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標題: 阿拉伯芥中兩個E3 RING finger基因之功能性分析及文心蘭C與E MADS box基因RNAi載體之構築
Functional analysis of two E3 RING finger genes in Arabidopsis thaliana and construction of RNAi constructs for C and E functional MADS box genes in Oncidium 'Gower Ramsey'
作者: 張廉仕
Chang, Lien-Shih
關鍵字: Arabidopsis;阿拉伯芥;ubiquitin;flowering gene;RING9;RING10;文心蘭;泛素;開花基因;RING9基因;RING10基因
出版社: 生物科技學研究所
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Ubiquitin (Ub) / 26S proteasome參與植物調節生長發育與荷爾蒙訊息等多方面生理功能的調控,而胺基酸的序列上有保守RING-finger domain的RING (Really Interesting New Gene) E3接合酶(E3 ligase)更是扮演重要的角色。本研究中從阿拉伯芥中選殖在演化樹上屬於同一個子群的AtRING9、10,進行功能性的分析。透過RT-PCR定量的方式發現AtRING9在14天幼年植株、腋葉、花苞及根部大量表現;而啟動子驅動GUS報導基因的表現,發現AtRING9主要表現在新生葉、小花苞、頂芽分生組織、葉脈組織及根部。AtRING10在RT-PCR定量中各部位都有表現,在幼年植株、新生花苞及根表現較多,而啟動子驅動GUS報導基因的表現,發現AtRING10主要表現在新生葉、小花苞、頂芽分生組織及根部。初步研究發現去除RING finger domain的AtRING9及全長之AtRING10,在接合GFP螢光分析結果中證實不會進核。透過異位大量表現AtRING10可以發現轉殖株的花序發育遭到抑制或造成終結花序,若異位大量表現反股AtRING10則發現轉殖植株失去頂芽優勢造成花序叢生及葉子捲曲。綜合上述,AtRING9、10對於新生組織特別是分生組織的發育應有一定的影響,而透過轉殖植物觀察進一步推論AtRING10對於分生組織及後續花序的發育扮演負調控的角色 (第一章)。
MADS box基因群對於植物花器生成佔重要的地位,而總共有五群MADS box基因依循一定模式相互協同調控一朵花的生成,所遵循的就是ABCDE model。在阿拉伯芥中,如果A功能基因失去功能會導致花器沒有花瓣與花萼,只有雄蕊跟雌蕊;若B功能基因失去功能會導致花器沒有雄蕊,只有花萼跟雌蕊;若C功能基因失去功能會導致花變成只具花萼及花瓣之重瓣花;E功能基因失去功能會導致所有花器被葉狀的萼片取代。本研究以阿拉伯芥MADS box基因的突變性狀為基準,透過RNAi技術抑制文心蘭C (OMADS4) 或E (OMADS6、7、11) 功能性MADS box基因表現,期望能在文心蘭當中看見重瓣花或是都是萼片的花,藉此創造新的文心蘭花型,也更進一步的確認實驗室所選殖到的OMADS box基因對文心蘭花器生成與調控所扮演真正的角色。本實驗因此對OMADS4設計其專一序列構築單一基因之RNAi構築體,而對E 功能之OMADS6、7、11則構築同時含三個基因專一序列之RNAi之構築體,以利後續文心蘭轉殖之實驗,期望能對多基因達到抑制效果 (第二章)。

E3s RING (Really Interesting New Gene) finger proteins contain a conserved RING motif and play key roles in ubiquitin-mediated proteolysis which has a central role in many processes of plant development. Two RING genes, AtRING9 and AtRING10, were isolated and characterized from Arabidopsis. AtRING9 mRNA is highly expressed in 14-d-old seedling, cauline leaf, flower buds and roots. Further promoter assay by transformed constructs containing AtRING9 promoter fused with report GUS gene indicated AtRING9 is expressed in young leaf, flower buds, SAM (shoot apical meristem), veins and roots. AtRING10 mRNA is expressed in all the organs tested with relatively high expression in seedling, flower buds and roots. Further promoter assay by transformed constructs fused the promoter of AtRING10 with report GUS gene indicated AtRING10 expressed in young leaf, flower buds, SAM and roots. Further analysis indicated that AtRING9 without RING finger domain and the full length of AtRING10 could not enter nucleus by fused with GFP. Ectopic expression of sense and anti-sense for AtRING10 was performed and phenotypic analyzed. The result indicated that sense transgenic plants delayed the flowering time and produced terminal flowers. The anti-sense transgenic plants lost apical dominance and produced curly leaves. These data indicated that AtRING9 and 10 may play roles in regulating SAM activity and controlling the inflorescence development (Chapter 1).
MADS box gene played a critical roles in floral organ development. ABCDE model predicts the formation of flower organ by the interaction of five classes of MADS box genes in plant. To generate transgenic flowers with novel phenotype of Oncidium ‘Gower Ramsey ’ with silence of the C (OMADS4) or E (OMADS6、7、11) function genes, RNAi strategy was performed. RNAi construct contained conserved region specific for OMADS4 was constructed. In addition, three conserved regions, specific for OMADS6, 7, 11 respectively, were constructed in the same RNAi construct. Further transformed these constructs into Oncidium in the future will expect to generate transgenic plants with novel flower phenotype (Chapter 2).
其他識別: U0005-1208200802332100
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