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標題: 阿拉伯芥中NAC-like基因之分子選殖與功能性分析
Molecular cloning and functional analysis of NAC-like genes from Arabidopsis thaliana
作者: 林建鑫
Lin, Jian-Shin
關鍵字: NAC
defective stamen
barren silique
出版社: 生物科技學研究所
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摘要: NAC-like基因群是植物中所特有的轉錄因子,它們是一群在蛋白質N端帶有150個高度保留氨基酸NAC(for NAM, ATAF1, 2, and CUC2)domain的基因群,並被認為會參與調控多種植物的發育過程。本研究在分析四個阿拉伯芥的NAC-like基因:AtNACL8、AtNACL10、AtNACL11及AtNACL12。經演化樹分析,它們同屬於NAC家族中NAC2的子群。在此四個基因表現量部份,植物生長早期隨著生長時期增長,mRNA表現有所提升,並在不同組織如:營養葉、腋生葉、花苞及花序中皆可偵測到mRNA表現。經過不同賀爾蒙及逆境處理發現這四個基因表現量分別會受到不同程度的影響。進一步構築此四個基因的promoter藉由驅動GUS報導基因的表現來呈現它們的表現位置。結果發現AtNACL8在根、子葉、頂芽分生組織、營養葉與腋生葉邊緣及花器皆有大量表現;AtNACL10則是只有在根、頂芽分生組織及花器有微量表現。AtNACL11及AtNACL12的表現很相似,在根、子葉及頂芽分生組織都可觀察到有大量表現。進一步透過GFP螢光分析結果證實AtNACL8及AtNACL10蛋白質能夠進入細胞核。另外在35S::AtNACL8轉基因植株中觀察到雄蕊發育異常及果莢不孕的現象。在35S::AtNACL8 antisense及35S::AtNACL10 antisense皆發現有花序生長變異,如:花序的同一節間位置長出多個次生花序。相似的花序生長變異同時也發現於AtNACL8 T-DNA insertion及AtNACL8與AtNACL10 RNA interference的突變株。經由軟體預測及實驗發現有四個基因會受到AtNACL8蛋白所調控。本研究並以yeast two-hybrid的系統分別鑑識出能與AtNACL8及AtNACL10相互作用之八個蛋白質。將來進一步的探討這些基因及蛋白質,所得結果將對NAC-like基因群在調控植物生長發育的功能上提供更深入的了解。
NAC-like genes, a class of plant-specific transcription factors, are characterized by having a highly conserved 150 amino acid NAC(for NAM, ATAF1, 2, and CUC2)domain at their N-terminal of proteins and have been thought to be involved in the regulation of diverse plant development processes. In this study, four Arabidopsis NAC-like genes AtNACL8, AtNACL10, AtNACL11 and AtNACL12 in NAC2 subgroup were isolated and analyzed. AtNACL8, AtNACL10, AtNACL11 and AtNACL12 mRNA were detected early and were increased gradually after germination. Their mRNA is also expressed in rosette leaves, cauline leaves, flowers and inflorescence. Different hormones and abioic stresses influenced their gene expression. Further promoter assay by transforming constructs fusing the promoter of these genes with report GUS gene in Arabidopsis indicated that AtNACL8 was highly expressed in roots, cotyledon, shoot apical meristem, margin of leaves and flowers whereas AtNACL10 was only weakly expressed in roots, shoot apical meristem and flowers. AtNACL11 and AtNACL12 showed similar expression pattern by highly expressing in roots, cotyledon and shoot apical meristem. Further analysis indicated that AtNACL8 and AtNACL10 proteins were able to enter nucleus by fusing with GFP protein. Defective stamen and barren siliques were observed in 35S::AtNACL8 transgenic plants. The alteration of shoot formation such as the production of more than one lateral organ on the same position of floral axis was found in 35S::AtNACL8 anti-sense and 35S::AtNACL10 anti-sense transgenic plants. Similar alteration of shoot formation was also observed in AtNACL8 T-DNA insertion, AtNACL8 and AtNACL10 RNA interference mutant. By way of the software prediction and the experiment analysis discoveried four genes to be able to regulated by AtNACL8 protein. To investigate the function of AtNACL8 and AtNACL10 action, yeast two-hybrid screening using AtNACL8 and AtNACL10 as a bait was performed. Eight proteins that interacted with AtNACL8 and AtNACL10 proteins were identified. Further analysis of these proteins should lead to a deeper understanding of the function for these NAC-like genes in regulating plant development.
其他識別: U0005-2408200706493700
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