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研究生: 林相頴
Lin, Hsiang-Yin
論文名稱: 蝴蝶蘭DP與E2F之功能探討和鑑定新穎RBR交互作用蛋白之技術建立
Functional characterization of PaE2F and PaDP genes and the identification of novel PaRBR-interacting proteins in Phalaenopsis aphrodite
指導教授: 方素瓊
Fang, Su-Chiung
共同指導教授: 劉景煌
Liu, Zin-Huang
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 熱帶植物科學研究所
Institute of Tropical Plant Sciences
論文出版年: 2014
畢業學年度: 103
語文別: 英文
論文頁數: 68
中文關鍵詞: G1和S過渡期細胞週期調控DP/E2FRBR
外文關鍵詞: G1-S transition, cell cycle regulation, DP/E2F, RBR
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  • 蘭屬品種繁多,每一種都具有很大的差異性,為極具生物多樣性的一屬。然而,細胞週期的調控如何配合蘭科特有性狀演化的研究仍非常缺乏。本論文著重於研究在細胞G1和S過渡期中辦演重要角色的細胞週期調控蛋白:正向調控的轉錄因子DP/E2F (E2 promoter binding Factor (E2F)-Dimerization Partner (DP))及轉錄抑制蛋白RBR (Retinoblastoma-Related)。在蝴蝶蘭基因資料庫裡,我們成功的找到四個E2F基因,兩個DP基因,及兩個RBR基因。利用酵母菌雙雜合及細胞定位的方法,我們確認了蘭花中不同的DP蛋白會與E2F蛋白相互做用。而這兩種蛋白之間的相互做用會幫助E2F轉譯蛋白從細胞質移至細胞核內。此外在阿拉伯芥轉植株中,表現蘭花E2F基因影響子葉及真葉,使其變的稍大。而在阿拉伯芥中受到E2F調控的基因也因為蘭花E2F的大量表現而影響其表現量。這些結果顯示蘭花的E2F蛋白可能影響細胞週期基因的轉錄調控。在論文的第二部份,我的研究著重於尋找和轉錄抑制蛋白RBR有相互做用並可能影響到植物發育的新穎蛋白。我用了生物資訊的方式選出了數組在蛋白序列裡含有會和RBR蛋白作用的LxCxE 序列的基因。並以雙分子螢光互補的方式證明其相互作用。這些蛋白分別為 SCARECROW Like 1 (SCRL1),Global Transcription Factor Group B1 Like (GTBL1) 及 ARF GAP Domain Like 2 (AGDL2) 。這樣的結果證實了利用生物資訊的平台可以篩選出可能在植物體內和RBR相互做用的發育調節因子。

    Orchidaceae belongs to one of the species-rich families with abundant floral diversity. However, how cell cycle program evolves to regulate the unique developmental program of orchids remains illusive. My study focuses on the cell cycle regulators required for G1 to S transition. Specifically, the positive regulators- heterodimerized E2 promoter binding Factor (E2F)-Dimerization Partner (DP) transcription factors and negative regulator- RETINOBLASTOMA-RELATED (RBR) proteins are the focus of my work. Four E2F, three DP, and two RBR genes have been identified from the Phalaenopsis aphrodite transcriptome database. The first part of the thesis focuses on functional characterization of Phalaenopsis E2Fs and DPs. Using Yeast two hybrid and sub-cellular localization of E2F and DP protein, the pairwise interactions of each Phalaenopsis E2F and DP proteins was confirmed. The physical interaction of Phalaenopsis E2F and DP proteins is important for nuclear translocation of E2F transcription factor. Moreover, overexpressing PaE2Fs in transgenic Arabidopsis plants increase the size of cotyledon and leaves and mis-regulates the expression of E2F-responsive genes. These results suggest that overexpression of PaE2Fs causes mis-regulation of the cell cycle genes in Arabidopsis and subsequently leads to abnormality in root length and size of cotyledons and leaves. For the second part of the project, I explored the potential RBR-interacting developmental regulators that may connect cell cycle program to plant development. I took the bioinformatic approach and identified orchid proteins containing the conserved RBR binding motif, LxCxE. Using Bimolecular Fluorescence Complementation (BiFC), the interaction of novel proteins such as Global Transcription Factor Group B1 Like (GTBL1) and ARF GAP Domain Like 2 (AGDL2) and RBR proteins were confirmed. This approach here provides a novel way to systemically isolate potential RBR-interacting proteins in plants.

    Table of Contents Chinese Abstract.......3 Abstract..........4 Index..........7 I INTRODUCTION I.1 Introduction........10 I.2 Cell cycle regulators ......10 I.2.1 Cyclin Dependent Kinases (CDKs) and Cyclins..10 I.2.2 Retinoblastoma-related (RBR) protein....11 I.2.3 E2F/DP transcription facrtor....13 I.3 The involvement of the cell cycle regulators during differentiation ......14 II MATERIALS AND METHODS II.1 Plant materials and growth conditions ...16 II.2 Agrobacterium-mediated transformation ....16 II.3 Yeast two hybrid assay .....17 II.4 Transient expression of PaDPs and PaE2Fs and microscopy .. 18 II.5 Measurement of protein concentration...19 II.6 Western blot analysis ......20 II.7 RNA extraction and quantitative RT-PCR ...21 II.8 Root length measurement ......21 II.9 Isolation of LxCxE containing proteins...22 II.10 BiFC assay and microscopy .....22 II.11 Phylogenetic analysis ......24 Part I III RESULTS III.1 Isolation of full-length Phalaenopsis E2F and DP cDNA ..24 III.2 Verification of the interaction between PaE2Fs and PaDP1 or PaDP2 .......24 III.3 Interaction of PaDPs with PaE2Fs stimulates nuclear translocation .......26 III.4 Generation of PaE2F overexpression plants ...26 III.5 Functional characterization of Phalaenopsis E2Fs ..27 IV DISCUSSION.......29 Part II V RESULTS V.1 Identification of potential RBR interacting proteins ..31 V.2 Verification of interaction between selected proteins and PaRBRs by BiFC ......31 VI DISCUSSION.......32 VII REFERENCES.......37 VIII TABLES .........44 Table 1. Constructs generated for experiments ....44 Table 2. Primers for PCR ......46 Table 3. Orchid gene ID from Orchidstra Phalaenopsis Genome Annotation Database .....49 Table 4.Gene/Protein ID used for analysis ....50 IX FIGURES Figure 1. The canonical RB pathway ....51 Figure 2. Protein sequence alignment of Phalaenopsis RBR, E2F and DP proteins .....52 Figure 3. Interaction assay between PaE2Fs and PaDPs ..55 Figure 4. Subcellular localization of PaDPs and PaE2Fs ..56 Figure 5. Effects of overexpressing PaE2Fs in Arabidopsis .57 Figure 6. Identification of potential RBR interacting protein.60 Figure 7. BiFC assay between RBR and candidates ..61 Figure 8. BiFC assay between RBR and candidates (single cell) ......62 Figure 9. BiFC assay with various markers ...63 Figure 10. Protein sequence alignment of GRAS and SCR.64 Figure 11. Domain structure and phylogenetic analysis of AGDL .......65 Figure 12. BiFC assay between PaRBR and PaAGDL2...67 Supplementary Figure S1. Phenotype of PaE2F overexpressors .68

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