Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97780
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dc.contributor楊長賢zh_TW
dc.contributorChang-Hsien Yangen_US
dc.contributor.author宮振庭zh_TW
dc.contributor.authorChen-Ting Kungen_US
dc.contributor.other生物科技學研究所zh_TW
dc.date2018zh_TW
dc.date.accessioned2019-03-22T06:05:12Z-
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The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana. The Plant journal : for cell and molecular biology 11, 213-226. Hellens, R.P., Brown, C.M., Chisnall, M.A.W., Waterhouse, P.M. and Macknight R.C. (2016). The Emerging World of Small ORFs.Trends in Plant Science.Volume 21, Issue 4, April 2016, Pages 317-328. Hiroko Ao-Kondo, Hiroko Kozuka-Hata and Masaaki Oyama. (2011). Emergence of the Diversified Short ORFeome by Mass Spectrometry-Based Proteomics. DOI: 10.5772/19433. Source:InTech. In book: Computational Biology and Applied Bioinformatics. Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K. (2001). The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. The Plant Cell 13, 2191–2209. Kraft, E., Stone, S.L., Ma, L., Su, N., Gao, Y., Lau, O.S., Deng, X.W., and Callis, J. (2005). Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 139, 1597-1611. Lam, Y.A., Lawson, T.G., Velayutham, M., Zweier, J.L., and Pickart, C.M. (2002). A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763-767. Lovering, R., Hanson, I.M., Borden, K.L., Martin, S., O'Reilly, N.J., Evan, G.I., Rahman, D., Pappin, D.J., Trowsdale, J., and Freemont, P.S. (1993). Identification and preliminary characterization of a protein motif related to the zinc finger. Proc Natl Acad Sci U S A 90, 2112-2116. Magori, S., and Citovsky, V. (2011). Hijacking of the Host SCF Ubiquitin Ligase Machinery by Plant Pathogens. Front Plant Sci 2, 87. Moriya, S., KhelI, N.B., and Parhar, IS. (2015). Cloning and serotonergic regulation of RING finger protein38 (rnf38) in the brain of medaka (Oryzias latipes). Neuroscience. Volume 294, 21 May 2015, Pages 109-115. Mueller, M.J. (1997). Enzymes involved in jasmonic acid biosynthesis. Physiologia Plantarum 100, 653-663. Ohi, M.D., Vander Kooi, C.W., Rosenberg, J.A., Chazin, W.J., and Gould, K.L. (2003). Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat Struct Biol 10, 250-255. Peng, Y.J., Shih, C.F., Yang, J.Y., Tan, C.M., Hsu, W.H., Huang, Y.P., Liao, P.C., and Yang, C.H. (2013). A RING-type E3 ligase controls anther dehiscence by activating the jasmonate biosynthetic pathway gene DEFECTIVE IN ANTHER DEHISCENCE1 in Arabidopsis. The Plant journal: for cell and molecular biology 74, 310-327. Pickart, C.M. (2001). Mechanisms underlying ubiquitination. Annu Rev Biochem 70, 503-533. Salinas-Mondragon R.E., Garcidueñas-Piña C., and Guzm´an P. (1999). Early elicitor induction in members of a novel multigene family coding fornhighly related RING-H2 proteins in Arabidopsis thaliana. Plant Molecular Biology .40, 579–590. Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.C., Lee, P.Y., Truong, M.T., Beals, T.P., and Goldberg, R.B. (1999). Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sexual Plant Reproduction 11, 297–322. Sanders, P.M., Lee, P.Y., Biesgen, C., Boone, J.D., Beals, T.P., Weiler, E.W., and Goldberg, R.B. (2000). The Arabidopsis DELAYED DEHISCENCE1 Gene Encodes an Enzyme in the Jasmonic Acid Synthesis Pathway. The Plant Cell 12, 1041-1061. Stone, S.L., Hauksdottir, H., Troy, A., Herschleb, J., Kraft, E., and Callis, J. (2005). Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol 137, 13-30. Turner, J.G., Ellis, C., and Devoto, A. (2002). The jasmonate signal pathway. Plant Cell 14 Suppl, S153-164. Vierstra, R.D. (1996). Proteolysis in plants: mechanisms and functions. Plant Mol Biol 32, 275-302. Vierstra, R.D. (2003). The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends in Plant Science 8, 135-142. Vierstra, R.D. (2009). The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol 10, 385-397. Wing, S.S. (2003). Deubiquitinating enzymes--the importance of driving in reverse along the ubiquitin-proteasome pathway. Int J Biochem Cell Biol 35, 590-605.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/97780-
dc.description.abstract阿拉伯芥中DAF 【DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor】 和DAFL1 (DAF-like gene 1) 皆屬於RING-finger E3 ligase蛋白,藉由形成泛素/26S蛋白酶體來調控蛋白質降解,而在植物生長發育、荷爾蒙調節、抵抗逆境及防禦機制中扮演很重要的角色。在前人研究中得知,DAF和DAFL1序列極為相似,但在植物中的表現位置卻有明顯差異,DAF主要表現在成熟花的雄蕊,而DAFL1則是專一表現於雌蕊。在阿拉伯芥中daf mutant突變株可以觀察到花藥不開裂的雄不稔情形,dafl1 mutant突變株則是胚珠發育受到影響使得果莢內種子無法正常形成,可知此兩個基因皆與花器發育相關。而在分析DAF和DAFL1序列時,在兩基因上游不遠處皆發現含有一小片段uORF (upstream Open Reading Frame)。文獻指出,當uORF存在時可能會造成gene splicing,藉以調控主要基因mORF (major Open Reading Frame) 之轉錄或轉譯作用。本實驗首先偵測在mRNA轉錄層次上,DAF、DAF的uORF小片段 (uDAF) 和DAFL1、DAFL1的uORF小片段 (uDAFL1) 在植物各部位之表現,發現皆在阿拉伯芥中成熟花中表現量最高。進一步選殖出uDAF、含有uORF和DAF的全基因片段uDAF+mDAF (F-DAF)和uORF起始密碼突變之uDAF(ATG→TTG)+mDAF (ns-FDAF)以及uDAFL1、包含uORF和DAFL1的全基因片段 uDAFL1+mDAFL1 (F-DAFL1)和uORF起始密碼突變之uDAFL1(ATG→TTG)+mDAFL1 (ns-FDAFL1),分別利用35S啟動子在阿拉伯芥中大量表現。結果發現,35S::uDAF和35S::F-DAF會造成不孕,觀察其花器發育,發現為花藥不開裂無法正常授粉而導致果莢無法延長,此現象與前人研究中DAF基因突變時的性狀相同。進一步偵測其mRNA表現量,發現35S::uDAF和35S::F-DAF轉殖株中內生性DAF的表現量皆明顯受到抑制,因此初步推測,當uDAF存在時會藉由抑制內生性DAF之表現而調控植株之花藥開裂。未來將持續觀察比較各構築載體之轉殖株性狀,藉以更加瞭解DAF和DAFL1基因上游小片段uORF調控植物花朵發育之機制。zh_TW
dc.description.tableofcontents前言................................................................................................................................ 1 一、Ubiquitin (Ub) /26S proteasome pathway ..................................................... 1 二、Ub/26S proteasome pathway 參與蛋白 ........................................................ 1 三、RING-finger domain ...................................................................................... 4 四、雄蕊發育與雄不稔 (Stamen development and male-sterile) ....................... 4 五、茉莉酸生合成 (Biosynthesis of jasmonic acid) ........................................... 5 六、實驗室前人相關研究.................................................................................... 5 七、上游開放閱讀框 (upstream Open Reading Frame) ..................................... 6 八、研究動機與目的............................................................................................ 6 材料與方法.................................................................................................................... 7 一、實驗材料........................................................................................................ 7 二、阿拉伯芥之種植............................................................................................ 7 三、瓊脂凝膠膠片之配製 (Preparation of agarose gel) ..................................... 7 四、瓊脂凝膠電泳 (Electrophoresis) .................................................................. 7 五、阿拉伯芥總體核醣核酸 (Total RNA) 之萃取 ........................................... 8 六、反轉錄增幅反應 (Reverse transcription, RT) .............................................. 8 七、聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) ................................... 8 八、DNA 片段之回收與純化 (Gel extraction) .................................................. 9 九、接合反應 (Ligation) ..................................................................................... 9 十、勝任細胞之製備 (Preparation of competent cell) ........................................ 9 十一、大腸桿菌之細胞轉型作用 (Transformation) ........................................ 10 十二、轉型細菌菌落進行聚合酶鏈鎖反應篩選 (colony PCR) ..................... 10 十三、高純度質體DNA 抽取 (Extraction of plasmid DNA) .......................... 10 十四、限制酶酵素截切 (Digestion of plasmid DNA) ...................................... 11 十五、農桿菌 (Agrobacterium tumefaciens) 勝任細胞之配製 ...................... 11 十六、農桿菌之細胞轉型作用.......................................................................... 11 十七、阿拉伯芥之基因轉殖 (Gene transformation) ........................................ 11 十八、基因轉殖株之篩選 (Screening of transgenic plants) ............................. 12 十九、阿拉伯芥染色體DNA 之萃取 (Extraction of genomic DNA) ............. 12 二十、即時定量聚合酶酵素連鎖反應 (Real-time PCR)................................. 12 二十一、亞歷山大染色 (Alexander's staining) ................................................ 13 二十二、共軛焦顯微鏡觀察 (Confocal microscopy) ....................................... 13 結果.............................................................................................................................. 14 一、 DAF 和DAFL1 上游小片段基因 (uORF) 和包含uORF 之全基因 片段之特性分析.................................................................................................. 14 二、 uDAF、DAF、uDAFL1 和DAFL1 於野生型阿拉伯芥之表現分析 15 三、大量表現uDAF、F-DAF 轉殖植株之功能性分析 .................................. 15 四、microRNA (miRNA) 結合區域之預測 ...................................................... 17 討論.............................................................................................................................. 19 參考文獻...................................................................................................................... 21 圖表.............................................................................................................................. 25 表1、本研究所使用的引子(primer)序列 ......................................................... 25 圖1、uDAF 編碼序列 (coding sequence) ......................................................... 27 圖2 、F-DAF 編碼序列 (coding sequence) ..................................................... 28 圖3、uDAFL1 編碼序列 (coding sequence) .................................................... 29 圖4、F-DAFL1 編碼序列 (coding sequence) ................................................... 30 圖5、uDAF、uDAFL1 及DAF、DAFL1 胺基酸序列比對 ............................ 31 圖6、uDAF、DAF 在野生型阿拉伯芥各部位表現量 .................................... 32 圖7、uDAFL1、DAFL1 在野生型阿拉伯芥各部位表現量............................ 33 圖8、大量表現 uDAF、F-DAF、nsF-DAF 載體構築示意圖 ....................... 34 圖9、35S::uDAF 載體構築與分子選殖 .......................................................... 35 圖10、35S::F-DAF 載體構築與分子選殖....................................................... 36 圖11、35S::ns-FDAF 載體構築與分子選殖 ................................................... 37 圖12、大量表現uDAFL1、F-DAFL1、nsF-DAFL1 載體構築示意圖 ......... 38 圖13、35S::uDAFL1 載體構築與分子選殖 .................................................... 39 圖14、35S::F-DAFL1 載體構築與分子選殖 ................................................... 40 圖15、35S::ns-FDAFL1 載體構築與分子選殖 ............................................... 41 圖16、大量表現uDAF 基因之轉殖株性狀 ..................................................... 42 圖17、大量表現F-DAF 基因之轉殖株性狀 ................................................... 43 圖18、大量表現uDAF 轉殖株表現量分析 ..................................................... 44 圖19、大量表現F-DAF 轉殖株表現量分析 ................................................... 45 圖20、大量表現uDAF 轉殖株之亞歷山大染色 ............................................. 46 圖21、大量表現F-DAF 轉殖株之亞歷山大染色 ........................................... 47 圖22、大量表現F-DAF 轉殖株之花藥內壁螢光圖 ....................................... 48 圖23、miRBase Database 搜尋可能參與調控DAF 的miRNA ...................... 49 圖24、可能參與調控DAF 的miRNA 結合區域示意圖 ................................ 50 圖25、miRBase Database 搜尋可能參與調控DAFL1 的miRNA ................. 51 圖26、可能參與調控DAFL1 的miRNA 結合區域示意圖 ............................ 52 附圖1、Ubiquitin (Ub) / 26S proteasome pathway 示意圖-1 ........................... 53 附圖2、Ubiquitin (Ub) / 26S proteasome pathway 示意圖-2 ........................... 54 附圖3、各種不同形式之E3 複合體示意圖 .................................................... 55 附圖4、RING-finger domain 示意圖 ............................................................... 56 附圖5、花藥開裂的過程示意圖....................................................................... 57 附圖6、茉莉酸生合成途徑............................................................................... 58 附圖7、花粉成熟,花藥開裂與開花的同步調控示意圖............................... 59 附圖8、DAF 調控阿拉伯芥花藥開裂模型示意圖 .......................................... 60 附圖9、DAFL1 調控阿拉伯芥胚珠發育.......................................................... 61 附圖10、uORF 主要存在於5'UTR 的形式 .................................................... 62 附圖11、uORF 主要調控下游基因的潛在形式 .............................................. 63 附圖12、阿拉伯芥中具有uORF 調控的相關基因 ......................................... 64 附圖13、pGEM ® -T Easy vector 之載體圖譜 .................................................. 65 附圖14、pEpyon 22K 之載體圖譜 ................................................................... 66zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2018-08-22起公開。zh_TW
dc.subject花藥不開裂zh_TW
dc.subject雄不稔zh_TW
dc.subjectDAFen_US
dc.subjectanther dehiscenceen_US
dc.subjectflower developmenten_US
dc.title基因上游小片段 (uORF) 調控DAF 和DAFL1 功能之探討zh_TW
dc.titleInvestigation of the function for upstream Open Reading Frame (uORF) in regulating DAF and DAFL1en_US
dc.typethesis and dissertationen_US
dc.date.paperformatopenaccess2018-08-22zh_TW
dc.date.openaccess2018-08-22-
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item.openairetypethesis and dissertation-
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item.languageiso639-1zh_TW-
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