請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55481
標題: | 菸草毛狀根中Root Locus B蛋白質交互作用之研究 Interactomic Study of Root Locus B in Tobacco Hairy Roots |
作者: | Sung-Hui Yi 易松輝 |
指導教授: | 李昆達(Kung-Ta Lee) |
關鍵字: | 毛狀根,根毛農桿菌,rolB,蛋白質交互作用體學,ABFs/ABI5轉錄因子, A. rhizogenes A4,hairy roots,rolB,Interactomic,ABFs/ABI5 protein family, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 毛狀根是快速生長的不定根組織,培養過程中會累積大量的植物二級代謝物。當植物受傷並受到根毛農桿菌感染後,根毛農桿菌 (Agrobacterium rhizogenes) 會將其Ri質體上的T-DNA隨機嵌入植物的染色體,誘導毛狀根的生成。然而,有許多植物並不受到根毛農桿菌的感染,限制了毛狀根組織的應用。如果能了解毛狀根分化過程的分子機制,將可提升更多藥用植物的應用性。在T-DNA當中,rolB被認為是影響毛狀根生成重要的關鍵。在我們的先前研究發現,缺失rolB的毛狀根生長速度明顯減緩,無法長期繼代培養,側根數量也會較野生型減少。為了了解RolB蛋白質在菸草毛狀根中的蛋白質交互作用之關係,我們透過酵母雙雜合法 (Y2H) 篩選,發現RolB蛋白質會和ORF13a、PHI-2與NtbZIP有交互作用。進一步於菸草BY2細胞中利用雙分子螢光互補系統確認其交互作用,發現RolB分別與PHI-2、NtbZIP交互作用於細胞內具有DNA的胞器中,而非細胞核。分別與黃色螢光蛋白融合連接後的RolB、 PHI-2、ORF13a皆同時表現於細胞核與細胞質中,NtbZIP則只在細胞核中被發現。ORF13a與RolB都是T-DNA上的基因;PHI-2與NtbZIP則為菸草中的兩個具有bZIP的轉錄因子,其序列比對為阿拉伯芥ABF轉錄因子家族中的一員。由於ABF轉錄因子在植物中受到ABA荷爾蒙的刺激下,會啟動ABA相關基因的表現,並抑制根的生長,我們推測,RolB是透過調控ABA的訊息傳遞路徑,進而促進毛狀根的生長。 Hairy roots are fast growing adventitious root tissues. They can accumulate high amount of plant secondary metabolites. Hairy roots are differentiated after Agrobacterium rhizogenes infects the wounded plant and transfers the T-DNA from its Ri plasmid randomly into the plant chromosome. However, some plants are resistant to the Agrobacterium-infection and therefore the utilization of hairy root culture in these plants is limited. Understanding the molecular mechanism during hairy root formation enables potential application of hairy root induction in medical herbs. In T-DNA, rolB has been considered to act the pivotal role during the differential process of hairy roots. In our previous studies, tobacco hairy roots deficient in rolB gene showed less lateral roots, less neoplastic growth and slower growth rate. To elucidate the protein-protein interactions of RolB protein in tobacco hairy roots, we conducted yeast two-hybrid screening. We found that RolB interacted with ORF13a, PHI-2 and NtbZIP. The potential protein-protein interactions were further studied by bimolecular fluorescence complementation (BiFC) assay in tobacco BY-2 cells. BiFC results showed that the interactions of RolB with PHI2 and NtbZIP were located in a DNA-containing organelle other than the nucleus. The localizations of RolB-YFP, ORF13a-YFP and PHI-2-YFP were found in both nucleus and cytosol, while NtbZIP-YFP was only detected in nucleus. ORF13a is encoded in the T-DNA as rolB. PHI-2 and NtbZIP are homologous to Arabidopsis ABA Responsive Element Binding Factors (ABFs) family. ABFs are known as the negative regulators in root growth upon ABA signaling. Our finding suggests that RolB interacts with ABFs family proteins and promotes hairy root growth possibly by regulating ABA signaling pathway. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55481 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 3.31 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。