by Li Wing Yen Francisca. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 94-105). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgemnets --- p.vii / Abbreviations --- p.ix / Table of contents --- p.xii / List of figures --- p.xvi / List of tables --- p.xvii / Chapter 1. --- General Introduction / Chapter 1.1 --- Introduction to oxidative stress / Chapter 1.1.1 --- Introduction to Reactive Oxygen Species --- p.1 / Chapter 1.1.2 --- Major sites of ROS production / Chapter 1.1.2.1 --- Chloroplast --- p.4 / Chapter 1.1.2.2 --- Mitochondria --- p.4 / Chapter 1.2 --- Regulation of intercellular ROS content in plant cells / Chapter 1.2.1 --- Enzymatic defense ofROS --- p.6 / Chapter 1.2.1.1 --- Superoxide dismutases --- p.6 / Chapter 1.2.1.2 --- "Ascorbate peroxidase, Glutathione reductase and the Ascorbate-Glutathione cycle" --- p.7 / Chapter 1.2.1.3 --- Catalase --- p.11 / Chapter 1.2.1.4 --- Alternative oxidase --- p.11 / Chapter 1.2.2 --- Non-enzymatic / Chapter 1.2.2.1 --- Ascorbate and Glutathione --- p.12 / Chapter 1.2.2.2 --- α-tocopherol --- p.12 / Chapter 1.3 --- "Salt, dehydration and oxidative stress" / Chapter 1.3.1 --- Oxidative stress is induced when plants were under salt stress --- p.13 / Chapter 1.3.2 --- Oxidative stress is induced when plants were under dehydration stress --- p.14 / Chapter 1.4 --- ROS scavenging: the road to achieve multiple-stress tolerance? --- p.16 / Chapter 1.5 --- Purple acid phosphatase and its relationship with oxidative stress in plants / Chapter 1.5.1 --- General introduction to plants purple acid phosphatase (PAP) --- p.20 / Chapter 1.5.2 --- Purple acid phosphatases that found to be involved in ROS scavenging in plants --- p.21 / Chapter 1.6 --- Previous studies in GmPAP3 --- p.23 / Chapter 1.7 --- Hypothesis and significance of this project --- p.25 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- "Plants, bacterial strains and vectors." --- p.26 / Chapter 2.1.2 --- Chemicals and reagents --- p.27 / Chapter 2.1.3 --- Commercial kits --- p.28 / Chapter 2.1.4 --- Primers and adaptors --- p.29 / Chapter 2.1.5 --- Equipments and facilities used --- p.31 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.31 / Chapter 2.1.7 --- Software --- p.31 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Molecular techniques / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.32 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.32 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5α, DE3 and Agrobacterium competent cells" --- p.33 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.36 / Chapter 2.2.1.5 --- DNA and RNA extraction --- p.37 / Chapter 2.2.1.6 --- Generation of single-stranded DIG-labeled PCR probes --- p.38 / Chapter 2.2.1.7 --- Testing the concentration of DIG-labeled probes --- p.40 / Chapter 2.2.1.8 --- Northern blot analysis --- p.40 / Chapter 2.2.1.9 --- PCR techniques --- p.41 / Chapter 2.2.1.10 --- Sequencing --- p.42 / Chapter 2.2.2 --- Plant cell culture and transformation / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.43 / Chapter 2.2.2.2 --- Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells --- p.44 / Chapter 2.2.3 --- Growth and treatment conditions for plants / Chapter 2.2.3.1 --- Growth and salt treatment condition of soybean samples for gene expression studies of GmPAPS --- p.45 / Chapter 2.2.3.2 --- Root assay of GmPAP3 transgenic Arabidopsis thaliana --- p.46 / Chapter 2.2.4 --- "Immunolabeling, mitochondria integrity, ROS detection and confocal microscopy" / Chapter 2.2.4.1 --- Immunolabeling of GmPAP3-T7 transgenic cell lines --- p.47 / Chapter 2.2.4.2 --- Mitochondria integrity --- p.48 / Chapter 2.2.4.3 --- Detection of Reactive oxygen species (ROS) --- p.48 / Chapter 2.2.4.4 --- Confocal microscopy --- p.49 / Chapter 2.2.4.5 --- Images processing and analysis --- p.49 / Chapter 2.2.5 --- Statistical analysis --- p.50 / Chapter 3. --- Results / Chapter 3.1 --- "Expression of GmPAP3 was induced by NaCl stress, oxidative stress, and dehydration stress" --- p.51 / Chapter 3.2 --- Establishment of GmPAP3-T7 fusion transgenic cell lines / Chapter 3.2.1 --- Subcloning of GmPAP3-T7 into the binary vector system W104 --- p.53 / Chapter 3.2.2 --- Transformation of W104-GmPAP3-T7 into tobacco BY-2 cells --- p.56 / Chapter 3.3 --- Establishment of GmPAP3 trangenic cell lines / Chapter 3.3.1 --- Subcloning of GmPAP3 into the binary vector system W104 --- p.58 / Chapter 3.3.2 --- Transformation of W104-GmPAP3 into tobacco BY-2 cells --- p.58 / Chapter 3.4 --- Establishment of GmPAP3 transgenic Arabidopsis thaliana / Chapter 3.4.1 --- Transformation of W104-GmPAP3 into Arabidopsis thaliana --- p.61 / Chapter 3.5 --- Colocalization of GmPAP3 with MitoTracker-orange --- p.66 / Chapter 3.6 --- Effect of expressing GmPAP 3 on mitochondria integrity of BY-2 cells under NaCl and dehydration stress. --- p.71 / Chapter 3.7 --- Effect of expressing GmPAP3 on ROS production in BY-2 cells under salt and PEG treatment --- p.75 / Chapter 3.8 --- Effect of expressing GmPAP3 in Arabidopsis thaliana under salt stress --- p.81 / Chapter 4. --- Discussion / Chapter 4.1 --- Gene expression profile of GmPAP3 --- p.83 / Chapter 4.2 --- Subcellular localization of GmPAP3 --- p.84 / Chapter 4.3 --- Functional tests of GmPAP 3 transgenic BY-2 cells / Chapter 4.3.1 --- GmPAP3 could protect the plant cells' mitochondria integrity when under salt and dehydration stress --- p.86 / Chapter 4.3.2 --- Expressing GmPAPS in tobacco BY-2 cells were able to reduce the production ofROS under salt and dehydration stresses --- p.88 / Chapter 4.4 --- Functional tests of GmPAP3 transgenic Arabidopsis --- p.91 / Chapter 5. --- Conclusion and perspectives --- p.92 / References --- p.94 / Appendix I: Restriction and modifying enzymes --- p.106 / Appendix II: Chemicals --- p.107 / Appendix III: Commercial kits --- p.111 / Appendix IV: Equipments and facilities used --- p.112 / "Appendix V: Buffer, solution, gel and medium formulation" --- p.113
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325249 |
| Date | January 2005 |
| Contributors | Li, Wing Yen Francisca., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvii, 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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