指導教授：李昆達臺灣大學：生化科技學系易松輝Yi, Sung-HuiSung-HuiYi2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261629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.謝誌 i 中文摘要 ii Abstract iii Contents iv Contents of tables and figures vii Chapter I Introduction 1 1.1 Hairy roots 2 1.2 Agrobacterium rhizogenes 3 1.3 rolB 4 1.4 Aim of this study 6 Chapter II Materials and Methods 7 2.1 Biological materials and growth conditions 8 2.1.1 Growth conditions of Saccharomyces cerevisiae 8 2.1.2 Cultures of tobacco hairy roots 8 2.1.3 Nicotiana tabacum cv. bright yellow 2 cell 9 2.2 Genetic materials 9 2.2.1 Gateway entry vectors construction 9 2.2.2 Constructions of Gateway destination vectors for Y2H 10 2.2.3 Constructions of Gateway destination vectors for BiFC 11 2.2.4 Constructions of Gateway destination vectors for YFP-fusion proteins 12 2.3 Yeast two-hybrid experiments 12 2.3.1 cDNA library construction 12 2.3.2 Yeast transformation 13 2.3.3 Yeast two-hybrid screening 13 2.3.4 Confirmation and sequencing of candidate cDNA fragments 14 2.3.5 Yeast two-hybrid of RolB with full-length candidate proteins 15 2.4 Bimolecular fluorescence complementation assay 15 2.4.1 Tobacco BY-2 cells protoplast isolation 15 2.4.2 Protoplast transformation 16 2.4.3 Confocal microscope observation 17 2.5 Subcellular Localization of RolB, PHI-2, NtbZIP, and ORF13a 17 Chapter III Results 18 3.1 Yeast two-hybrid screening 19 3.2 Full-length cDNA cloning 20 3.3 Yeast two-hybrid assay of RolB with full-length proteins 21 3.4 BY-2 protoplasts isolation 21 3.5 Bimolecular fluorescence complementation 22 3.6 Subcellular localization of RolB, PHI-2, NtbZIP, and ORF13a 22 Chapter IV Discussion 23 4.1 Yeast two-hybrid screening 24 4.2 ORF13a is an open reading frame on T-DNA 24 4.3 phi-2 is a phosphate-induced gene-2 25 4.4 NtbZIP regulates the biosynthesis of chlorophyll 26 4.5 Possible roles of PHI-2 and NtbZIP are ABA-responsive element binding factors 27 4.6 NtbZIP has similar function as Arabidopsis LONG HYPOCOTYL5 29 4.7 Localizations of the protein-protein interactions in BY-2 cells 30 4.8 Possible regulatory mechanism of RolB in hairy roots 31 Chapter V Conclusion 33 Tables 35 Figures 39 References 563393520 bytesapplication/pdf論文使用權限：同意有償授權(權利金給回饋本人)毛狀根根毛農桿菌rolB蛋白質交互作用體學ABFs/ABI5轉錄因子thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261629/1/ntu-103-R01b22008-1.pdf