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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃鵬林(Pung-Ling Huang) | |
dc.contributor.author | Yu-Fen Huang | en |
dc.contributor.author | 黃郁芬 | zh_TW |
dc.date.accessioned | 2021-06-15T05:58:56Z | - |
dc.date.available | 2015-08-20 | |
dc.date.copyright | 2010-08-20 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-17 | |
dc.identifier.citation | 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47418 | - |
dc.description.abstract | 為了瞭解香蕉ABC轉運蛋白MhPDR1及MhPDR2在生理及生化的功能,本研究針對過量表達香蕉ABC轉運蛋白的轉殖株Nt-MhPDR1及Nt-MhPDR2進行試驗。以不同濃度之重金屬銅、鎘和鋅處理,再進行生理生化及植體內重金屬含量分析,結果顯示,與未轉殖株相比,轉殖株對銅、鎘和鋅離子有較高的抗性,進一步分析顯示,轉殖株體內含較低量的重金屬銅、鎘和鋅離子,且體內總葉綠素含量較未轉殖株高,轉殖株受脂質過氧化程度較未轉殖株低,因此推測MhPDR1及MhPDR2扮演對重金屬銅、鎘和鋅離子輸出幫浦 (efflux pump) 的角色。另一試驗以營養元素氮素、磷酸、鉀和鐵離子進行處理,結果顯示,轉殖株體內營養元素含量都高於未轉植株,因此推測MhPDR1及MhPDR2扮演對大量元素運移的角色。而為了瞭解ABC轉運蛋白MhPDR1及MhPDR2在細胞中表現的位置,將MhPDR1及MhPDR2分別與綠色螢光蛋白 (green fluorescent protein, GFP) 基因融合,構築為表現載體,利用PEG法轉殖到阿拉伯芥 (Arabidopsis thaliana L. ecotype Columbia) 之原生質體 (protoplast) ,透過觀察融合蛋白 (fusion protein) 的位置,了解香蕉ABC轉運蛋白在植物中表達位置,結果顯示MhPDR1位在液胞膜,而MhPDR2則是位在細胞膜上。 | zh_TW |
dc.description.abstract | In order to understand the function of banana ABC transporter MhPDR1 and MhPDR2, ABC transporter (MhPDR1 or MhPDR2) overexpressed tobacco plants (Nt-MhPDR1 or Nt-MhPDR2) have been tested by several treatments. The results of heavy metals treatments indicated that Nt-MhPDR1 and Nt-MhPDR2 have higher tolerance to copper, cadmium, and zinc ions than untransformed plants. In heavy metal content analysis, the results showed that Nt-MhPDR1 and Nt-MhPDR2 accumulated lower concentrations of copper, cadmium, and zinc. In chemical component analysis, the results showed that Nt-MhPDR1 and Nt-MhPDR2 have higher chlorophyll and lower lipid peroxidation than untransformed plants. Therefore, banana ABC transporter MhPDR1 and MhPDR2 are suggested to play role as efflux pump of the copper, cadmium, and zinc ions. Furthermore, transgenic tobacco Nt-MhPDR1 and Nt-MhPDR2 accumulated higher nitrate, phosphate, potassium, and iron contents than untransformed plants. Therefore, MhPDR1 and MhPDR2 may also possibly play the role as a transporter for these elements. To investigate the cellular localization of ABC transporter MhPDR1 and MhPDR2, coding regions of MhPDR1 and MhPDR2 were fused with the green fluorescent protein (GFP) gene and transiently introduced into Arabidopsis protoplasts by PEG method. The result indicated that MhPDR1 and MhPDR2 are located at tonoplast and plasma membrane, respectively. | en |
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dc.description.tableofcontents | 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰
目錄 中文摘要..............................I 英文摘要................................II 壹、 前言..............................1 貳、 前人研究............................2 一、 ABC轉運蛋白 (ATP-binding cassette transporter)..........2 二、 植物ABC轉運蛋白之結構...................2 三、 植物ABC轉運蛋白之分類...................3 四、 整元體植物ABC轉運蛋白...................4 (一) MRP次家族.......................4 (二) MDR / PGP次家族....................5 (三) PDR次家族.......................6 五、植物轉運蛋白.....................9 (一) 硝酸根轉運蛋白.....................9 (二) 磷酸根轉運蛋白.....................9 (三) 鉀離子轉運蛋白.....................9 六、植物重金屬轉運蛋白相關研究................10 七、香蕉ABC轉運蛋白....................10 (一) Mh-ABC1基因啟動子分析.................10 (二) 香蕉ABC轉運蛋白cDNA的選殖..............11 (三) 香蕉ABC轉運蛋白功能分析................11 (四) 香蕉ABC轉運蛋白定位分析................11 参、 材料與方法..........................13 一、 試驗材料..........................13 (一) 植物材料............................................13 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 (二) 轉殖質體...............................13 (三) 菸草之穩定性轉殖........................13 1. 菸草之種植..........................13 2. 菸草葉圓片之轉殖...................................................................................14 3. 光合作用光反應曲線測定.......................................................................15 4. GUS活性組織化學染色...........................................................................15 5. 啟動子誘導物分析試驗...........................................................................15 6. 南方氏雜交分析.......................................................................................16 (1) 基因組DNA之抽取.............................................................................16 (2) 探針之製備...........................................................................................16 (3) 預雜合與雜合反應...............................................................................17 (四) 蛋白質定位分析.....................................................................................17 1. 阿拉伯芥原生質體分離..................17 2. 阿拉伯芥原生質體轉殖..................18 (五) 基因過量表達之轉殖株分析.................................................................18 1. 重金屬試驗.......................18 2. 葉綠素含量測定.....................19 3. 蛋白質含量測定.....................19 4. 脯胺酸含量測定.....................20 5. 脂質過氧化程度之測定..................20 6. Superoxide dismutase活性分析.................................................................21 7. Glutathione reductase活性分析.................................................................21 8. Ascorbate peroxidase活性分析.................................................................22 9. Catalase活性分析......................................................................................22 10. 菸草元素含量測定和統計方法...............................................................23 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 11. 菸草重金屬含量測定和統計方法...........................................................23 肆、 結果............................26 一、營養元素誘導香蕉ABC轉運蛋白基因啟動子活性分析.......26 二、菸草轉殖株生理測試.....................26 三、營養元素處理菸草轉殖株之生理分析...............26 四、重金屬處理菸草轉殖株之生理及生化分析..................................................27 (一) 單一重金屬處理菸草轉殖株之生理分析............27 (二) 雙重重金屬處理菸草轉殖株之生理分析............28 (三) 單一重金屬處理菸草轉殖株之生化分析............30 (四) 雙重重金屬處理菸草轉殖株之生化分析............31 五、蛋白質定位分析.......................34 (一) 阿拉伯芥原生質體暫時性表達分析.........................................................34 (二) 菸草轉殖株穩定性表達分析.....................................................................34 伍、討論...............................................................87 參考文獻.............................93 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 圖索引 圖一、五種含不同香蕉ABC轉運蛋白基因啟動子與報導基因GUS質體之構築圖示..................................................................................................................................35 圖二、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株之光合作用、氣孔導度、蒸散作用及細胞間隙二氧化碳濃度分析..................................................................................................................................36 圖三、不同濃度營養元素誘導菸草pBI121Bid轉殖株之GUS活性表現... 37 圖四、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與未轉殖株幼苗處理不同濃度氮素之表型..................................................................38 圖五、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與 未轉殖株幼苗處理不同濃度氮素之鮮重、乾重及氮素含量..................................39 圖六、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與 未轉殖株幼苗處理不同濃度磷酸之表型..................................................................40 圖七、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與未轉殖株幼苗處理不同濃度磷酸之鮮重、乾重及磷酸含量..................................41 圖八、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與未轉殖株幼苗處理不同濃度鉀離子之表型.............................................................42 圖九、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與未轉殖株幼苗處理不同濃度鉀離子鮮重、乾重及鉀離子含量.............................43 圖十、 過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與未轉殖株幼苗處理不同濃度亞鐵離子之表型......................................................44 圖十一、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1和Nt-MhPDR2與未轉殖株幼苗處理不同濃度亞鐵離子鮮重、乾重及亞鐵離子含量.................45 圖十二、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 之外表型差異............................46 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 圖十三、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 之鮮重、乾重及植體內銅離子含量分析..........................................................................................................................47 圖十四、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬鎘離子 (4, 40, 400 μM) 之外表型差異............................48 圖十五、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬鎘離子 (4, 40, 400 μM) 之鮮重、乾重及植體內鎘離子含量分析..........................................................................................................................49 圖十六、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬鋅離子 (4, 40, 400 μM) 之外表型差異.............................50 圖十七、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬鋅離子 (4, 40, 400 μM) 之鮮重、乾重及植體內鋅離子含量分析.........................................................................................................................51 圖十八、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子 (4 μM) 之外表型差異..................................................................................................................................52 圖十九、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子 (4 μM) 之鮮重、乾重及植體內銅及鎘離子含量分析..........................................................................................53 圖二十、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子 (40 μM) 之外表型差異..................................................................................................................................54 圖二十一、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子 (40 μM) 之鮮重、乾重及植體內銅及鎘離子含量分析..........................................................................................55 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 圖二十二、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子 (400 μM) 之外表型差異..................................................................................................................................56 圖二十三、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子 (400 μM) 之鮮重、乾重及植體內銅及鎘離子含量分析......................................................................................57 圖二十四、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (4 μM) 之外表型差異..................................................................................................................................58 圖二十五、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (4 μM) 之鮮重、乾重及植體內銅及鋅離子含量分析..........................................................................................59 圖二十六、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (40 μM) 之外表型差異.................................................................................................................................60 圖二十七、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (40 μM) 之鮮重、乾重及植體內銅及鋅離子銅含量分析.....................................................................................61 圖二十八、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (400 μM) 之外表型差異..................................................................................................................................62 圖二十九、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及 Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (400 μM) 之鮮重、乾重及植體內銅及鋅離子含量分析..................................................................63 圖三十、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析......................................................................................64 圖三十一、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 之抗氧化酵素活性分析............65 圖三十二、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR 與未轉殖株處理重金屬鎘離子 (4, 40, 400 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析.....................................................................................66 圖三十三、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬鎘離子 (4, 40, 400 μM) 之抗氧化酵素分析....................67 圖三十四、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR 與未轉殖株處理重金屬鋅離子 (4, 40, 400 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析......................................................................................68 圖三十五、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬鋅離子 (4, 40, 400 μM) 之抗氧化酵素分析.....................69 圖三十六、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 及鎘離子 (4 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析......................................................70 圖三十七、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子(4 μM) 之抗氧化酵素分析..................................................................................................................................71 圖三十八、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2 與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 及鎘離子 (40 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析...............................................72 圖三十九、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2 與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子(40 μM) 之抗氧化酵素分 析..................................................................................................................................73 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 圖四十、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 及鎘離子 (400 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析...................................................74 圖四十一、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2 與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鎘離子(400 μM) 之抗氧化酵素分 析..................................................................................................................................75 圖四十二、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 及鋅離子 (4 μM) 之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析.....................................................76 圖四十三、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子(4 μM) 之抗氧化酵素分析.................................................................................................................................77 圖四十四、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 及鋅 (40 μM) 離子之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析..................................................78 圖四十五、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子(40 μM) 之抗氧化酵素分析..................................................................................................................................79 圖四十六、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅離子 (4, 40, 400 μM) 及鋅離子 (400 μM)之可溶性蛋白質、葉綠素、脯胺酸及脂質過氧化程度之分析.................................................80 圖四十七、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1及Nt-MhPDR2與未轉殖株處理重金屬銅 (4, 40, 400 μM) 及鋅離子 (400 μM) 之抗氧化酵素分析..................................................................................................................................81 圖四十八、MhPDR1 : GFP或MhPDR2 : GFP及液胞膜螢光蛋白分子標誌於阿拉 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 伯芥原生質體之蛋白質定位分析..............................................................................82 圖四十九、MhPDR1 : GFP或MhPDR2 : GFP及原生質膜螢光蛋白分子標誌於阿拉伯芥原生質體之蛋白質定位分析..........................................................................83 圖五十、過量表達香蕉ABC轉運蛋白之菸草轉殖株Nt-MhPDR1 : GFP、 Nt-MhPDR2 : GFP及未轉殖株葉片之GUS活性組織化學染色分析.....................84 圖五十一、2xCaMV 35Spro::MhPDR1: GFP 菸草擬轉殖株之基因完整性分析.............................................................................................................................85 圖五十二、2xCaMV 35Spro::MhPDR2 : GFP 菸草擬轉殖株之基因完整性分析..............................................................................................................................86 國立台灣大學生物資源暨農學院園藝學系 黃郁芬撰 表索引 表一、 重金屬試驗濃度.......................24 表二、 感應耦合電漿原子發射光譜分析儀 (ICP-AES) 分析各元素所使用之波長................................25 | |
dc.language.iso | zh-TW | |
dc.title | 香蕉ABC轉運蛋白基因之生理及生化功能分析 | zh_TW |
dc.title | Studies on Physiological and Biochemical Functions of Banana ABC (ATP-Binding Cassette) Transporter Genes | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 杜宜殷(Yi-Yin Do) | |
dc.contributor.oralexamcommittee | 羅筱鳳,洪傳揚 | |
dc.subject.keyword | ABC轉運蛋白,重金屬,蛋白質定位, | zh_TW |
dc.subject.keyword | ABC transporter,heavy metal,protein localization, | en |
dc.relation.page | 100 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-17 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
顯示於系所單位: | 園藝暨景觀學系 |
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