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研究生:蘇源霖
研究生(外文):Yuan-LinSu
論文名稱:轉殖似轉錄激活因子蛋白核酸酶(TALEN)來改變菸草葉綠體 DNA 序列之研究
論文名稱(外文):A study to modify tobacco chloroplast DNA sequence by nuclear transformation of transcription activator like effector nuclease (TALEN) gene
指導教授:張清俊張清俊引用關係
指導教授(外文):Ching-Chun Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:103
中文關鍵詞:葉綠體同源性重組
外文關鍵詞:chloroplast DNA editingtranscription activator-like effector nuclease
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在先前研究中,為了修飾葉綠體基因序列,基因轟擊法是最常使用在將表現載體送入葉綠體中,並利用葉綠體之基因組同源重組機制導致基因序列改變,而此方法須經多次之篩選及再生過程可得到穩定之轉殖株,然而,其為耗時且不經濟之方法。為了專一且更方便的進行葉綠體基因修飾,在本研究利用細胞核表現一對帶有葉綠體轉運胜肽的 Transcription activator-like effector nuclease (TALEN) ,暫時性或穩定表現,並經由葉綠體的 TOC-TIC 轉運機制運送入葉綠體中,進而辨認葉綠體目標 DNA 序列,造成 DNA 雙股斷裂,利用葉綠體 DNA 之修復機制,預期將造成葉綠體 DNA 缺失或插入。研究結果顯示,將煙草 (Nicotiana tabacum) 暫時表現含有一對重組 TALEN 蛋白後,其 DNA 經 HRM (High resolution melting) 分析,暫時性表現 TALEN 之煙草目標 DNA 序列與對照組有顯著上之差異,表示 TALEN 重組蛋白能在葉綠體中進行作用。
To edit the plastid DNA, biolistic bombardment is the most common used method to deliver expression vectors into plastid, and then the transgene could integrate into plastid genome via homologous recombination. Under subsequently long selection and regeneration process, the transplastomic plant could be obtained. However, biolistic bombardment is costly and selection/regeneration process is time consuming. To specifically and easily edit the plastid DNA, in this study, a pair of transcription activator like effector nuclease (TALENs) was stablely or transiently expressed in the nucleus, and the fusion TALEN proteins were targeted to tobacco chloroplasts through TOC-TIC translocons. After agroinfiltration to transiently express TALENs in tobacco chloroplasts, high resolution melting analysis of PCR amplified chloroplast DNA revealed the presence of mutagenic DNA from the preliminary results.
中文摘要 I
英文摘要 II
誌謝 VI
目錄 VII
表目錄 X
圖目錄 XI
縮寫表 XIII
一、研究背景 1
1-1 葉綠體 1
1-2 葉綠體基因轉殖技術之發展與應用 2
1-3 特定位之專一性基因修飾 4
1-4 鋅指核酸酶 6
1-5 CRISPR/Cas-9系統 7
1-6 似轉錄活化因子樣蛋白核酸酶 9
1-7 TALEN 進行基因編輯之優點 11
1-8 TALEN 在植物生物技術之應用 12
1-9 TALEN 對生物體胞器的應用 12
1-10 葉綠體 DNA 的修復方式 13
1-11 研究目的 14
二、材料與方法 16
2-1 使用之材料 16
2-2 植物基因轉殖載體之構築 16
2-3 構築細胞核表現之載體 17
2-4 構築表現載體所使用之方法 22
2-5 於煙草葉片組織暫時性表現外源基因 27
2-6 植物葉片組織 DNA 之萃取 29
2-7 以 PCR 確認 TALEN 基因是否存在於轉殖煙草的基因體中 29
2-8 高解析度解離分析 30
2-9 煙草轉殖植株之獲得 31
2-10 煙草種子之播種 32
2-11 西方墨點法偵測外源基因在植物組織的表現量 33
2-12 西方墨點轉印分析法 34
2-13 葉綠體暫時性表現 35
2-14 煙草組織中螢光蛋白酶活性分析 36
三、結果 38
3-1 構築表現載體 38
3-2 暫時性表現 TALEN 於煙草 38
3-3 檢測農桿菌浸潤感染煙草後的葉綠體 DNA之變化 39
3-4 高解析度解離分析 40
3-5 獲得表現TALEN之煙草基因轉殖品系 41
3-6 PCR 確認 TALEN-L 基因是否存在於轉殖煙草的基因組中 42
3-7 PCR 確認 TALEN-R 基因是否存在於轉殖煙草的基因組中 42
3-8 PCR 確認 TALEN-L、R 基因是否有存在於轉殖煙草的基因組中 43
3-9 西方點墨法偵測轉殖植株之 TALEN 蛋白的表現量 44
3-10 暫時性表現不同葉綠體基因表現載體 45
3-11 總結 45
四、討論 47
4-1 暫時性表現 TALEN 對於植物葉綠體基因之修飾 47
4-2 利用農桿菌轉殖成功建立表現 TALEN 之煙草轉殖植株 48
參考文獻 52
附錄 94

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