蛋白質分泌及胞吐作用是指蛋白質在內質網(ER)中合成後前往質膜(PM) ， 隨後， 被分泌到達細胞外的過程。 而細胞分泌路徑是指蛋白質途經數個含有膜包被的細胞器後運送到细胞之外。 這些細胞器，包括內質網， 高爾基體， 反式高爾基網絡(TGN) 及質膜。 分泌蛋白分泌出细胞之外後， 在细胞外基質中進行其功能。 / 為達到這項研究的目的，我們結合了細胞、分子和生物化學上的方法， 來對蛋白質運輸路徑及參與蛋白質分泌的細胞器進行研究。首先，通過MALDI-MS/MS對煙草懸浮BY-2細胞中的原態分泌性蛋白質進行分析。第二，把已識別的分泌蛋白包括陽離子過氧化物酶同工酶40K（40K）和N1過氧化物酶（N1），透過轉基因細胞的GFP融合表達方式、及應用特異性抗體於免疫螢光和膠體金免疫電鏡上的測定來對其特性作進一步分析。 第三，總合以上的研究，煙草懸浮BY-2細胞的典型蛋白質分泌路徑次序為質網 - 高爾基體 - 反式高爾基網絡 - 質膜。 / Protein secretion or exocytosis is the process by which proteins synthesized in the endoplasmic reticulum (ER) travel to the plasma membrane (PM) for their subsequent secretion outside of the cell. The secretory pathway responsible for protein secretion contains several membrane-bounded organelles such as the ER, Golgi apparatus, trans-Golgi Network (TGN), and PM. The secreted proteins move outside of the cell and perform their functions in the extracellular matrix. / The general objective of this study was to examine the transport pathways and organelles involved in protein secretion in plant cells using a combination of cellular, molecular and biochemical approaches. First, major native secreted proteins in suspension cultures of tobacco BY-2 culture cells were identified via MALDI-MS/MS analysis. Second, the identified secreted proteins, cationic peroxidase Isozyme 40K (40K) and peroxidase N1 (N1), were further characterized by examining the GFP fusion expression of transgenic cell lines and by generating specific antibodies in immunofluorescent and immunogold electron microscope (EM) studies. Third, throughout all of these studies, a typical ER-Golgi-TGN-PM pathway was mapped for protein secretion in tobacco BY-2 cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lam, Chun Kok. / "December 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 79-84). / Abstracts also in Chinese. / Thesis /Assessment Committee --- p.i / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Secreted protein --- p.1 / Chapter 1.2. --- Secretory Pathway --- p.1 / Chapter 1.3. --- Protein secretion --- p.2 / Chapter 1.4. --- Plant Peroxidases --- p.3 / Chapter 1.5. --- Project Objective --- p.4 / Chapter 1.6. --- Significance --- p.4 / Chapter Chapter 2 --- Materials and Methods --- p.6 / Chapter 2.1. --- Mass spectrometry analysis --- p.6 / Chapter 2.2. --- Generation of 40K/N1-GFP construct --- p.7 / Chapter 2.2.1. --- For transient expression --- p.7 / Chapter 2.2.2. --- For stable expressing constructs --- p.7 / Chapter 2.3. --- Transient expression of 40K/N1-GFP --- p.7 / Chapter 2.4. --- Generation of transgenic cell lines --- p.8 / Chapter 2.5. --- Fluorescence microscopic screening --- p.9 / Chapter 2.6. --- Generation and characterization of antibodies specific for 40K/N1 peroxidase --- p.9 / Chapter 2.7. --- Confocal immunofluorescence studies --- p.10 / Chapter 2.8. --- (TIRF) Total internal reflection fluorescence microscopy --- p.11 / Chapter 2.9. --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.11 / Chapter 2.10. --- Drug Treatment --- p.12 / Chapter 2.10.1. --- Dexamethasone (dex) --- p.12 / Chapter 2.10.2. --- Brefeldin A (BFA)/Concanamycin A (ConcA) --- p.12 / Chapter 2.11. --- Salt treatment (plasmolysis) --- p.13 / Chapter 2.12. --- EM (electron microscopy) study --- p.13 / Chapter Chapter 3 --- Results --- p.14 / Chapter 3.1. --- Protein secretion from tobacco BY2 cells --- p.14 / Chapter 3.2. --- Western blot analysis --- p.15 / Chapter 3.3. --- Protein expression in tobacco plant tissues --- p.15 / Chapter 3.4. --- EM labeling on the wild type BY-2 cells --- p.16 / Chapter 3.5. --- Localization in the tobacco root tip apoplast --- p.17 / Chapter 3.6. --- 40K/N1 peroxidase transient/stable cell line expression --- p.18 / Chapter 3.7. --- Time course study of 40K/N1 peroxidase-GFP cell line expression after induction --- p.18 / Chapter 3.8. --- Plasmolysis (salt treatment analysis) --- p.20 / Chapter 3.9. --- Brefeldin A (BFA) and concanamycin A (ConcA): Trafficking through the Golgi and TGN --- p.20 / Chapter 3.10. --- Examining exocytosis by total internal reflectance fluorescence (TIRF) --- p.22 / Chapter 3.11. --- Immunolabeling study --- p.23 / Chapter 3.12. --- EM study on transgenic 40K & N1 peroxidase-GFP cell lines --- p.24 / Chapter Chapter 4 --- Discussion --- p.26 / Chapter 4.1. --- Trafficking from the ER to the extracellular matrix --- p.26 / Chapter 4.2. --- Secretion through PM by exocytosis --- p.28 / Chapter 4.3. --- Time required for the secretory pathway --- p.29 / Chapter 4.4. --- Similarities of 40K and N1 --- p.30 / Chapter 4.5. --- Future perspectives --- p.30 / References --- p.79
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328739 |
| Date | January 2013 |
| Contributors | Lam, Chun Kok., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiv, 84 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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