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dc.contributor.authorKao, Nai-Hsuanen_US
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dc.description.abstract對植物來說,許多重要的生理發育過程是由MADS box基因所調控。而大部分影響花器形成的ABCDE基因皆屬於此類MADS box基因,其蛋白質結構中皆具有一段保守的DNA結合區域(MADS box domain),後有一段和蛋白質相互作用相關的K domain。為了能更清楚了解蘭科植物花器形成機制,本研究從文心蘭(Oncidium Gower Ramsey)中選殖出OMADS8及OMADS9兩個基因進行功能性分析。OMADS8可轉譯出一個含210個胺基酸的蛋白質,其序列比對結果發現,和阿拉伯芥B class中之PI基因有高度相似性,屬於PI同源性基因。OMADS8在文心蘭葉、根及花皆有表現。進一步觀察花器內部表現,發現OMADS8在花萼及花瓣表現量較雄蕊、心皮來的高。將OMADS8基因大量表現於野生型阿拉伯芥中,發現會使植株矮小提早開花,並造成花萼轉變成似花瓣的構造。OMADS9可轉譯出一個含222個胺基酸的蛋白質,其屬於B class中之PaleoAP3族基因,例如鴿石斛蘭(Dendrobium)的DcOAP3B和文心蘭的OMADS5皆屬於此類。OMADS9C端部分具有兩個完整的paleoAP3演化分支特有的motif — PI-derived motif及paleoAP3 motif。OMADS9在文心蘭花器中表現量以花瓣及唇辦中最高。大量表現OMADS9於阿拉伯芥,觀察到早開之花朵白化但較晚開的花和野生型無異。此外,透過酵母菌雙雜交系統(yeast two hybrid),來分析所知的文心蘭B群基因的蛋白質間的相互關係,結果顯示,OMADS5及OMADS9有形成homodimer的能力,也會相互作用形成 heterodimer。OMADS8無法形成同質二聚體,也無法形成異質二聚體。zh_TW
dc.description.abstractMADS box genes controlled many important developmental processes in plants. Most ABCDE class genes regulating flower formation are MADS genes that contained a DNA-binding domain (MADS box domain) and a protein interaction domain (K domain) in their encoded proteins. To investigate the flower formation in orchid, two B class MADS box genes, OMADS8 and OMADS9 from Oncidium Gower Ramsey, were isolated and characterized in this research. OMADS8 encoded a 210 amino acid protein showed high sequence homology to Arabidopsis B class gene PI. OMADS8 is expressed in all the organs tested including leaf, root and flower. In flowers, OMADS8 mRNA was especially detected higher in the sepal and petal than in stamen and carpel. Ectopic expression of OMADS8 significantly reduced the plant size and promoted flowering time in transgenic Arabidopsis. The conversion of sepal to petal-like structure was also observed in 35S::OMADS8 transgenic plants. OMADS9 encoded a 222 amino acid protein and showed sequence homology to PaleoAP3 lineage of B group MADS box gene such as DcOAP3B in Dendrobium and OMADS5 in Oncidium. In its C-terminal region, two completely consensus motifs such as PI-derived and paleoAP3 motifs which are unique to paleoAP3 lineage were identified. OMADS9 is highly expressed in petal and lip petal in flowers. The early developed flowers are whitened, but the late developed flowers in transgenic Arabidopsis ectopically expressing OMADS9 are indistinguishable from that of wild-type plants. Furthermore, to investigate the interaction among the Oncidium B class proteins such as OMADS8, OMADS9 and OMADS5, yeast two hybrid analysis was performed. The result indicated that OMADS5 and OMADS9 were able to form homodimer and heterodimer to each other. OMADS8 was not able to form either homodimer or heterodimer.en_US
dc.description.tableofcontents壹 、前言 1 貳 、材料與方法 9 参 、結果 ㄧ、文心蘭B群功能性基因之選殖 23 二、文心蘭中OMADS8及OMADS9之演化樹分析 23 三、文心蘭OMADS8及OMADS9在植物體各部位表現情形 25 四、OMADS8、OMADS8△M、OMADS9、OMADS9△M構築體之構築與轉殖阿拉伯芥植株之篩選 25 五、35S:OMADS8之轉基因阿拉伯芥植物性狀分析 26 六、35S:OMADS8△M 之轉基因阿拉伯芥植物性性分析 26 七、35S:OMADS9 之轉基因阿拉伯芥植物性狀分析 27 八、35S:OMADS9△M 之轉基因阿拉伯芥植物性狀分析 27 九、利用yeast two hybrid方法以探討OMADS5、OMADS8、OMADS9 之間相互作用關係 27 十、文心蘭OMADS5 RNAi 建置 29 肆 、討論 一、文心蘭OMADS8、OMADS9轉基因阿拉伯芥植物探討 30 二、利用酵母菌雙雜交系統來探討OMADS5、OMADS8、OMADS9之 間相互作用的關係 32 三、文心蘭MADS box B群基因OMADS5、OMADS8、OMADS9可能功 能之探討 33 伍 、參考文獻 36 陸 、圖表 表1.本論文中聚合酶連鎖反應所使用之引子之序列 41 表2.本論文相關基因基因庫編碼 43 圖1.文心蘭B 群功能性基因之選殖 45 圖2.文心蘭OMADS8 之cDNA序列及相對應胺基酸序列 46 圖3.文心蘭OMADS9 之cDNA序列及相對應胺基酸序列 47 圖4.文心蘭OMADS8、OMASDS9演化樹分析 48 圖5.文心蘭OMADS8與其他物種B功能性PI同源基因之胺基酸序列比 對分析 49 圖6.文心蘭OMADS9與其他物種B功能性paleoAP3及euAP3分支基因 之胺基酸序列比對分析 50 圖7.OMADS5、 OMADS8 、 OMADS9 在文心蘭各部位之表現量分析 51 圖8.文心蘭OMADS8、OMASDS9各種不同基因片段之構築與鑑定 52 圖9.35S:OMADS8轉基因阿拉伯芥植物抽出花序天數及營養葉數統計圖 53 圖10.轉殖35S:OMADS8之轉基因阿拉伯芥植物之性狀分析 54 圖11.利用RT-PCR鑑定35S:OMADS8轉基因阿拉伯芥植物中OMADS8 之表現 55 圖12.35S:OMADS8轉基因阿拉伯芥植物T2代抽出花序天數及營養葉數 統計圖 56 圖13.以SEM掃描式電子顯微鏡觀察35S::OMADS8阿拉伯芥轉基因植 物花器上之表皮細胞結構 57 圖14.轉殖35S:OMADS8 及OMADS8△M 轉基因植物抽出花序天數及 營養葉數統計圖 59 圖15.轉殖35S: OMADS8△M大量表現之轉基因植物性狀分析 60 圖16.轉殖35S:OMADS9之轉基因阿拉伯芥植物性狀分析 61 圖17.35S:OMADS9及35S:OMADS9△M 轉基因阿拉伯芥植物抽出花序 天數及營養葉數統計圖 62 圖18.以酵母菌雙雜交系統之ONPG分析方式分析OMADS5、OMADS8 、OMADS9之間相互作用方式 63 圖19.利用營養篩選實驗方式證實OMADS5、OMADS8、OMADS9之間 相互作用的方式 64 圖20.文心蘭 OMADS5 RNAi構築 65 圖21.文心蘭OMADS8蛋白質K box區域胺基酸序列比對及結構預測 66 圖22.文心蘭OMADS9、OMADS5蛋白質K box區域胺基酸序列比對 及結構預測 67 附圖1.花器形成之四聚體模式(Quartet model) 68 附圖2.植物MADS box基因MIKC-type蛋白質結構圖 69 附圖3.植物MADS box B群功能性基因 70 附圖4. modified ABC模式 71 附圖5. Yeast two-hybrid系統原理示意圖 72 附圖6. pGEM®-T Easy 載體圖譜及限制酶酵素切位 73 附圖7.轉基因表現用之載體圖譜及限制酶切位 74 附圖8.加強蛋白質表現所用之載體圖譜及限制酶切位 75 附圖9. pGADT7載體圖譜 76 附圖10. pGBKT7載體圖譜 77 附圖11. pBlueACTi轉接載體圖譜 78zh_TW
dc.subjectMADS boxen_US
dc.subjectB-function genesen_US
dc.title文心蘭中B功能性MADS Box基因之選殖與特性分析zh_TW
dc.titleMolecular Cloning and Characterization of B function MADS Box Genes from Orchid (Oncidium Gower Ramsey)en_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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