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dc.contributor.authorChiu, Yi-Fengen_US
dc.identifier.citation陸、參考文獻 徐杏芬 (2003) 文心蘭花朵發育相關之MADS box基因之選殖及功能分析。國立中興生物科技研究所大學碩博士論文。 吳家偉 (2006) 文心蘭中B和E功能性之MADS box開花基因之選殖與特性分析。國立中興生物科技研究所大學碩士論文。 Albert, V.A., Oppenheimer, D.G., and Lindqvist, C. (2002). Pleiotropy, redundancy and the evolution of flowers. Trends in Plant Science 7, 297-301. Blázquez, M.A., Soowal, L. N., Lee, I., and Weigel, D. (1997). LEAFY expression and flower initiation in Arabidopsis. Development 124, 3835 -3844. Blázquez, M.A. (2000). Flower development pathways. Journal of Cell Science. 113, 3547-3548. Blázquez, M.A., and Weigel, D. (2000). Integration of floral inductive signals in Arabidopsis. Nature 404, 889-892. Blázquez, M. A., Ferrandiz, C., Madueno, F., and Parcy, F. (2006). How floral meristems are built. Plant Mol. Biol. 60, 855-870. Bomblies, K., Wang, R.-L., Ambrose, B. A., Schmidt, R. J., Meeley, R. B., and Doebley, J. (2003) Duplicate FLORICAULA/LEAFY homologs zfl1 and zfl2 control inflorescence architecture and flower patterning in maize. 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dc.description.abstract高等植物開花過程是由營養組織轉變到花分生組織,進而進入一連串花之形成和花器發育過程,最終構成一朵完整的花。本研究之目的是針對調節文心蘭開花時期相關基因之選殖、功能性分析及應用,並進一步去了解如何達到提升文心蘭花卉品質及產量的目標。由文心蘭中選殖到三個與開花相關基因包括LEAFY (LFY)、MADS Box gene OMADS10 和 OMADS11,三者均是花分生組織形成及發育重要的調控者。OnLFY,序列上跟阿拉伯芥中的LFY具有同源性,由467 個胺基酸所組成並且和水稻的OsRFL有58%的相似性。RT-PCR分析顯示出OnLFY 高度表現於初期的花分生組織之原基(primordia)。大量表現OnLFY可促進阿拉伯芥野生種提早開花,OMADS10 相似於阿拉伯芥中A群MADS Box基因的 AP1, RT-PCR分析結果顯示此基因於所有的發育時期與器官皆可偵測到其表現,而OMADS11 相似於阿拉伯芥中E 群MADS Box 基因的SEP1/2,分析結果顯示OMADS11只表現於文心蘭中花器的部分。在35S::OMADS10 轉基因阿拉伯芥中觀察到整個植株會有提早開花並且有終結花(terminal flowers)產生的現象,而35S::OMADS11轉基因植株中會有植株矮小,產生腋生葉,極度早開花並且失去無限花序(inflorescence indeterminacy)等現象。本研究結果証明了在文心蘭中OnLFY 、OMADS10 和 OMADS11這三個基因在調控開花啟始及發育形成,皆扮演著重要的角色。未來將把基因大量表現於文心蘭,並利用RNA 干擾(RNAi)之方式阻斷基因表現,以便作更進一步功能性探討。zh_TW
dc.description.abstractFlowering in higher plants involves the transition of vegetative development into floral initiation and formation and results in the production of flowers. The goal for this study is to clone and characterize orchid (Oncidium Gower Ramsey) genes regulating flowering time for further practical application of these genes in floral quality improvement. LEAFY (LFY) and MADS Box gene APETALA1 (AP1) are specific meristem identity regulator in controlling floral transition in plant. We report here the isolation of OnLFY, the orthologue of LFY from O. Gower Ramsey that encods a 467 amino acids protein and share 58% identity to rice OsRFL. RT-PCR analysis indicated that OnLFY was expressed in young flower primordia. Ectopic expression of OnLFY promoted flowering time in transgenic Arabidopsis. To investigate the flower formation of orchid, two MADS box genes, OMADS10 and OMADS11, were isolated from O. Gower Ramsey and functionally characterized in this research. OMADS10 showed a high similarity to the A function MADS Box gene AP1. RT-PCR analysis indicated that OMADS10 was expressed in all the organs and developmental stages tested. OMADS11, with high similarity to the E function MADS Box genes such as SEP1/2, was only expressed in flower and in all different floral parts. 35S::OMADS10 transgenic Arabidopsis plants flowered extremely early and produced terminal flowers. Transgenic Arabidopsis plants ectopically expressing OMADS11 also showed novel phenotypes resembling 35S::OMADS10 plants by significantly reducing plant size, producing curly leaves, flowering early, and losing inflorescence indeterminacy. Our result provided evidence to show that OnLFY, OMADS10 and OMADS11 play multiple roles in regulating both floral transition and formation in orchid. The RNA interference technology was applied to generate transgenic mutants to further investigate the function for these genes.en_US
dc.description.tableofcontents目錄 壹、緒言 一、探討植物開花 (一)開花誘導 1 (二)花器之啟始 3 (三)花器形成與發育 3 二、開花基因之功能性探討 (一)LEAFY(LFY) 5 (二)AP1/SQUA次家族(subfamily)MADSbox基因 7 (三)SEP次家族(subfamily)MADSbox基因 8 三、文心蘭(Oncidium) 10 四、研究動機及目的 11 貳、材料與方法 12 參、結果 23 一、文心蘭OnLFY (一)文心蘭中開花基因OnLFY之選殖 23 (二)OnLFY基因序列之分析 24 (三)OnLFY演化樹之分析 24 (四)OnLFY各部位之表現分析 24 (五)35S::OnLFY轉殖載體之構築 25 (六)35S::OnLFY轉基因阿拉伯芥植株之分析與表現鑑定 26 (七)OnLFY RNAi之載體構築 27 二、洋桔梗EgLFY (一)洋桔梗開花基因EgLFY之選殖 27 (二)EgLFY基因序列之分析 28 (三)EgLFY演化樹之分析 28 (四)洋桔梗各部位之表現分析 28 三、文心蘭OMADS10及OMADS11 (一)開花相關基因OMADS10、OMADS11之選殖 29 (二)文心蘭OMADS10基因序列之分析 29 (三)文心蘭OMADS11基因序列之分析 30 (四)35S::OMADS10及35S::OMADS11轉殖載體之構築 31 (五)OMADS10及OMADS11在文心蘭中各部位之表現 31 (六)轉基因植物之分析 31 (七)轉基因植物之OMADS10及OMADS11表現分析 32 肆、討論 34 伍、結論 42 陸、參考文獻 43 圖1、退化引子(Degenerate primer) 位置示意圖 51 圖2、OnLFY 全長cDNA之選殖 52 圖3、文心蘭OnLFY cDNA序列及其相對應胺基酸序列 53 圖4、OnLFY 胺基酸序列比對 54 圖5、OnLFY之演化樹圖譜 56 圖6、OnLFY 在文心蘭中營養組織之表現 57 圖7、OnLFY 在文心蘭中生殖組織之表現 58 圖8、35S::OnLFY正股選殖之確認 59 圖9、35S::OnLFY之阿拉伯芥轉殖株(T1)之性狀分析 60 圖10、35S::OnLFY阿拉伯芥轉殖株(T1)之性狀分析 61 圖11、轉殖35S::OnLFY 阿拉伯芥植株(T1)之OnLFY表現量分析 62 圖12、OnLFY RNAi 正股及反股之確認 63 圖13、OnLFY RNAi 構築示意圖 64 圖14、洋桔梗EgLFY cDNA序列及其相對應胺基酸序列 65 圖15、EgLFY 胺基酸序列比對 66 圖16、EgLFY之演化樹圖譜 67 圖17、EgLFY 在洋桔梗中之表現量偵測 69 圖18、OnAP1及OnSEP1 全長cDNA之選殖 70 圖19、OnAP1及OnSEP1 全長cDNA之選殖 71 圖20、文心蘭OMADS10 cDNA序列及其相對應胺基酸序列 72 圖21、OMADS10與其他物種植物A功能性基因之胺基酸比對分析 73 圖22、OMADS10與已知A群同源基因之演化樹分析 74 圖23、文心蘭OMADS11 cDNA序列及其相對應胺基酸序列 76 圖24、OMADS11與其他物種植物E功能性基因之胺基酸比對分析 77 圖25、OMADS11與已知E群同源基因之演化樹分析 78 圖26、35S::OMADS10、35S::OMADS11之構築 80 圖27、OMADS10 及 OMADS11 在文心蘭中各部位之表現 81 圖28、轉殖35S::OMADS10之阿拉伯芥轉殖株(T1)之性狀分析 82 圖29、轉殖35S::OMADS11 之阿拉伯芥轉殖株(T1)之早期性狀分析 83 圖30、轉殖35S::OMADS11之阿拉伯芥轉殖株(T1)之晚期性狀分析 84 圖31、轉殖35S::OMADS11之阿拉伯芥轉殖株(T2)之性狀 85 圖32、35S::OMADS10 、35::OMADS11 轉殖阿拉伯芥植株中 OMADS10及OMADS11之表現量分析 86 圖33、文心蘭Gower Ramsey 花器發育之ABCDE模式 87 附圖1、植物生長週期 88 附圖2、阿拉伯芥花朵發育基因調控路徑示意圖 89 附圖3、模式植物阿拉伯芥花部示意圖 90 附圖4、阿拉伯芥決定花器形成之ABCDE model 91 附圖5、MADS-box proteins之結構圖 92 附圖6、花器形成之四聚體模式(quartet model) 93 附圖7、LFY為成花發育過程中重要的關鍵基因 94 附圖8、花器分生組織基因互相作用概要圖 95 附圖9、文心蘭Oncidium Gower Ramsey花序及花朵外觀 96 附圖10、pGEM®-T Easy vector之載體圖譜 97 附圖11、pBI-mGFP之載體圖譜 98 附圖12、pBlueACTi之載體圖譜 99 表1、 本論文所使用引子之序列 100 表2、文心蘭Gower Ramsey MADS Box Genes 102 附錄1、LFY/FLO同源基因 103 附錄2、MADS box A群同源基因 104 附錄3、MADS box E群同源基因 105zh_TW
dc.subjectFloral Initiationen_US
dc.titleMolecular Cloning and Characterization of Genes Regulating Floral Initiation and Formation from Orchid (Oncidium Gower Ramsey)en_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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