial rhodopsins. In a previous study, we have isolated a haloarchaea from Beiman saltern in southern Taiwan and and identified it as a new strain; it was temporarily named as Ht. In this study, further growth condition experiments suggested Ht as a new strain; we therefor name it Haloarcula taiann. A total of four microbial rhodopsins were successfully cloned and sequenced from Ht. Protein sequence alignment showed only 87-93% similarity to any known microbial rhodopsins; it further supported H. taiann as a new species. Among those four rhodopsins, we found sensory rhodopsin I, HtSRI, to be unusually more stable and was surprisingly responsive to light of > 650 nm, a feature not being observed in any other know SRI proteins. In addition to biological, biochemical and biophysical studies, we also tried to grow protein crystal, a procedure failed in all other SRI due to stability issue. Since SRI is the only microbial rhodopsin that still lacks atomic structure, we have list it as a goal in this study. We expected that solving protein structure for HtSRI can shine light on protein science. We also cloned its transducer, a cognate partner protein when SRI exerting positive phototaxis, and engineered them together as another strategy to further stabilize HtSRI for crystallographic study. Hence all the evidences supported H. taiwanenesis a new species from Taiwan, we further conducted whole genomic sequencing and gene annotation. Now we have obtained all the rhodopsins exiting in H. taiwanenesis and full length gene sequences. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:47:41Z (GMT). No. of bitstreams: 1 ntu-104-R02b22053-1.pdf: 2470404 bytes, checksum: c16adc66f25334f48dc4f502d974ce49 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 目錄
目錄 i 圖目錄 iv 表目錄 vi 摘要 vii Abstract ix 第一章 緒論 1 第一節 古細菌的分類地位與嗜鹽古細菌之介紹 1 第二節 微生物視紫蛋白質 (microbial rhodopsin) 2 第三節 微生物光趨性視紫蛋白質 (Sensory rhodopsin, SR) 5 第五節 研究目的、動機與設計 12 第二章 材料與方法 16 第一節 實驗材料與藥品 16 1.1 菌種 16 1.2 質體 17 1.3 DNA引子 17 1.4 實驗藥品 17 第二節 實驗儀器與設備 20 2.1核酸電泳設備 20 2.2 蛋白質電泳設備與轉印設備 20 2.3離心機 20 2.4分光光度計 20 2.5 酸鹼度計 20 2.6光趨性實驗設備 20 2.7 光電流實驗設備 21 2.8 光週期實驗設備 21 2.9 其他 21 第三節 實驗方法 22 3.1嗜鹽古細菌之生長曲線 22 3.2 核酸定序分析 22 3.3 H. taiwanensis全基因體中微生物視紫蛋白質之可能產物與其序列分析 23 3.4 古細菌之視紫蛋白質與transducers之生物資訊學分析與結構預測 25 3.5 Ht之生化特性分析 26 3.6質體建構 27 3.7 視紫蛋白質之表現與純化 28 3.8 視紫蛋白質之分析 29 3.9 H. taiwanensis之全基因體定序實驗 31 第三章 結果與討論 32 第一節 菌種鑑定 32 1.1 菌種分離與細胞型態 32 1.2 不同鹽濃度下之生長曲線 33 第二節 生物資訊系統分析 35 2.1 可能微生物視紫蛋白質之核酸定序分析 35 2.2 感光型視紫蛋白質之分析 37 2.3 感光型視紫蛋白質訊息傳遞複合體交互作用蛋白質觸發器之分析 40 第三節 菌體之光趨性 (phototaxis) 泳動分析 42 第四節 感光型視紫蛋白質之功能性鑑定 44 4.1 設計以E.coli進行蛋白質異源表現與純化 44 4.2 不同物種間之SRI純化量 比較 46 4.3 不同菌種間之Sensory rhodopsin之蛋白質特徵吸收峰光譜分析 47 4.4 HtSRI和HtSRI-HtHtrI fusion 蛋白質 UV/Vis 光譜與counterion之pKa 測量 48 4.5 光趨性視紫蛋白質I 之穩定性測試 52 4.6 蛋白質光週期測試 56 4.7 HtSRI-HtHtrI fusion protein 受紅光誘導之光譜分析 58 4.8 Ht之感光型視紫蛋白質氫離子傳遞機制 62 第五節 全基因體定序分析 66 第四章 總結 71 第五章 未來展望 72 參考文獻 73 附錄 79 圖目錄 圖 1:二十株已全基因體定序的嗜鹽古細菌完成菌種分離地點在地圖上的分布。 10 圖 2 本實驗之實驗設計流程圖 15 圖 3 :ITO 電化裝置系統架設示意圖 35。 30 圖 4 :不同鹽濃度與光照與否對嗜鹽古細菌的生長曲線影響。 34 圖 5:利用H. marismortui之六個不同rhodopsins相對應的引子,以H. taiwanensis全基因體為模板之PCR產物電泳圖。 36 圖 6:利用軟體MegAlign 進行 H. taiwanensis之SRI (A) 和SRII (B)與NCBI資料庫中已知序列的差異性比對。 38 圖 7:利用軟體MegAlign 以Clustal W演算法建立 H. taiwanensis之感光型視紫蛋白質I (A) 和II (B)與NCBI資料庫中已知序列間的演化樹圖。 39 圖 8:H. taiwanensis之transducer I 蛋白質序列分析。 41 圖 9:利用swarm plate分析菌體之泳動能力。 42 圖 10:H. taiwanwnsis在波長505 nm之光源下的光趨性泳動分析。 43 圖 11: HtSRI-HtrI fusion 蛋白質之設計1, 2 45 圖 12:H. taiwanensis與不同物種間之 sensory rhodpsins的UV/Vis光譜。 47 圖 13: SRI蛋白質序列比對與counterion位置資訊。 49 圖 14: H. taiwanensis之sensory rhodpsins的UV/Vis光譜。 49 圖 15: Sensory rhodopsin I (SRI) 與SRI-HtrI fusion protein在不同pH值下之最高吸收峰值。 50 圖 16: HtSRI隨時間改變之可見光譜分析。 53 圖 17: HtSRI, HmSRI 與HsSRI 於不同鹽濃度緩衝溶液下之特徵吸收峰圖譜。 55 圖 18 HtSRI (上), HmSRI (中) 與HsSRI (下) 在不同鹽濃度下的最大吸收峰值。 55 圖 19: HtSRI和HtSRII之光週期。 57 圖 20:利用儀器偵測可激發HtSRI-HtrI fusion protein之光線的全波長光譜圖。 59 圖 21: SRI-HtrI fusion protein在黑暗中與紅光激發下的蛋白質顏色變化。 60 圖 22: SRI-HtrI fusion protein在黑暗中與紅光激發下之UV/Vis光譜分析圖。 60 圖 23:HtSRI-HtHtrI fusion protein之280 nm的吸收值調整為1。將黑暗中的光譜吸收值分別減去紅光激發後的吸收值後作圖,其最大吸收峰為602 nm。 61 圖 24:利用H. salinarum、H. marismortui和H. taiwanenesis之sensory rhodopsins 最大吸收峰標示蛋白質可偵測之光波長示意圖。 61 圖 25: HtSRI (A) 和HtSRII (B) 蛋白質層面之光電流訊號。 62 圖 26: HtSRI (A) 和HtSRII (B) 全細胞光電流訊號偵測。 63 圖 27:光驅動氫離子幫浦活性測試結果。 65 圖 28:視紫蛋白質在H. salinarum (A)、H. marismortui (B) 和H. taiwanensis (C) 全基因體中之基因圖譜示意圖。 67 圖 29:HsSRI (A)、HmSRI (B) 和HtSRI (C)上下游之基因圖譜。 67 圖 30:利用SOSUI預測HtSRI之二級結構與序列穿膜區段。 68 圖 31:利用軟體MegAlign進行HtSRI, HmSRI和HsSRI胺基酸序列比對。 69 表目錄 表 1:利用 E. coli 異源系統表現將H. marismortui 六個視紫蛋白質,並嘗試確認其功能14。 4 表 2 :目前已完成基因體定序之嗜鹽古細菌中,opsin 與其可能的transducer 基因標註整理 6 表 3:目前已完成基因體定序之嗜鹽古細菌具有視紫蛋白質的種類。 11 表 4:整理Halobacterium sp. NRC-1、Haloarcula marismortui、Haloarcula vallismortis與H. taiwanensis (Ht) 相關資訊。 14 表5:本研究所需之藥品、培養基與相關試劑。 17 表 6: Hm六個視紫蛋白質之引子序列 24 表 7:利用H. marismortui之rhodopsins相對應引子,以H. taiwanensis全基因體為模板擴增之DNA產物與H. marismortui視紫蛋白質基因序列比較表。 36 表 8:各菌種SR之相關蛋白質純化量。 46 表 9: 不同物種間之 SRI和SRI-HtrI fusion protein的UV/Vis光譜與其pKa相關資訊。 51 表 10:整理HtSRI蛋白質相關資訊與特性。 70 | |
dc.language.iso | zh-TW | |
dc.title | 從台灣分離之嗜鹽古細菌光趨性分析與其感受型視紫蛋白質之特性 | zh_TW |
dc.title | A Study on Sensory Rhodopsins from a Native Halobacterium Isolated from Taiwan | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許瑞祥,吳韋訥,李昆達,林晉玄 | |
dc.subject.keyword | 光趨視紫蛋白質,光趨性,光週期, | zh_TW |
dc.subject.keyword | haloarchaea,Haloarcula,senosory rhodopsin,phototaxis,photocycle, | en |
dc.relation.page | 81 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-16 | |
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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