Lo Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 108-115). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.viii / Table of content --- p.x / List of tables --- p.xv / List of figures --- p.xvi / List of abbreviations --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The secretory pathway --- p.2 / Chapter 1.1.1 --- Endoplasmic reticulum --- p.2 / Chapter 1.1.2 --- Golgi complex --- p.3 / Chapter 1.1.3 --- Vacuoles --- p.3 / Chapter 1.1.4 --- Prevacuolar compartment --- p.4 / Chapter 1.2 --- The secretory pathway in plant cells --- p.5 / Chapter 1.2.1 --- The secretory pathway in yeast and mammalian cells --- p.7 / Chapter 1.2.2 --- The lytic pathway in plant cells --- p.8 / Chapter 1.2.3 --- The protein storage vacuole pathway in plant cells --- p.10 / Chapter 1.3 --- Dynamic studies between organelles --- p.12 / Chapter 1.4 --- Objectives of this thesis research --- p.13 / Chapter Chapter 2 --- Development of Transgenic Cell Lines Expressing PVC and Golgi Markers --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Putative PVC marker --- p.16 / Chapter 2.1.2 --- Golgi marker --- p.17 / Chapter 2.1.3 --- Dynamic studies --- p.18 / Chapter 2.1.4 --- Cell culture study --- p.18 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Plant material --- p.21 / Chapter 2.2.2 --- Construction of fusion reporters --- p.22 / Chapter 2.2.2.1 --- Cloning materials --- p.22 / Chapter 2.2.2.2 --- Vector preparation --- p.22 / Chapter 2.2.2.3 --- Cloning of pGFP-BP-80K and pGFP-BP-80H --- p.24 / Chapter 2.2.2.4 --- Cloning of pGFP-α-TIPH --- p.28 / Chapter 2.2.3 --- Transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.3.1 --- Agrobacterium transformation --- p.30 / Chapter 2.2.3.2 --- BY-2 cell transformation --- p.30 / Chapter 2.2.4 --- Screening of transgenic BY-2 cells --- p.31 / Chapter 2.2.4.1 --- Killing curve study --- p.31 / Chapter 2.2.4.2 --- Antibiotic selection --- p.32 / Chapter 2.2.4.3 --- Fluorescence microscopy screening (For single-construct cell lines) --- p.33 / Chapter 2.2.4.4 --- Confocal laser scanning microscopy (CLSM) screening (For double-construct cell lines) --- p.33 / Chapter 2.2.5 --- Detection of fluorescent protein expression --- p.35 / Chapter 2.2.5.1 --- Confocal imaging --- p.35 / Chapter 2.2.5.2 --- Protein extraction and subcellular fractionation --- p.36 / Chapter 2.2.5.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.36 / Chapter 2.2.5.4 --- Western blot analysis --- p.37 / Chapter 2.2.5.5 --- Cell culture study --- p.37 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Hygromycin concentration at 50 mg/L was optimal for selection --- p.39 / Chapter 2.3.2 --- Lower transformation efficiency for double-construct cell lines --- p.40 / Chapter 2.3.3 --- Screening of transgenic cell lines --- p.41 / Chapter 2.3.4 --- Both pGFP-BP-80K and pGFP- a -TIPH expressed as punctate signals in single-construct cell lines --- p.45 / Chapter 2.3.5 --- Weak punctate or diffuse signals were detected from PVC markers in double-construct cell lines --- p.47 / Chapter 2.3.6 --- GFP reporters were successfully transformed into BY-2 cells --- p.51 / Chapter 2.3.7 --- Profiles of fluorescent signals in transgenic cells during cell culture --- p.53 / Chapter 2.4 --- Discussion --- p.59 / Chapter 2.4.1 --- Abnormal cell growth might be due to high selection pressure --- p.59 / Chapter 2.4.2 --- Double-construct cell lines developed were not yet suitable for further study --- p.60 / Chapter 2.4.3 --- Single-construct cell lines expressing putative PVC markers were developed --- p.62 / Chapter 2.4.4 --- 2- to 3-day-old cells were more suitable for subsequent studies --- p.63 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Expressing Reporters for Distinct Prevacuolar Compartments --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.1.1 --- Wortmannin --- p.69 / Chapter 3.1.2 --- Brefeldin A --- p.70 / Chapter 3.1.3 --- FM4-64 --- p.71 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Plant material --- p.73 / Chapter 3.2.2 --- Confocal immunofluorescence studies --- p.73 / Chapter 3.2.3 --- Drug treatment studies --- p.74 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.74 / Chapter 3.2.3.2 --- BFA treatment --- p.75 / Chapter 3.2.4 --- FM4-64 uptake study --- p.76 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Organelles marked by GFP- a -TIP CT reporters did not localize at Golgi compartment --- p.78 / Chapter 3.3.2 --- Wortmannin induced GFP- a -TIP marked organelles to vacuolated --- p.81 / Chapter 3.3.3 --- GFP- a -TIP CT reporters partially colocalized with VSRin wortmannin-treated cells --- p.83 / Chapter 3.3.4 --- BFA induced GFP- a -TIP marked organelles to form BFA- induced compartments --- p.88 / Chapter 3.3.5 --- GFP-α -TIP CT reporter colocalized with internalized FM4-64 --- p.91 / Chapter 3.4 --- Discussion --- p.94 / Chapter 3.4.1 --- GFP- α -TIP CT reporter was a putative PVC marker --- p.94 / Chapter 3.4.2 --- GFP- a -TIP marked organelles behaved differently from lytic PVCs --- p.95 / Chapter 3.4.3 --- GFP- a -TIP marked organelles were not lytic PVCs --- p.96 / Chapter 3.4.4 --- FM4-64 uptake study reveals a new PVC marker --- p.98 / Chapter Chapter 4 --- Summary and Future Prospects --- p.100 / Chapter 4.1 --- Summary --- p.101 / Chapter 4.1.1 --- Hypothesis --- p.101 / Chapter 4.1.2 --- Development of transgenic cell lines --- p.102 / Chapter 4.1.3 --- Characterization of organelles marked by GFP- a -TIP CT reporter --- p.103 / Chapter 4.2 --- Future prospects --- p.106 / Reference --- p.108
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324714 |
| Date | January 2004 |
| Contributors | Lo, Sze Wan., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xx, 115 leaves : ill. (some col.) ; 30 cm. |
| 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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