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研究生: 曾文宏
Zeng, Wun-Hong
論文名稱: 蝴蝶蘭葉綠體基因RNA編輯之分析
Analysis of chloroplast RNA editing in Phalaenopsis orchid
指導教授: 張清俊
Chang, Ching-Chun
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 生物科技研究所
Institute of Biotechnology
論文出版年: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 170
中文關鍵詞: 葉綠體蝴蝶蘭
外文關鍵詞: RNA editing, Phalaenopsis aphrodite, moth orchid, chloroplast
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  • 由於蝴蝶蘭(台灣阿嬤)葉綠體基因體之定序已經完成,由之前本實驗室的實驗結果顯示,蝴蝶蘭的轉錄子rpl2藉由RNA編輯而修復了轉譯起始密碼,我們進一步針對蝴蝶蘭(台灣阿嬤)葉綠體可轉譯形成蛋白的76個轉錄子進行定序工作,分析其發生RNA編輯的位置,並且與其他物種做一分析比較。至目前為止,在蝴蝶蘭葉綠體的24個轉錄子發現了44個RNA編輯位置,7個屬於不完全編輯,2個發生於不轉譯區域,發生RNA編輯的機率佔所有定序分析序列的比例為0.07%,蝴蝶蘭所發現的44個RNA編輯數目,為目前種子植物中所發現最高的。在這些RNA編輯位置中,藉由RNA編輯作用產生了一個基因之轉譯起始密碼,但是並沒有任何停止密碼被修復。我們也發現了所有的RNA編輯都屬於胞嘧啶轉換成尿嘧啶,但是並沒有尿嘧啶轉換成胞嘧啶之情形發生,並且以發生於密碼子的第二個位置的機率最高(90.5%)。另外,在蝴蝶蘭葉綠體的ndh轉錄子並沒有藉由RNA編輯來修復其不完整的轉譯讀碼框,此將更加地確定了在蝴蝶蘭葉綠體中ndh基因群為沒有功能的偽基因。將蝴蝶蘭RNA編輯圖譜與其他3種單子葉植物,3種雙子葉植物,以及一種裸子植物做一比較,發現分別有13個RNA編輯位置與三種單子葉植物中的任一種相同,15個RNA編輯位置與三種雙子葉植物中的任一種相同,2個RNA編輯位置與裸子植物相同,而其中有 21個RNA編輯位置是屬於蝴蝶蘭所特有的,而被子植物之間有一個共同的RNA編輯位置。

    The complete chloroplast genome sequence of Taiwan moth orchid (Phalaenopsis amabilis var. formosa) have been determined in our lab. In a previous study, we verified the presence of an RNA editing system in Phalaenopsis aphrodite to repair the start codon by a C to U conversion in the chloroplast rpl2 transcripts. We further extend the study to entire transcripts of 76 protein-coding genes to search the RNA editing sites in the chloroplasts of P. aphrodite and compare them with those of other seed plants. The presence of RNA editing was detected in twenty four transcripts. The total number of editing sites is 44, the highest reported in seed plants and they represent an average of 0.07% of the nucleotides in the Phalaenopsis chloroplast genome. Seven of these forty-four editing sites are edited partially. One initiation codon is created but no internal stop codon was repaired by RNA editing. All editing is C to U conversion, and 42 sites of editing bring about the change in amino acids which occurred mostly in the second codon position (90.5%). The consequence of RNA editing in codon position mostly restores the conservation of amino acids. One of the remaining two editing sites occurs in the transcripts of ndhB pseudogene, and another in the 5’ untranslated region of psbH transcripts. However, RNA editing did not restore the continuous ORF in the frameshifted ndh genes, further confirming that they are pseudogenes. On comparing the editing sites of Phalaenopsis orchid with three monocots, three dicots and one gymnosperm, thirteen, fifteen and two sites are shared between orchid and any monocots, between orchid and any dicots, and between orchid and the gymnosperm, respectively, while twenty one sites are unique to P. aphrodite and one common editing site between angiosperm.

    第一章、前言……………………………………………………………..1 第二章、文獻探討………………………………………………………..3 1. 葉綠體及其基因體之介紹…………………………………………...3 2. RNA編輯(RNA editing)的介紹……………………………………..3 3. RNA編輯的發現歷史………………………………………………..5 4. 目前在哪些物種有發現RNA編輯的現象……...……………….…..5 4.1 核甘酸的插入或移除(insertion/deletion)..........…….…..………..6 4.2 核甘酸的轉換(conversion)………….….…..………………...…..6 5. RNA編輯影響植物葉綠體以及粒線體基因表現的方式.................10 5.1 RNA編輯影響葉綠體基因表現的方式………....…...........……10 5.2 RNA編輯影響粒線體基因表達的方式………………………...15 6. RNA編輯之機制…………….... …..………...…………..…………16 6.1 cis-acting elements………………………………………….…….16 6.2 trans-acting factors……………………………………..…………18 6.3 catalytic editing enzymes…………………………………………20 7. 比較不同種植物之間葉綠體的RNA編輯…...…………………….20 7.1 核甘酸轉換方式………………..……………………………….20 7.2 數目多寡…………………..………………………….…………21 7.3 在codon position的發生頻率…………..………………….……22 7.4 胺基酸序列改變的趨勢…………………..…………………….22 7.5 發生RNA編輯次數最多的基因……………..…………………23 8. RNA編輯之特性……………………………...……………………24 8.1 植物組織之生長分化與RNA編輯之關係……...….…..……..24 8.2 環境因子與RNA編輯之相關性………………....……………24 9. RNA編輯與蛋白質功能之關係……………...……………………25 10. 演化上發生RNA編輯的可能原因………...……………………..26 11. RNA編輯與物種演化之相關性…………......….…….……………26 12. 蝴蝶蘭的特性……………...………………….……………………28 第三章、材料與方法……………………………………………………30 1. 實驗材料…….…………………………………………...30 2. 實驗方法………………………………………………………….30 2.1 蝴蝶蘭RNA的製備………………….………………………..30 2.2 選殖蝴蝶蘭葉綠體cDNA………….…….…..………..………33 2.3 葉綠體基因RNA編輯之分析…………………….......………38 第四章、結果……………………...…………………………………….41 1. 台灣阿嬤蝴蝶蘭(TS-97)葉綠體蛋白質轉錄基因之RNA定序… ……………………………..…………………………………...……41 2. 蝴蝶蘭葉綠體蛋白轉錄子之RNA編輯分析……………...…..41 2.1 發生於有關Transcription/translation apparatus genes………… 轉錄子的RNA編輯…………..……………....………….……43 2.2 發生於ribosomal protein 轉錄子的RNA編輯……..………46 2.3 發生於與Photosynthesis相關的轉錄子之RNA編輯…...….47 2.4 發生於其他蛋白質轉錄子的RNA編輯………….….…........51 2.5 發生於ndhs 轉錄子的RNA編輯…………….……………..53 3. 部分編輯(partial editing)…………...….……………………….53 4. 蝴蝶蘭葉綠體RNA編輯的核甘酸轉換型式…………....…….54 5. 蝴蝶蘭葉綠體RNA編輯發生於密碼子(codon)的位置…….…54 6. 蝴蝶蘭葉綠體RNA編輯造成胺基酸改變的趨勢…………….55 7. 比對蝴蝶蘭葉綠體基因不同RNA編輯位置其周圍之序列….56 8. 比較RNA編輯位置的前一個以及後一個核甘酸序列…….…57 9. 比較蝴蝶蘭與其他種子植物間相同RNA編輯位置的數目….58 第五章、討論…………………………………….…..………………….59 1. RNA編輯與蛋白質功能……………………………………..…59 1.1 RNA編輯與accD基因功能之分析………..….…………..….59 1.2 RNA編輯與psbF基因之功能分析…………...…..…………..60 1.3 RNA編輯與petB基因之功能分析…………......……………..61 1.4 RNA編輯與matK基因之功能分析……….…….…….………61 2. RNA編輯與RNA二級結構之變化……………………………62 3. RNA編輯與frameshifted ndh基因………….…..……………..63 3.1 蝴蝶蘭葉綠體ndh基因…………….………………………….63 3.2 發生於frameshifted ndhB基因的RNA編輯……….…..…….65 3.3 蝴蝶蘭ndh基因群轉錄子不具RNA編輯的可能原因…....…67 4. 蝴蝶蘭葉綠體中發現最多RNA編輯位置的基因…….………68 5. 演化上相鄰近之物種具有類似的RNA編輯圖譜…..…….…..69 6. 蝴蝶蘭葉綠體轉錄子rpl20不具有RNA編輯現象….....…….70 7. RNA編輯於葉綠體基因遺失所扮演角色…………….……….70 第六章、文獻參考………………………...…………………………….72 圖目錄 圖一、葉綠體rpoA基因RNA編輯的分析............................................88 圖二、葉綠體rpoB基因RNA編輯的分析………………….….…......90 圖三、葉綠體rpoC1基因RNA編輯的分析..........................................92 圖四、葉綠體rpoC2基因RNA編輯的分析...........................................94 圖五、葉綠體rpl2基因RNA編輯的分析............................................96 圖六、葉綠體rpl23基因RNA編輯的分析...........................................98 圖七、葉綠體rps2基因RNA編輯的分析...........................................100 圖八、葉綠體rps8基因RNA編輯的分析……………………….……102 圖九、葉綠體rps14基因RNA編輯的分析..........................................104 圖十、葉綠體rps16基因RNA編輯的分析...........................................106 圖十一、葉綠體psaI基因RNA編輯的分析.........................................108 圖十二、葉綠體ycf3基因RNA編輯的分析.........................................110 圖十三、葉綠體psbF基因RNA編輯的分析........................................112 圖十四、葉綠體psbH基因RNA編輯的分析.......................................115 圖十五、葉綠體petB基因RNA編輯的分析.........................................117 圖十六、葉綠體petL基因RNA編輯的分析.........................................119 圖十七、葉綠體atpA基因RNA編輯的分析........................................121 圖十八、葉綠體atpB基因RNA編輯的分析........................................123 圖十九、葉綠體atpF基因RNA編輯的分析........................................125 圖二十、葉葉綠體atpI基因RNA編輯的分析.....................................127 圖二十一、葉綠體accD基因RNA編輯的分析...................................129 圖二十二、葉綠體clpP基因RNA編輯的分析....................................131 圖二十三、葉綠體matK基因RNA編輯的分析...................................134 圖二十四、葉綠體ndhB基因RNA編輯的分析………..…………….135 圖二十五、RNA編輯位置之間cis-acting elements的比對分析…….138 圖二十六、發生於蝴蝶蘭葉綠體atpA、clpP、ndhB、psbF、rpoA、rps8等轉錄子中,屬於部分編輯(partial editing)的RNA編輯位置…..…..140 圖二十七、蝴蝶蘭葉綠體RNA編輯位置之U_A context分析...........141 表目錄 表一、蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)葉綠體基因RNA編輯的情形.....................................................................142 表二、七種被子植物以及一種裸子植物之間的葉綠體RNA編輯圖譜…………………………………….……………………….143 表三、八種不同植物之間葉錄體RNA編輯之情形..........................147 表四、蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)以及其他七種種子植物之間RNA編輯造成胺基酸改變之情形...................148 表五、蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)以及其他七種種子植物之間相同RNA編輯位置的數目………………..….149 附圖目錄 附圖一、Ndh複合體蛋白於葉綠體中所扮演可能角色.......................150 附圖二、RT-PCR之電泳結果圖.................................................151 附圖三、colony PCR電泳結果圖................................................153 附圖四、葉綠體基因轉殖載體pNIC101-LIS.......................................169 附表目錄 附表一、蝴蝶蘭葉綠體76個蛋白轉譯基因........................................155 附表二、本論文所使用到的PCR引子.............................................157 附表三、蝴蝶蘭(Phalaenopsis aphrodite subsp. formosana)與其他七種種子植物之RNA編輯圖譜...................................................160

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