請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57436
標題: | 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 A Study on the Functionality and Assistant Role in Phylogenetic Analysis of the Bacteriorhodopsin Cloned from a Haloarcula vallismortis New Strain Candidate |
作者: | Yi-An Han 韓怡安 |
指導教授: | 楊啟伸(Chii-Shen Yang) |
關鍵字: | 嗜鹽古細菌,Haloarcula,古細菌視紫紅質 (BR),氫離子幫浦, haloarchaea,Haloarcula,bacteriorhodopsin (BR),proton pump, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57436 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 8.46 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。