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

Phang Tsui-Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 146-162). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / Abbreviations --- p.viii / Table of contents --- p.xii / List of figures --- p.xviii / List of tables --- p.xx / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Formation of saline soil --- p.1 / Chapter 1.3 --- Urgent need to reclaim saline lands --- p.2 / Chapter 1.4 --- Cellular routes for Na+ uptake --- p.2 / Chapter 1.4.1 --- Carriers involved in K+ and Na+ uptake --- p.2 / Chapter 1.4.2 --- Channels involved in K+ and Na+ uptake --- p.4 / Chapter 1.5 --- Adverse effects of high salinity --- p.5 / Chapter 1.5.1 --- Hyperosmotic stress --- p.5 / Chapter 1.5.2 --- Ionic stress --- p.6 / Chapter 1.5.2.1 --- Deficiency of K+ --- p.6 / Chapter 1.5.2.2 --- Perturbation of calcium balance --- p.7 / Chapter 1.5.3 --- Toxicity of specific ions --- p.7 / Chapter 1.5.4 --- Oxidative stress --- p.10 / Chapter 1.6 --- Mechanisms of salt stress adaptation in plants --- p.11 / Chapter 1.6.1. --- Maintenance of ion homeostasis --- p.12 / Chapter 1.6.1.1 --- Regulation of cytosolic Na+ concentration --- p.12 / Chapter 1.6.1.2 --- SOS signal transduction pathway --- p.15 / Chapter 1.6.2 --- Dehydration stress adaptation --- p.17 / Chapter 1.6.2.1 --- Aquaporins ´ؤ water channel proteins --- p.17 / Chapter 1.6.2.2 --- Osmotic adjustment --- p.20 / Chapter 1.6.2.2.1 --- Genetic engineering of glycinebetaine biosynthesis --- p.23 / Chapter 1.6.2.2.2 --- Genetic engineering of mannitol biosynthesis --- p.27 / Chapter 1.6.3 --- Morphological and structural adaptation --- p.28 / Chapter 1.6.4 --- Restoration of oxidative balance --- p.29 / Chapter 1.6.5 --- Other metabolic adaptation --- p.31 / Chapter 1.6.5.1 --- Induction of Crassulacean acid (CAM) metabolism --- p.34 / Chapter 1.6.5.2 --- Coenzyme A biosynthesis --- p.34 / Chapter 1.7 --- Soybean as a target for studying salt tolerance --- p.36 / Chapter 1.7.1 --- Economical importance of soybean --- p.36 / Chapter 1.7.2 --- Reasons for studying salt stress physiology in soybeans --- p.38 / Chapter 1.7.3 --- Salt tolerant soybean in China --- p.39 / Chapter 1.7.4 --- Exploring salt tolerant crops by genetic engineering --- p.41 / Chapter 1.8 --- Significance of this project --- p.47 / Chapter 2. --- Materials and methods --- p.48 / Chapter 2.1 --- Materials --- p.48 / Chapter 2.1.1 --- Plant materials used --- p.48 / Chapter 2.1.2 --- Bacteria strains and plasmid vectors --- p.48 / Chapter 2.1.3 --- Growth media for soybean --- p.48 / Chapter 2.1.4 --- Equipment and facilities used --- p.48 / Chapter 2.1.5 --- Primers used --- p.48 / Chapter 2.1.6 --- Chemicals and reagents used --- p.49 / Chapter 2.1.7 --- Solutions used --- p.49 / Chapter 2.1.8 --- Commercial kits used --- p.49 / Chapter 2.1.9 --- Growth and treatment condition --- p.49 / Chapter 2.1.9.1 --- Characterization of salt tolerance of Wenfeng7 --- p.49 / Chapter 2.1.9.2 --- Samples for subtractive library preparations --- p.50 / Chapter 2.1.9.3 --- Samples for slot blot and northern blot analyses --- p.50 / Chapter 2.1.9.4 --- Samples for gene expression pattern analysis --- p.50 / Chapter 2.2. --- Methods --- p.52 / Chapter 2.2.1 --- Total RNA extraction --- p.52 / Chapter 2.2.2 --- Construction of subtractive libraries --- p.53 / Chapter 2.2.3 --- Cloning of salt-stress inducible genes --- p.53 / Chapter 2.2.3.1 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.53 / Chapter 2.2.3.2 --- Ligation of candidate DNA fragments with T-vector --- p.53 / Chapter 2.2.3.3 --- Transformation --- p.54 / Chapter 2.2.3.4 --- PCR screening --- p.54 / Chapter 2.2.4 --- Preparation of recombinant plasmid for sequencing --- p.55 / Chapter 2.2.5 --- Sequencing of differentially expressed genes --- p.55 / Chapter 2.2.6 --- Homology search of differentially expressed genes --- p.56 / Chapter 2.2.7 --- Expression pattern analysis --- p.56 / Chapter 2.2.7.1 --- Preparation of single-stranded DIG-labeled PCR probes --- p.56 / Chapter 2.2.7.2 --- Preparation of cRNA DIG-labeled probes --- p.57 / Chapter 2.2.7.3 --- Testing the concentration of DIG-labeled probes --- p.58 / Chapter 2.2.7.4 --- Slot blot --- p.58 / Chapter 2.2.7.5 --- Northern blot --- p.59 / Chapter 2.2.7.6 --- Hybridization --- p.60 / Chapter 2.2.7.7 --- Stringency washed --- p.60 / Chapter 2.2.7.8 --- Chemiluminescent detection --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Characterization of salt tolerance of Wenfeng7 --- p.61 / Chapter 3.2 --- Identification of salt-stress induced genes from Wenfeng7 --- p.70 / Chapter 3.2.1 --- Screening subtractive libraries of Wenfeng 7 for salt inducible genes --- p.70 / Chapter 3.2.1.1 --- Results of homology search for salt inducible genes --- p.71 / Chapter 3.2.1.2 --- Northern blot showing the salt inducibility of selected clones --- p.72 / Chapter 3.3 --- Genes expression pattern of selected salt inducible genes --- p.104 / Chapter 3.3.1 --- Expression of genes related to dehydration adjustment --- p.104 / Chapter 3.3.1.1 --- Dehydration responsive protein RD22 (Clone no.: HML806) --- p.104 / Chapter 3.3.1.2 --- Beta-amylase (Clone no.: HML767) --- p.104 / Chapter 3.3.2 --- Expression of genes related to structural modification --- p.105 / Chapter 3.3.3 --- Expression of genes related to metabolic adaptation --- p.105 / Chapter 3.3.3.1 --- Subgroup 1: Gene related to protein synthesis --- p.105 / Chapter 3.3.3.1.1 --- Translational initiation factor nsp45 (Clone no.: HML1042) --- p.105 / Chapter 3.3.3.1.2 --- Seed maturation protein PM37 (Clone no.: HML931) --- p.106 / Chapter 3.3.3.2 --- Subgroup 2: Genes related to phosphate metabolism (Clone no.: HML1000) --- p.107 / Chapter 3.3.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.107 / Chapter 3.3.3.3.1 --- Vegetative storage protein A (Clone no.: HML762) --- p.107 / Chapter 3.3.3.3.2 --- Cysteine proteinase (Clone no.: HML928) --- p.107 / Chapter 3.3.3.4 --- Subgroup 4: Genes related to senescence --- p.109 / Chapter 3.3.4 --- Expression of genes encoding novel protein (Clone no.: HML782) --- p.109 / Chapter 4. --- Discussion --- p.125 / Chapter 4.1 --- Evaluation of salt tolerance of Wenfeng7 --- p.125 / Chapter 4.2 --- Isolation of salt inducible genes in Wenfeng7 --- p.127 / Chapter 4.2.1 --- Genes associated with dehydration adaptation --- p.129 / Chapter 4.2.1.1 --- Dehydration responsive protein RD22 --- p.129 / Chapter 4.2.1.2 --- Beta-amylase --- p.130 / Chapter 4.2.2 --- Genes associated with structural adaptation --- p.132 / Chapter 4.2.3 --- Genes associated with metabolic adaptation --- p.133 / Chapter 4.2.3.1 --- Subgroup 1: Genes related to protein synthesis --- p.133 / Chapter 4.2.3.2 --- Subgroup 2: Genes related to phosphate metabolism --- p.137 / Chapter 4.2.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.138 / Chapter 4.2.3.4 --- Subgroup 4: Genes related to senescence --- p.140 / Chapter 4.2.4 --- Novel genes --- p.142 / Chapter 4.3 --- Brief summary --- p.142 / Chapter 5. --- Conclusion and perspectives --- p.144 / References --- p.146 / Appendix I: Screening for salt tolerant soybeans --- p.163 / Appendix II: Major equipment and facilities used --- p.165 / Appendix III: Major chemicals and reagents used in this research --- p.166 / Appendix IV: Major common solutions used in this research --- p.168 / Appendix V: Commercial kits used in this research --- p.170