Water scarcity induced by drought, temperature, and salinity has plagued agricultural sustainability in recent years with unprecedented revenue losses, raising concerns for worldwide food security. Recent studies have revealed unique botanical response mechanisms to combat water related stress, namely the expression of proteins known as the dehydrins. Dehydrin proteins have been shown to serve various intracellular protective functions. The gene for a SK5 type dehydrin from the arctic plant Cerastium arcticum (CaDHN) was introduced into tobacco plants and water deficit tolerance was evaluated. Plants overexpressing CaDHN displayed improved tolerance to salt stress, but no improvement was observed under drought stress. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

A proteômica expandiu-se dentro da área biológica a partir da década de 90 com o desenvolvimento de técnicas de ionização branda que permitem analisar macromoléculas por espectrometria de massas (MS) e é uma nova estratégia na busca de genes de interesse e de informações sobre o controle da expressão gênica para a manipulação genética de plantas. Na busca por genes diferencialmente expressos durante o desenvolvimento da madeira, foi estabelecida uma plataforma 2D-LC-MS/MS (eletroforese bidimensional seguida de cromatografia líquida e espectrometria de massas em tandem) e identificadas 72 proteínas da região cambial de Eucalyptus grandis de árvores de 2 anos e 8 meses. A identificação foi feita por homologia com o banco de proteínas SwissProt, com um banco de ESTs específico de Eucalyptus spp. (GENOLYPTUS) e com o NCBI. Os dados de proteínas também foram confrontados com a expressão dos transcritos correspondentes obtidos a partir do mesmo tecido por SAGE (Serial Analysis of Gene Expression). Dentre as proteínas identificadas, a maior parcela (24,1 %) tem função relacionada com resposta a estresse, 20,5 % pertence a metabolismo de energia, 17,8 % são componentes estruturais, 14,3 % participam em metabolismo de macromoléculas e 15,2 % em metabolismos básicos como de nitrogênio, aminoácidos, nucleotídeos, lipídeos e metabolismo secundário. / Proteomics impacted the biological sciences since the 90´s after the development of soft ionization techniques that allow to analyse macromolecules by mass spectrometry (MS). It became a new strategy to identify genes and obtain information about the molecular control of gene expression, of importance to genetic manipulation of plants. In order to search for genes involved in wood quality and to understand gene expression during wood development, a 2D-LC-MS/MS system (bidimensional electrophoresis followed by liquid cromatography coupled to mass spectrometry in tandem) was stablished. 72 proteins from the cambial region of Eucalyptus grandis trees at the age of 2 years and 8 months were identified. The MS/MS spectra were processed using the SwissProt databank, an EST (Expressed Sequence Tags) bank of Eucalyptus spp. (GENOLYPTUS) and the NCBI. A comparation of gene and protein expression was carried out using a databank constructed in our laboratory using SAGE (Serial Analysis of Gene Expression) obtained from the same cambial tissue. Most identified proteins are related to stress response (24.1 %), 20.5 % participate in energy metabolism, 17.8 % are important to the cell as structural components, 14,3 % are related to macromolecular metabolism and 15,2 % to basic metabolism of nitrogen, amino acids, nucleotides, lipids and secondary metabolism.

Cheung Ming-yan. / Thesis submitted in 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-185). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / List of figures --- p.xii / List of tables --- p.xv / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction - Using transgenic plants as bioreactor --- p.1 / Chapter 1.1 --- Plant as Bioreactor --- p.1 / Chapter 1.1.1 --- Plant transformation historical milestones --- p.1 / Chapter 1.1.2 --- Applications of transgenic plants --- p.5 / Chapter 1.1.2.1 --- Examples of in situ Application --- p.5 / Chapter 1.1.2.2 --- Examples of ex situ application of transgenic plant --- p.9 / Chapter 1.2 --- Plant Hosts for Transformation: Arabidopsis thaliana and Glycine max --- p.18 / Chapter 1.2.1 --- Essential components for plant transformation --- p.18 / Chapter 1.2.1.1 --- Marker genes --- p.18 / Chapter 1.2.1.2 --- Promoters --- p.18 / Chapter 1.2.2 --- Arabidopsis thaliana --- p.20 / Chapter 1.2.2.1 --- Agrobacterium-mediated transformation --- p.20 / Chapter 1.2.2.2 --- Transformation methods for A. thaliana --- p.21 / Chapter 1.2.3 --- Glycine max (soybean) --- p.22 / Chapter 1.2.3.1 --- Soybean cultivars for transformation --- p.23 / Chapter 1.2.3.2 --- Soybean regeneration systems --- p.24 / Chapter 1.2.3.3 --- Soybean transformation systems --- p.26 / Chapter 1.3 --- Target Pharmaceutical and Agricultural Proteins: Lymphocytic choriomeningitis virus and Goldfish Growth hormones I and II --- p.29 / Chapter 1.3.1 --- Production of pharmaceutical proteins --- p.29 / Chapter 1.3.1.1 --- Lymphocytic choriomeningitis virus --- p.30 / Chapter 1.3.1.2 --- Nucleoprotein of LCMV --- p.33 / Chapter 1.3.2 --- Agricultural protein category --- p.34 / Chapter 1.3.2.1 --- Carassius auratus --- p.34 / Chapter 1.3.2.2 --- Growth hormones I and II --- p.35 / Chapter 1.4 --- Hypothesis and Objectives --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.38 / Chapter 2.1.2 --- Chemicals and Regents --- p.43 / Chapter 2.1.3 --- Commercial kits --- p.44 / Chapter 2.1.4 --- Primers and Adaptors --- p.45 / Chapter 2.1.5 --- Equipments and Facilities used --- p.47 / Chapter 2.1.6 --- "Buffer, solution and medium" --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Molecular Techniques --- p.48 / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.48 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5a, DE3 and Agrobacterium competent cells" --- p.49 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.52 / Chapter 2.2.1.5 --- "DNA, RNA and protein extractions" --- p.53 / Chapter 2.2.1.6 --- Generation of cRNA probes for Southern and Northern blot analyses --- p.56 / Chapter 2.2.1.7 --- Southern blot analysis --- p.56 / Chapter 2.2.1.8 --- Northern blot analysis --- p.57 / Chapter 2.2.1.9 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) in bacterial system --- p.58 / Chapter 2.2.1.10 --- Western blot analysis for LCMV NP --- p.59 / Chapter 2.2.1.11 --- Protein dot blot for detecting the presence of recombinant LCMV-NP generated from transgenic plants --- p.62 / Chapter 2.2.1.12 --- PCR techniques --- p.62 / Chapter 2.2.1.13 --- Sequencing --- p.63 / Chapter 2.2.2 --- Plant tissue culture and transformation --- p.64 / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.64 / Chapter 2.2.2.2 --- Soybean --- p.65 / Chapter 2.2.3 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.68 / Chapter 2.2.3.1 --- In vitro transcription of target genes with with Ribomix large scale RNA production systems-T7 and SP6 (Promega) --- p.68 / Chapter 2.2.3.2 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract --- p.69 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1 --- Expression of LCMV-NP in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1.1 --- Cloning of the gene encoding LCMV NP into the binary vector system W104 --- p.71 / Chapter 3.1.1.2 --- Transformation of W104-LCMV-NP into the Agrobacterium GV3101/pMP90 --- p.78 / Chapter 3.1.1.3 --- Transformation of LCMV-NP cDNA into Arabidopsis thaliana --- p.80 / Chapter 3.1.1.4 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.83 / Chapter 3.1.1.5 --- Production of recombinant LCMV-NP protein in DE3 cells --- p.90 / Chapter 3.1.1.6 --- Detection of recombinant LCMV-NP protein in transgenic A.thaliana --- p.98 / Chapter 3.1.2 --- Expression of goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.105 / Chapter 3.1.2.1 --- "Screening of homozygous lines of goldfish, Carassius auratus, growth hormones transgenic Arabidopsis thaliana" --- p.105 / Chapter 3.1.2.2 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.109 / Chapter 3.1.2.3 --- Detection of recombinant GHI and GHII from transgenic plant --- p.112 / Chapter 3.2 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.117 / Chapter 3.2.1 --- Subcloning of target genes in pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.1 --- Subcloning of LCMV-NP fragment into pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.2 --- Subcloning of goldfish GHI and GHII fragments into pGEM-3Zf(+) vector --- p.120 / Chapter 3.2.2 --- In vitro transcription of target genes with Ribomix large scale RNA production systems-T7 and SP6 --- p.125 / Chapter 3.2.3 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract systems --- p.128 / Chapter 3.3 --- Establishment of Glycine max regeneration and transformation systems --- p.130 / Chapter 3.3.1 --- The Establishment of soybean regeneration system --- p.130 / Chapter 3.3.2 --- Establishment of soybean transformation system --- p.133 / Chapter 3.3.2.1 --- Definition of transformation efficiency --- p.133 / Chapter 3.3.2.2 --- Effects of plant hosts --- p.136 / Chapter 3.3.2.3 --- Effects of Agrobacterium strains --- p.138 / Chapter 3.3.2.4 --- The application of vacuum infiltration --- p.139 / Chapter 3.3.2.5 --- Effect of kanamycin --- p.140 / Chapter 3.3.2.6 --- Effect of cocultivation duration and light/ dark treatment during germination --- p.141 / Chapter 3.3.2.7 --- Application of the detergent Silwet-77 --- p.142 / Chapter 3.3.3 --- Verification of transformation results by PCR screening --- p.143 / Chapter Chapter 4 --- Discussion --- p.147 / Chapter 4.1 --- "Expression of LCMV-NP, GHI and GHII in A. thaliana" --- p.148 / Chapter 4.2 --- Establishing a soybean transformation system --- p.157 / Chapter 4.2.1 --- Plant hosts and explants --- p.158 / Chapter 4.2.2 --- Regeneration of explants --- p.159 / Chapter 4.2.3 --- Agrobacterium strains --- p.161 / Chapter 4.2.4 --- Bacteria-plant interaction --- p.161 / Chapter 4.2.5 --- Transient versus stable transformation --- p.165 / Chapter 4.3 --- Conclusion and perspective --- p.167 / References --- p.169 / Appendix --- p.186

Sha Ou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 167-189). / Abstracts in English and Chinese. / abstract --- p.i / 中文摘要 --- p.iv / acknowledgements --- p.vii / Chapter chapter 1. --- introduction --- p.1 / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Ribosome-inactivating proteins (RIPs) --- p.1 / Chapter 1.2.1 --- Classification --- p.2 / Chapter 1.2.2 --- Structure --- p.3 / Chapter 1.2.3 --- Enzymatic activities --- p.3 / Chapter 1.3 --- Type II RIPs --- p.5 / Chapter 1.3.1 --- Ricin --- p.5 / Chapter 1.3.2 --- Ricinus communis agglutinin (RCA) --- p.6 / Chapter 1.3.3 --- Intracellular mechanism --- p.7 / Chapter 1.3.4 --- Application of RIPs in neuroscience research: suicide axonal transport --- p.10 / Chapter 1.4 --- Type I RIPs --- p.12 / Chapter 1.4.1 --- Trichosanthin (TCS) --- p.12 / Chapter 1.4.2 --- Ricin A chain (RTA) --- p.15 / Chapter 1.4.3 --- Medical applications: immunolesioning and immunotherapy --- p.16 / Chapter 1.5 --- The types of Cell death --- p.17 / Chapter 1.5.1 --- Necrosis --- p.18 / Chapter 1.5.2 --- Apoptosis --- p.18 / Chapter 1.6 --- Inflammations --- p.21 / Chapter 1.6.1 --- Acute inflammation --- p.21 / Chapter 1.6.2 --- Chronic inflammation --- p.22 / Chapter 1.6.3 --- Retinitis --- p.22 / Chapter 1.7 --- Eye model for neurotoxicity studies in CNS --- p.23 / Chapter 1.8 --- Objective of present study --- p.24 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Plan of this chapter --- p.25 / Chapter 2.2 --- Toxins and methods used --- p.25 / Chapter 2.3 --- Animals --- p.26 / Chapter 2.4 --- Preparation of toxin solutions --- p.27 / Chapter 2.4.1 --- RIP solutions --- p.27 / Chapter 2.4.2 --- Labeling type I RIPs with fluorescence --- p.27 / Chapter 2.4.3 --- Control solutions --- p.29 / Chapter 2.5 --- Administrations of solutions --- p.30 / Chapter 2.5.1 --- Basic procedures of vitreous chamber injection --- p.30 / Chapter 2.5.2. --- Injection of trichosanthin (TCS) --- p.31 / Chapter 2.5.3 --- Injection of ricin A chain (RTA) --- p.31 / Chapter 2.5.4 --- Injection of ricinus communis agglutinin (RCA) --- p.32 / Chapter 2.5.5 --- Administration of FITC-TCS --- p.33 / Chapter 2.5.6 --- Administration of FITC-RTA --- p.33 / Chapter 2.6 --- Retinal tissue processing --- p.33 / Chapter 2.6.1 --- Paraffin method --- p.34 / Chapter 2.6.2 --- Cryostatic method --- p.35 / Chapter 2.6.3 --- Electron microscopic method --- p.35 / Chapter 2.7 --- General effects of RIPs on rat retinas --- p.36 / Chapter 2.7.1 --- Hematoxylin-and-eosin staining --- p.36 / Chapter 2.7.2 --- Retinal thickness --- p.37 / Chapter 2.7.3 --- Pathological changes --- p.38 / Chapter 2.7.4 --- Dosage study on TCS --- p.39 / Chapter 2.7.5 --- Statistics --- p.40 / Chapter 2.8 --- Mechanisms of cell death --- p.40 / Chapter 2.8.1 --- Terminal dUTP nick-end labeling (TUNEL) --- p.40 / Chapter 2.8.2 --- Immunohistochemistry for caspase-3 --- p.42 / Chapter 2.8.3 --- Double staining of cleaved caspase-3 and TUNEL --- p.42 / Chapter 2.8.4 --- Electronic microscope observation --- p.43 / Chapter 2.9 --- Entry of type I RIPs into cells --- p.43 / Chapter 2.9.1 --- Propidium iodide staining --- p.43 / Chapter 2.9.2 --- Immunohistochemical localization of Muller cells --- p.44 / Chapter 2.9.3 --- Double staining of Muller cells and TUNEL --- p.44 / Chapter 2.9.4 --- Confocal microscope --- p.44 / Chapter 2.10 --- Reactions of glial cells --- p.45 / Chapter CHAPTER 3. --- RESULTS --- p.47 / Chapter 3.1 --- Preparation of fluorescein-type I RIP conjugates --- p.47 / Chapter 3.1.1 --- Conjugate of FITC-TCS --- p.47 / Chapter 3.1.2 --- Conjugate of FITC-RTA --- p.47 / Chapter 3.2 --- Effects of TCS on retina --- p.47 / Chapter 3.2.1 --- Retina cell count - a dose-dependence study --- p.48 / Chapter 3.2.2 --- Retinal thickness measurement - a time-course study --- p.49 / Chapter 3.2.3 --- Pathological changes --- p.50 / Chapter 3.3 --- Effects of RTA on retina --- p.51 / Chapter 3.3.1 --- Retinal thickness measurement - a time-course study --- p.51 / Chapter 3.3.2 --- Pathological changes --- p.53 / Chapter 3.4 --- Effects of RCA on retina --- p.54 / Chapter 3.4.1 --- Retinal thickness measurement --- p.54 / Chapter 3.4.2 --- Pathological changes --- p.55 / Chapter 3.5 --- Summary of results: general effects of RIPs --- p.56 / Chapter 3.6 --- Cell death - TUNEL method --- p.56 / Chapter 3.6.1 --- TCS experiment --- p.57 / Chapter 3.6.2 --- RTA experiment --- p.58 / Chapter 3.6.3 --- RCA experiment --- p.58 / Chapter 3.7 --- Cell death 一 cleaved caspase-3 immunohistochemistry --- p.58 / Chapter 3.7.1 --- TCS experiment --- p.59 / Chapter 3.7.2 --- RTA experiment --- p.59 / Chapter 3.8 --- EM observation --- p.59 / Chapter 3.8.1 --- TCS experiment --- p.59 / Chapter 3.8.2 --- RTA experiment --- p.60 / Chapter 3.9 --- Summary of results: mode of cell death --- p.60 / Chapter 3.10 --- Localisation of type I RIPs --- p.61 / Chapter 3.10.1 --- FITC-TCS --- p.62 / Chapter 3.10.2 --- FITC-TCS and Muller cell double staining --- p.63 / Chapter 3.10.3 --- Muller cell and TUNEL double staining --- p.64 / Chapter 3.10.4 --- FITC-RTA --- p.64 / Chapter 3.10.5 --- Summary of results: route of intoxication --- p.65 / Chapter 3.11 --- Glial cell reactions after RIP treatment --- p.65 / Chapter 3.11.1 --- TCS experiment --- p.65 / Chapter 3.11.2 --- RTA experiment --- p.66 / Chapter 3.11.3 --- RCA experiment --- p.67 / Chapter 3.11.4 --- Summary of results: glial reactions --- p.67 / Chapter CHAPTER 4. --- DISCUSSION --- p.69 / Chapter 4.1 --- General effects of RIPs on rat retinas --- p.69 / Chapter 4.1.1 --- Effects of trichosanthin (TCS) --- p.69 / Chapter 4.1.2 --- Effects of ricin A chain (RTA) --- p.71 / Chapter 4.1.3 --- Effects of ricinus communis agglutinin (RCA) --- p.73 / Chapter 4.2 --- The mechanisms of cell death --- p.74 / Chapter 4.2.1 --- Cell death caused by TCS --- p.75 / Chapter 4.2.2 --- Caspase-3 and the retina of RCS rat --- p.77 / Chapter 4.2.3 --- Cell death caused by RTA --- p.78 / Chapter 4.2.4 --- Cell death caused by RCA --- p.80 / Chapter 4.2.5 --- Mechanism of RTA - induced necrosis --- p.81 / Chapter 4.3 --- The mechanisms of type I RIPs entering cells --- p.82 / Chapter 4.3.1 --- Transport of TCS in retinal cells --- p.82 / Chapter 4.3.2 --- The uptake of Pure FITC by rat retina --- p.85 / Chapter 4.4 --- Reactions of glial cells --- p.85 / Chapter 4.4.1 --- Glial cell reactions in TCS experiment --- p.86 / Chapter 4.4.2 --- Glial cell reactions in RTA and RCA experiments --- p.87 / Chapter 4.5 --- Possible applications of RIPs on retinal studies --- p.88 / Chapter 4.5.1 --- Potential applications of TCS --- p.88 / Chapter 4.5.2 --- Possible uses of RTA and RCA --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS --- p.91 / "FIGURES, TABLES, GRAPHS, AND LEGENDS" --- p.93 / APPENDICES --- p.154 / Appendix A Source of materials --- p.154 / Appendix B Dosages for vitreous chamber injection --- p.156 / Appendix C Protocol of conjugate fluorescein to proteins --- p.157 / Appendix D Electronic Microscope methods --- p.160 / Appendix E Histological methods --- p.162 / Appendix F Protocols of TUNEL --- p.163 / Appendix G Protocols of Immunohistochemistry staining --- p.165 / REFERENCES --- p.167

Au Yeung, Wan Kin. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 89-103). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.v / General abbreviations --- p.vi / Abbreviations of chemicals --- p.vii / List of figures --- p.ix / List of figures in Appendix VI --- p.xii / List of tables --- p.xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The significance of studying rice disease resistance --- p.1 / Chapter 1.1.1 --- Economic importance of rice --- p.1 / Chapter 1.1.2 --- Diseases caused by pathogens virulent to rice --- p.1 / Chapter 1.1.2.1 --- Bacterial leaf blight diseases --- p.1 / Chapter 1.1.2.2 --- Fungal blast diseases --- p.2 / Chapter 1.1.3 --- Approach to enhance resistance of crops towards pathogens --- p.2 / Chapter 1.2 --- Literature review on plant immunity system --- p.3 / Chapter 1.2.1 --- Pathogen associated molecular patterns (PAMP) and PAMP -triggered immunity (PTI) --- p.4 / Chapter 1.2.2 --- Pathogen effectors and effector-triggered immunity (ETI) --- p.5 / Chapter 1.2.3 --- Roles of phytohormones in plant defense responses --- p.6 / Chapter 1.2.4 --- G protein signaling and plant defense responses --- p.9 / Chapter 1.3 --- Literature review on FK506 binding proteins (FKBPs) --- p.10 / Chapter 1.4 --- Background information of this study - origin of the clone chosen for study in this project --- p.11 / Chapter 1.5 --- Hypothesis and Objectives --- p.12 / Chapter Chapter 2 --- Materials and Methods --- p.13 / Chapter 2.1 --- Materials --- p.13 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.13 / Chapter 2.1.2 --- Chemicals and Regents --- p.18 / Chapter 2.1.3 --- Commercial kits --- p.18 / Chapter 2.1.4 --- Primers and Adaptors --- p.19 / Chapter 2.1.5 --- Equipments and facilities used --- p.23 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.23 / Chapter 2.2 --- Methods --- p.24 / Chapter 2.2.1. --- Bacterial and yeast cultures --- p.24 / Chapter 2.2.2 --- Plant growth conditions and treatments --- p.25 / Chapter 2.2.2.1 --- Surface sterilization of J. thaliana seeds --- p.25 / Chapter 2.2.2.2 --- Environmental conditions of A. thaliana for germination of seeds and growing of seedlings --- p.26 / Chapter 2.2.2.3 --- Environmental conditions of A. thaliana for growing of plants --- p.26 / Chapter 2.2.2.4 --- Pathogen inoculation test of A. thaliana with Pst DC3000 --- p.27 / Chapter 2.2.3 --- Cloning and subcloning of OsFKBP 12 and OsUCCl --- p.27 / Chapter 2.2.3.1 --- Sub-cloning of OsFKBP12 to pGEX-4T-l and pMAL-c2 --- p.27 / Chapter 2.2.3.2 --- Cloning of OsUCCl to pGEX-4T-l --- p.29 / Chapter 2.2.4 --- "DNA, RNA and protein extractions" --- p.29 / Chapter 2.2.4.1 --- Plasmid extraction from bacterial cells --- p.29 / Chapter 2.2.4.2 --- Genomic DNA extraction from plant through CTAB method --- p.29 / Chapter 2.2.4.3 --- RNA extraction from plant tissues --- p.30 / Chapter 2.2.4.4 --- Protein extraction from plant tissues --- p.31 / Chapter 2.2.4.5 --- Fusion protein extraction from E. coli --- p.31 / Chapter 2.2.5 --- Western blot analyses --- p.32 / Chapter 2.2.5.1 --- Western blot analysis of GST tag and MBP tag fusion proteins --- p.32 / Chapter 2.2.5.2 --- Western blot analysis native OsYchFl proteins --- p.33 / Chapter 2.2.6 --- Real-time PCR study --- p.33 / Chapter 2.2.6.1 --- cDNA synthesis --- p.33 / Chapter 2.2.6.2 --- Real-time PCR --- p.34 / Chapter 2.2.7 --- Yeast two hybrid --- p.35 / Chapter 2.2.7.1 --- Screening of OsFKBP 12 interaction protein partners by yeast mating --- p.35 / Chapter 2.2.7.2 --- Identification of positive interacting protein partners by extracting DNA plasmid from yeast --- p.35 / Chapter 2.2.7.3 --- Re-transformation of pGBKTl-OsFKBP 12 with their interacting partner clones into yeast (AH 109) by co-transformation --- p.36 / Chapter 2.2.8 --- In vitro pull down assay of OsFKBP 12 with their putative protein interacting partner --- p.36 / Chapter 2.2.8.1 --- In vitro pull down of native OsYchFl by MBP-His-OsFKBP12 --- p.36 / Chapter 2.2.8.2 --- In vitro pull down of GST-AtYchF 1 by MBP-His-OsFKBP12 --- p.37 / Chapter 2.2.8.3 --- In vitro pull down of MBP-His-OsFKBP12 by GST-OsUCCl --- p.37 / Chapter 2.2.8.4 --- In vitro pull down of MBP-His-OsFKBP12 by GST-OsYchFl G domain --- p.38 / Chapter 2.2.9 --- GTPase assay ofOsYchF with OsFKBP12 --- p.38 / Chapter 2.3.0 --- Phylogenetic analysis and sequence alignment --- p.39 / Chapter Chapter 3 --- Results --- p.40 / Chapter 3.1 --- Identification of OsFKBP 12 encoding a FKBP (FK506 binding protein)-domain containing protein in Oryza sativa (rice) --- p.40 / Chapter 3.2 --- OsFKBP12 was down-regulated in the pathogen-inoculated Xal4 rice line CBB14 --- p.47 / Chapter 3.3 --- Ecotpic expression of OsFKBP 12 repressed the expression of defense marker genes in transgenic A. thaliana --- p.50 / Chapter 3.4 --- Expressing OsFKBP 12 in transgenic A. thaliana enhanced the susceptibility to the bacterial pathogen Pst DC3000 --- p.54 / Chapter 3.5 --- OsFKBP 12 protein interacted with a putative defense-related G-protein and a copper binding protein --- p.57 / Chapter 3.6 --- "OsFKBP 12 protein interacted with the G domain of defense-related G protein, OsYchFl" --- p.69 / Chapter 3.7 --- OsFKBP 12 protein enhanced the in vitro phosphate release of OsYchFl --- p.72 / Chapter Chapter 4 --- Discussion --- p.74 / Chapter 4.1 --- The identification and characterization of OsFKBP 12 --- p.74 / Chapter 4.2 --- Expression pattern of OsFKBP 12 upon biotic stress in bacterial blight resistant near isogenic line (NIL) --- p.75 / Chapter 4.3 --- OsFKBP 12 repressed the expression of SA-regulated defense marker genes when ectopically expressed in A. thaliana --- p.75 / Chapter 4.4 --- Ectopic expression of OsFKBP 12 enhanced susceptibility towards Pst DC3000 in transgenic A. thaliana --- p.76 / Chapter 4.5 --- The interacting partners of OsFKBP 12 in relation to plant defense response --- p.78 / Chapter 4.6 --- The specific biochemical interaction of OsFKBP 12 with OsYchFl --- p.80 / Chapter 4.7 --- Future perspectives --- p.85 / Chapter Chapter 5 --- Conclusion --- p.87 / References --- p.89 / Appendix --- p.104

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2011 / Soybean is one of the most cultivated legume plants in developing countries. Nodule senescence is a major limitation in producing high yields of soybean as it coincides with the pod filling stage. Delaying nodule senescence could be a way of increasing the yield of soybean therefore determination of the role of cysteine protease in soybean is of vital importance. In this study, soybean plants were grown under controlled temperature and light conditions. Leaves and root crown nodules were collected at 4, 6, 10, 12 and 16 weeks of age. In a comparative 1-dimensional SDS-PAGE analysis of soybean nodule proteomes as the plant matured, it showed differences in proteins expressed as shown by different banding patterns with less variation between the younger soybean nodule extracts (4, 6 and 10 weeks old) as compared to the older ones (12 and 16 weeks old). As determined by azocasein assay and protease zymography, the protease activity of the nodule extracts generally decreased with an increase in the age of the nodules whereas that of the leaves increased as the plants grew older. Cysteine proteases in the soybean nodule extracts readily cleaved the Z-Arg-Arg-AMC substrate with the highest activity shown in the younger nodules as compared to the older ones. In the leaf extracts, cysteine protease activity increased with age of the leaves. DCG-04, a biotinylated irreversible inhibitor, proved to be an effective label in profiling of activity of cysteine proteases in 1-dimensional and 2-dimensional systems. The labelling was inhibited specifically by cysteine protease inhibitor, E-64. In root nodules, the DCG-04 probing demonstrated that the expression of cysteine proteases is higher in early stages of development of the soybean nodules as compared to the later stages whereas in the leaves, there is higher expression of cysteine proteases in the old leaves (16 weeks). Using 2-dimensional polyacrylamide gel electrophoresis, five cysteine protease isoforms were visualised with the size ranging from approximately 25 to 30 kDa and a pI range of 4-6. In older nodules (12 and 16 weeks old) the higher pI isoforms are down-regulated with the 26 kDa and pI 4.5 protease being the predominant isoform. Affinity precipitation of the cysteine proteases yielded a strong band with the size of about 26 kDa. All assays used show that while in leaves, the expected trend of high expression of cysteine proteases in senescing leaves is observed, in soybean nodules the expression of cysteine proteases decreases with senescence. There is, therefore, no correlation between senescence and cysteine proteases in nodules. The highly expressed cysteine protease in young nodules could play a developmental or regulatory role during the early stages of development.

Les enjeux de santé publique et de développement durable conduisent à intensifier l’utilisation de protéines végétales notamment dans les secteurs de biens de consommation comme l’industrie pharmaceutique,l’agro-alimentaire et les cosmétiques. La levée récente de certains verrous technologique permet aujourd’hui la purification industrielle de protéines végétales issus de tourteaux provenant de la production d’huiles végétales. Ces protéines sont valorisables comme substituts aux graisses saturées dans la structuration d’huiles à destination de la consommation humaine. Le manque de texture d’huiles végétales insaturées peut être compensé par ces protéines jouant le rôle de stabilisants et de gélifiants dans les émulsions. Nous nous intéressons aux propriétés fonctionnelles des protéines de blé, de tournesol et de colza, en volume et aux interfaces. Nous avons montré que des gels de protéines de tournesol avec des élasticités modulables sont obtenus par dénaturation thermique. La dynamique de formation de films protéique aux interfaces fluides a été étudiée en combinant des mesures de tensiométrie, de viscoélasticité dilatationnelle et d’ellipsométrie. Les mesures sur plusieurs ordres de grandeurs en concentrations et en temps mettent en évidence différents régimes de structuration associés à différentes dynamiques d’adsorption pour les trois protéines de blé, de tournesol et de colza étudiées. Nous discutons également le rôle de la flexibilité des protéines dans ces différents régimes de structuration. / Challenges of public health and sustainable development trend to intensify the use of vegetables proteins, particularly in consumer goods sectors such as pharmaceutical, food and cosmetics industries. The recent overcome of technical limitation allows the industrial purification of vegetables proteins derived from meal made by vegetable oils production. These proteins are valuable as substitutes for saturated fats in structuring oils for human consumption. The lack of unsaturated vegetable oil texture can be reduced by these proteins acting as stabilizers and gelling agents in emulsions. We are interested in the functional properties of wheat, sunflower and rapeseed proteins, by volume and at interfaces. We have shown that sunflower protein gels with modulable elasticities are obtained by thermal denaturation. In addition, we studied the dynamics of protein film formation at fluid interfaces by combining measurements of tensiometry, dilatational viscoelasticity and ellipsometry. We highlight different structuring regimes and discuss the role of protein flexibility in this structuring.

Bibliography: leaves 138-158. / x, 158 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study identified and investigated the properties, functions and patterns of accumulation of prominent berry proteins associated with white wine haze. Detailed analysis was conducted on two PR-like proteins of V. vinifera, VVPR-4a and VVTL1. In vitro fungal growth inhibition assays suggested that berry PR-like proteins may play an important role in plant defence, particularly against fungal attack. Results of this study also have future implications for controlling the ripening process of grapes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1999

xxi, 299 leaves : ill. ; 25 cm / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1976) from the Dept. of Plant Physiology, University of Adelaide

Bibliography: leaves 138-158. This study identified and investigated the properties, functions and patterns of accumulation of prominent berry proteins associated with white wine haze. Detailed analysis was conducted on two PR-like proteins of V. vinifera, VVPR-4a and VVTL1. In vitro fungal growth inhibition assays suggested that berry PR-like proteins may play an important role in plant defence, particularly against fungal attack. Results of this study also have future implications for controlling the ripening process of grapes.