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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
dc.contributor.author | Yi-An Han | en |
dc.contributor.author | 韓怡安 | zh_TW |
dc.date.accessioned | 2021-06-16T06:46:01Z | - |
dc.date.available | 2016-08-01 | |
dc.date.copyright | 2014-08-13 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-28 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57436 | - |
dc.description.abstract | 嗜鹽古細菌在全世界分佈甚廣,且大多數菌體內皆有視紫紅質蛋白質做為對光能之利用和感受。本論文欲研究位處亞熱帶的臺灣嗜鹽古細菌之視紫紅質的性質,和已知者做比較。因此,希望能先證實確可取得臺灣地區之菌株,再研究其上之視紫紅質。首先,本研究從臺南井仔腳鹽田的鹽結晶中分離出一株紅色嗜鹽菌,先初步經由 16S rRNA 定序結果得知其為一株 Haloarcula 屬的嗜鹽古細菌,暫名之 Ht。接著,多項生理生化測試,以及傅立葉轉換紅外線光譜 (FT-IR) 分析其菌膜成份,發現 Ht 和具有高 16S rRNA 相似度之 Haloarcula marismortui 和 Haloarcula vallismortis 之間,具有不一樣的細胞膜成分。因此,暫以可能為臺灣之特殊菌株視之。接著,由氫離子幫浦活性測試結果顯示,Ht 和大多數嗜鹽古細菌一樣,在激發光照射下,會產生胞外環境 pH 值下降的情形,代表 Ht 可能具有氫離子幫浦 bacteriorhodopsin (BR),符合臺灣特殊株上視紫紅質之研究目的。進一步利用 H. marismortui 的六個視紫紅質 (rhodopsins) 引子,以 Ht 全基因體為模板進行聚合酶連鎖反應,得到 HtBR 之近全長擴增產物,再利用特異性引子從 Ht 全基因體找到 HtBR 的全長序列。此 HtBR DNA 序列和目前已知 BR 序列不完全相同,和 H. vallismortis cruxrhodopsin-3 (cop3) 有 93% 相似度,和 H. marismortui bacteriorhodopsin (bop) 有 91% 相似度。雖然 Ht 和許多已知 Haloarcula 屬嗜鹽古細菌雖具有高 16S rRNA 相似度,然而 Ht 中的 BR 序列卻和已知 BR 序列有所不同。以上結果顯示,本研究從臺灣井仔腳鹽田分離出一株具有 BR 之 Haloarcula 屬嗜鹽古細菌,可能為新菌株 (strain)。最後,本研究也將 HtBR 異源表現於大腸桿菌並純化出來,進行基本的功能性鑑定。發現其 HtBR 的光電流訊號反轉點,是比死海之 BR 蛋白質更偏鹼的 6.0。再由菌體生長環境 pH 值為 8.0 之資訊,初步發現,BR 蛋白質光驅動質子外送能力之最佳 pH 值,的確和其原菌體生長環境有關。 | zh_TW |
dc.description.abstract | The main aim of this study was to investigate whether there exists a region-dependent property adaptation in bacteriorhodopsin proteins from haloarchaea. In order to achieve this goal, new strain determination of a halobacteria isolated from saline crystals collected from Beimen saltern in southern Taiwan was conducted. The isolate was first subject to 16S rRNA sequencing for phylogenetic analysis. A comparison of 16S rRNA sequences revealed the isolate to be placed in the radiation of genus Haloarcula. Basic local alignment search tool (BLAST) analysis showed 98% identity between the isolate and some Haloarcula species, and a working name “Ht” was given to this isolate. Fourier transform infrared spectroscopy (FT-IR) was performed to analyze the membrane composition in addition to biochemical and physiological studies. An unique profile suggested membrane from Ht was different from at least Haloarcula marismortui and Haloarcula vallismortis. Positive light-driven proton pump activity at the whole cells indicated Ht potentially has light-driven proton pump protein, bacteriorhodopsin (BR). Using primers of six rhodopsins in H. marismortui and ATP synthase specific primers, the full DNA sequence of BR (HtBR) from Ht was cloned and it showed 93% similarity to the closest known BR, H. vallismortis cruxrhodopsin-3 gene (cop3) and 91% similarity to H. marismortui BR gene (bop). Although Ht showed 98% high similarity of 16S rRNA to recognized Haloarcula species, the HtBR DNA sequence showed lower similarity to any known BR sequences. These results suggest the Ht is potentially a new strain and it belongs to the genus Haloarcula. The HtBR is the candidate of the first Taiwan local new BR protein. Overexpression of HtBR was therefore executed and the purifed protein showed similar maximum absorbance and light-driven proton pump activity, but different in pH-dependent photocurrent measurements, reversed at pH 6.0, when compared to HmBRⅠ. Since Ht was isolated from slatern with pH 8.0, those results thefore suggested a local adaption in BR proteins could indeed happen, at least in this HtBR protein. | en |
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dc.description.tableofcontents | 目錄
目錄 i 圖目錄 iv 表目錄 vi 摘要 viii Abstract ix 第一章 緒論 1 第一節 嗜鹽古細菌 (Haloarchaea) 1 第二節 嗜鹽古細菌之鑑定與分類研究 2 第三節 微生物視紫紅質 (microbial rhodopsin) 5 第四節 古細菌視紫紅質 (bacteriorhodopsin, BR) 7 第五節 臺灣嗜鹽古細菌及古細菌視紫紅質之研究現況 8 第六節 傅立葉轉換紅外光譜 (FT-IR) 9 第七節 研究動機、目的與設計 10 第二章 材料與方法 13 第一節 實驗材料與藥品 13 1.1 菌種 13 1.2 質體 14 1.3 核酸引子 14 1.4 藥品 15 第二節 實驗儀器與設備 16 2.1 核酸電泳設備 16 2.2 蛋白質電泳與轉印設備 16 2.3 離心機 16 2.4 氫離子幫浦實驗設備 16 2.5 光電流實驗設備 17 2.6 傅立葉轉換紅外光譜實驗設備 17 2.7 其他 17 第三節 實驗方法 18 3.1 單一菌種分離與細胞形態觀察 18 3.2 生化特性分析 19 3.3 核酸定序分析 19 3.4 序列之生物資訊學分析與結構預測 24 3.5 HtBR 和 HvCR-3 之質體建構 26 3.6 HtBR 和 HvCR-3 膜蛋白質之表現與純化 27 3.7 氫離子幫浦活性測試 29 3.8 HtBR 和 HvCR-3 膜蛋白質分析 30 3.9 傅立葉轉換紅外光譜測定 (FT-IR) 33 第三章 結果與討論 34 第一節 單一菌種分離 34 1.1 單一菌種分離與細胞形態觀察 34 1.2 16S rRNA 核酸定序分析 36 1.3 新設計 16S rRNA 引子之特異性 40 第二節 生化特性分析 44 2.1 生長曲線 44 2.2 泳動性測試 45 第三節 傅立葉轉換紅外光譜測定 (FT-IR) 47 第四節 感光蛋白質是否存在及成為分類輔佐依據可能性之探究 50 4.1 嗜鹽古細菌之氫離子幫浦活性測試 50 4.2 Ht 可能微生物視紫紅質之核酸定序分析 52 4.3 感光蛋白質之功能探究 55 第五節 感光蛋白質之功能性鑑定 62 5.1 HtBR 和 HvCR-3 基因選殖、重組及表現蛋白質 62 5.2 大腸桿菌表現之氫離子幫浦重組蛋白質活性測試 63 5.3 蛋白質電泳與西方墨點法 64 5.4 感光蛋白質之特徵吸收峰光譜鑑定 65 5.5 光電流訊號測試 67 第四章 總結 70 第五章 未來展望 71 參考文獻 72 圖目錄 圖 1:本研究之實驗設計流程圖。 12 圖 2:ITO 電化裝置系統架設示意圖。 33 圖 3:Halomedium 固態培養基上菌落形態比較。 34 圖 4:光學顯微鏡下,細菌外型比較。 35 圖 5:Ht 在 Halomedium agar 和 Halomedium agarose 固態培養基上的生長情形。 35 圖 6:嗜鹽古細菌 16S rRNA PCR 電泳結果圖。 36 圖 7:利用軟體 MegAlign 進行 H. marismortui ATCC 43049 和 H. hispanica ATCC 33960 以及 Ht 之 16S rRNA 序列差異性比對。 37 圖 8:利用軟體 MegAlign 以 Clustal W 演算法建立 H. marismortui ATCC 43049 和 H. hispanica ATCC 33960 以及 Ht 16S rRNA 之間的演化樹圖。 38 圖 9:利用軟體 MegAlign 以 Clustal W 演算法建立 Haloarcula 屬嗜鹽古細菌和 Ht 16S rRNA 之間的演化樹圖。 40 圖 10:利用 MegAlign 比對H. marismortui ATCC 43049, H. hispanica ATCC 33960 和 Ht 的 16S rRNA,找出差異最多的片段設計成引子。 41 圖 11:嗜鹽古細菌的 16S rRNA 特異性引子之 PCR 電泳結果圖。 42 圖 12: 16S rRNA 特異性引子在 Ht 全基因體中,擴增出的目標序列 (Ht-type1 primers-product, Ht-type2 primers-product) 和 Ht 16S rRNA 524 - 1133 bp 相符。 43 圖 13:嗜鹽古細菌在 pH 5.5 – 7.5 環境下的生長曲線比較。 45 圖 14:嗜鹽古細菌在 pH 5.0 – 6.0 環境下的生長曲線比較。 45 圖 15:利用 swarm plates 測試嗜鹽古細菌泳動性。 46 圖 16:嗜鹽古細菌之 FT-IR 光譜。 48 圖 17:將 FT-IR 光譜進行群集分析。 48 圖 18:Ht 和 H. vallismortis 之 FT-IR 光譜。 49 圖 19:嗜鹽古細菌 Ht 之氫離子幫浦活性測試結果圖。 51 圖 20:嗜鹽古細菌 Hs, Hm, Hv, Ht 之氫離子幫浦活性測試結果圖。 52 圖 21:Hm 六個視紫紅質引子之 PCR 電泳結果圖。 53 圖 22:從網站 KEGG 查詢 Hmbop 上下游的相關基因,設計三對特異性引子。 56 圖 23:利用 Hmbop 上下游基因為依據所設計的特異性引子之 PCR 電泳結果圖。 57 圖 24:HtBR DNA 序列和其他已知 BR DNA 序列之間的演化樹圖。 59 圖 25:HtBR 胺基酸序列和其他已知 BR 胺基酸序列之間的演化樹圖。 59 圖 26:TMHMM 及 SOSUI 預測 HtBR 穿膜區數量。 60 圖 27:藉由 SWISS-MODEL 模擬 HtBR 三級結構,具有七個 α-helices 穿膜區。 61 圖 28:誘導表現 HtBR和 HvCR-3 蛋白質的 E. coli C43 (DE3) 菌塊顏色及純化後之蛋白質顏色。 62 圖 29:HtBR 和 HvCR-3 之 E. coli 表現株之氫離子幫浦活性測試結果圖。 64 圖 30:HtBR 和 HvCR-3蛋白質電泳以 CBR 染色及西方墨點法免疫染色結果圖。免疫染色使用抗 His-tag 之抗體。 65 圖 31:HtBR 和 HvCR-3 可見光/UV光譜之特徵吸收峰結果圖。 66 圖 32:HtBR 在 pH 4 - 6 下的光電流訊號。 68 圖 33:HvCR-3 在 pH 4 - 6下的光電流訊號。 68 圖 34:HtBR 與 HvCR-3 在 pH 4 - 6 下的光電流訊號比較圖。 69 表目錄 表 1:嗜鹽古細菌 Haloarcula 屬已發表且有完整鑑定分類之菌種共有九株。 3 表 2:紅外光譜中,五個用來鑑定細菌成分的主要區域。 9 表 3:本研究所需之藥品、培養基與相關試劑。 15 表 4:Halomedium 成分表。 18 表 5:Transformation buffer 成分表。 21 表 6:Haloarcula 16S rRNA 特異性引子序列。 22 表 7:Hm 六個視紫紅質引子序列。 23 表 8:依據 Hmbop 上下游的相關基因所設計之引子序列。 24 表 9:HtBR、HvCR-3 質體建構之引子序列。 27 表 10:Ht rrnA 與 NCBI 資料庫中 Haloarcula 屬嗜鹽古細菌 16S rRNA 一致性比對結果。 39 表 11:Ht rrnB 與 NCBI 資料庫中 Haloarcula 屬嗜鹽古細菌 16S rRNA 一致性比對結果。 39 表 12:利用 Hm 六個視紫紅質引子從 Ht 全基因體中擴增的 DNA 產物與相對應的 Hm 視紫紅質比較表。 54 表 13:以 Hmbop 引子從 Ht 全基因體中擴增之 DNA 產物和 NCBI 已知序列比較表。 54 表 14:以 Hmhop 引子從 Ht 全基因體中擴增之 DNA 產物和 NCBI 已知序列比較表。 55 表 15:以 Hmsop Ⅰ 引子從 Ht 全基因體中擴增之 DNA 產物和 NCBI 已知序列比較表。 55 表 16:以 Hmsop Ⅱ 引子從 Ht 全基因體中擴增之 DNA 產物和 NCBI 已知序列比較表。 55 表 17:HtBR DNA 序列和 NCBI 已知序列比較表。 58 表 18:HtBR 胺基酸序列和 NCBI 已知序列比較表。 58 表 19:TMHMM 及 SOSUI 預測 HtBR 穿膜區之胺基酸序列。 60 表 20:本實驗室純化之膜蛋白質的特徵吸收峰75。 67 表 21:BR 光電流訊號反轉點比較表。 69 | |
dc.language.iso | zh-TW | |
dc.title | 一嗜鹽角形古菌屬候選新菌株之古細菌視紫紅質功能及其在演化分析輔助角色之探討 | zh_TW |
dc.title | A Study on the Functionality and Assistant Role in Phylogenetic Analysis of the Bacteriorhodopsin Cloned from a Haloarcula vallismortis New Strain Candidate | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 賴美津(Mei-Chin Lai),張麗冠(Li-Kwan Chang),許瑞祥(Ruey-Shyang Hseu),林晉玄(Ching-HsuanLin) | |
dc.subject.keyword | 嗜鹽古細菌,Haloarcula,古細菌視紫紅質 (BR),氫離子幫浦, | zh_TW |
dc.subject.keyword | haloarchaea,Haloarcula,bacteriorhodopsin (BR),proton pump, | en |
dc.relation.page | 80 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-07-28 | |
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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