Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90092
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dc.contributor楊長賢zh_TW
dc.contributor.author林哲逸zh_TW
dc.contributor.authorJhe-Yi Linen_US
dc.contributor.other生物科技學研究所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-09T02:07:12Z-
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dc.identifier.urihttp://hdl.handle.net/11455/90092-
dc.description.abstract真核生物中其中一種主要的分解蛋白質途徑就是泛素/26S 蛋白酶系統,是一種藉由連接泛素當作標記,形成一種蛋白質降解訊號,在細胞內部此標記的蛋白質得以被辨識,因而控制細胞內蛋白質的生命週期。阿拉伯芥RING finger gene DAF-Like gene 1 (DAFL1) 預測是一個E3 protein ligase,在26S proteosome pathway系統中扮演了重要的角色。在演化樹分析中DAFL1與實驗室前人研究的DAF (DAD1-Activating Factor)基因親緣關係非常接近,但這兩個基因表現的位置卻有明顯的差異。在阿拉伯芥發育早期的花器裡,DAF在雄蕊中表現,而DAFL1專一地表現在雌蕊與胚珠中,推測DAFL1可能與DAF的功能有所不同。異位表現DAFL1 RNAi與antisense的基因轉殖植株可觀察到不孕的性狀,是由於花藥不開裂造成花粉無法正常授粉,異位表現DAFL1-H135A點突變的基因轉殖植株同樣也有此性狀。將野生型阿拉伯芥花粉與不孕植株的雌蕊雜交,能得到正常授粉而延長的果莢,不過果莢中的種子只有野生型的一半左右。另外構築可以專一表現在雌蕊與胚珠的載體,使得基因轉殖植株可以專一地表現DAFL1-H135A點突變蛋白質,其花藥可以正常開裂,果莢中的種子數量同樣只有大約野生型的一半左右。利用酵母菌雙雜交系統,藉由營養篩選方法從阿拉伯芥的花苞cDNA library中去找出能與DAFL1有交互作用的蛋白質,經過高嚴謹度篩選後得到了數個可能是DAFL1的候選受質。其中CAD1經由啟動子活性分析實驗得知其表現位置在胚珠,與DAFL1啟動子表現的部位有重疊,異位表現CAD1的轉基因植株也觀察到種子數目減半的性狀。CAD1已被證實為合成木質素重要成分的關鍵酵素,並參與果莢木質化的過程,與DAFL1同樣表現在雌蕊非木質化的部位。因此推測在雌蕊發育早期的階段,胚珠藉由DAFL1降解CAD1避免木質素過早合成。zh_TW
dc.description.abstractIn eukaryotes, the ubiquitin/26S proteasome system is one of the major mechanisms that act on the protein degradation. Proteins are tagged with ubiquitin as the signals to be degraded. In the cells, the ubiquitin-tagged proteins can be recognized and to regulate the life cycle of proteins. It was proposed that RING finger gene DAF-Like gene 1 (DAFL1) encodes an E3 protein ligase that plays a key role in 26S proteasome pathway in Arabidopsis thaliana. Based on phylogenetic analysis, DAFL1was closely related to DAF (DAD1-Activating Factor); however, the spatial specificities of DAFL1 and DAF gene expression patterns in Arabidopsis thaliana were different. In the early age of floral organs of Arabidopsis thaliana, DAFL1 specifically accumulated in the pistil and the ovule whereas DAF was expressed in stamen. It was suggested that DAFL1 and DAF might behave divergently in regulating plant development. Transgenic Arabidopsis plants that ectopically expressing DAFL1 RNAi and antisense caused indehiscent anthers and resulted in the unreleased pollen from the anthers. Similarly, ectopic expression of DAFL1-H135A caused indehiscent anthers in transgenic Arabidopsis plants. Pollens of wild-type Arabidopsis were crossed to the stigmas of sterile transgenic plants. At 7 days after pollination, the siliques of sterile transgenic plants developed and elongated. However, the amount of seeds of the transgenic plants is about a half of that of the wild-type Arabidopsis. Furthermore, transgenic plants that specifically expression of DAFL1-H135A in the pistil and the ovule produced dehiscent anthers, but the amount of seeds of the transgenic plants is about a half of that of the wild-type Arabidopsis. To discover the proteins that might interact with DAFL1, yeast-two hybrid system was performed to screen putative ones from flower buds cDNA library of Arabidopsis. One of the candidate genes, CAD1, was applied in the promoter assay. The result indicated that the gene expression patterns of CAD1 and DAFL1 was the same in ovule. In addition, transgenic plants that ectopic expressing CAD1 produced the amount of seeds is about a half of that of the wild-type Arabidopsis. CAD1 is a key enzyme in the process of lignin synthesis and has been thought to be involved in the lignification of siliques. Interestingly, DAFL1 and CAD1 also highly accumulated in the non-lignification site such as in the carpel. Our results revealed that DAFL1 degrades CAD1 and inhibits lignin biosynthesis at the early stage of pistil development.en_US
dc.description.tableofcontents中文摘要............................................................................................................................i Abstract...........................................................................................................................ii 前言..................................................................................................................................1 材料與方法.......................................................................................................................8 結果 1. DAFL1之分子選殖與特異性分析.....................................................................19 2. DAFL1之功能性分析.........................................................................................20 3. 篩選DAFL1之受質基因...................................................................................25 4. DAFL1受質候選基因之分子選殖與特異性分析............................................27 5. DAFL1受質候選基因之功能性分析................................................................28 討論.................................................................................................................................31 參考文獻.........................................................................................................................36 圖表 表1. 本實驗使用之引子序列................................................................................41 表2. cDNA library篩選結果 (1st)..........................................................................44 表3. DAFL1可能有交互作用之受質....................................................................45 圖1. DAFL1 (AT3G10910) 之編碼序列 (coding sequence, CDS) 及胺基酸序 列.................................................................................................................46 圖2. DAF與DAFL1胺基酸序列比對...................................................................47 圖3. DAFL1啟動子活性分析之載體與分子鑑定................................................48 圖4. DAFL1啟動子轉殖植株之GUS活性分析及組織切片...............................49 圖5. 異位表現DAFL1 RNAi載體之分子鑑定....................................................50 圖6. DAFL1基因分子選殖、異位表現載體之構築與分子鑑定..........................52 圖7. 異位表現DAFL1 RNAi、DAFL1反股基因轉殖植株之性狀......................54 圖8. 異位表現DAFL1-H135A點突變基因載體之構築與分子鑑定..................56 圖9. 異位表現DAFL1-H135A點突變基因轉殖植株之性狀分析......................58 圖10. 異位表現DAFL1反股、RNAi及點突變基因轉殖植株與野生型花粉雜 交實驗之分析.................................................................................................60 圖11. 專一表現DAFL1-H135A載體組合之構築與分子鑑定............................62 圖12. 專一表現DAFL1-H135A基因轉殖植株之性狀分析................................64 圖13. 專一表現DAFL1-H135A轉殖植株花粉活性與種子之性狀分析............66 圖14. 阿拉伯芥花苞cDNA library之酵母菌雙雜交系統篩選 (1st)..................67 圖15. 構築DAFL1雙雜交系統載體與酵母菌質體上cDNA library之序列分 析.....................................................................................................................68 圖16. DAFL1受質候選基因分子選殖與酵母菌雙雜交載體之構築..................69 圖17. 蛋白質交互作用強度量化之ONPG assay.................................................70 圖18. 阿拉伯芥花苞 cDNA library之酵母菌雙雜交系統篩選 (2nd)................71 圖19. CAD1 (At4g39330) 之編碼序列及胺基酸序列.........................................72 圖20 . SHM4 (At4g13930) 之編碼序列及胺基酸序列........................................73 圖21. CAD1胺基酸序列與酵母菌質體上library cDNA序列之比對.................74 圖22. SHM4胺基酸序列與酵母菌載體上library cDNA序列之比對.................75 圖23. CAD1、SHM4啟動子之分子選殖與載體構築............................................76 圖24. CAD1啟動子轉殖植株之GUS活性分析...................................................77 圖25. SHM4啟動子轉殖植株之GUS活性分析...................................................78 圖26. 異位表現CAD1、SHM4載體之構築與分子鑑定.......................................79 圖27. 異位表現CAD1基因轉殖植株之性狀分析...............................................80 圖28. 異位表現SHM4基因轉殖植株之性狀分析...............................................81 圖29. DAFL1 與CAD1基因功能假說圖..............................................................82 附圖 附圖1. Ubiquitin / 26S proteasome pathway..........................................................83 附圖2. 各類型E3之功能與機轉..........................................................................84 附圖3. RING-finger motif結構示意圖..................................................................85 附圖4. DAF 反股與DAF RNAi異位表現基因轉殖植株之花器........................86 附圖5. DAF 反股與DAF RNAi異位大量表現轉基因植株之雄不稔性狀........87 附圖6. DAF與DAFL1在阿拉伯芥各部位表現量與花器的GUS活性分析......88 附圖7. 21個阿拉伯芥RING-H2 type E3演化樹親緣關係分析..........................89 附圖8. pBI101載體圖譜........................................................................................90 附圖9. 野生型阿拉伯芥胚珠發育各時期之特徵................................................91 附圖10. pBI-mGFP1載體圖譜..............................................................................92 附圖11. pEpyon 32K載體圖譜..............................................................................93 附圖12. DAF和DAF (C132S) 點突變蛋白質之泛素化酵素活性分析.............94 附圖13. pGEM-T Easy Vector圖譜.......................................................................95 附圖14. 附圖14. pBroly-HXV載體圖譜.............................................................96 附圖15. pEpyon72K載體圖譜...............................................................................97 附圖16. 酵母菌雙雜交法原理..............................................................................98 附圖17. Dualsystems Biotech公司販售之阿拉伯芥花苞cDNA library..............99 附圖18. pGAD-HA載體圖譜..............................................................................100 附圖19. pGBKT7載體圖譜.................................................................................101 附圖20. 酵母菌菌種AH109和Y187內含之報導基因....................................102 附圖21. pGADT7載體圖譜.................................................................................103 附圖22. 不同阿拉伯芥組織中九個CAD基因cDNA數量之百分比...............104 附圖23. 果莢中AtCAD1啟動子之活性分析.....................................................105 附圖24. pEpyon01K載體圖譜.............................................................................106 附圖25. eFP Browser網站DAFL1、CAD1及SHM4在cDNA微矩陣資料庫中 各部位表現量差異.......................................................................................107 附圖26. DNA Ladder............................................................................................108zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2018-02-04起公開。zh_TW
dc.subject泛素zh_TW
dc.subject接合酶zh_TW
dc.subject胚珠發育zh_TW
dc.subjectubiquitinen_US
dc.subjectE3en_US
dc.subjectRING fingeren_US
dc.subjectovule developmenten_US
dc.titleCharacterization and Functional Analysis of a RING-Type E3 Protein Ligase in Regulating Ovule Development in Arabidopsis thalianaen_US
dc.title阿拉伯芥中參與胚珠發育之一個RING-type E3蛋白質接合酶之功能性分析zh_TW
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-02-04zh_TW
dc.date.openaccess2018-02-04-
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