by Cheng Man Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 71-76). / Abstracts in English and Chinese. / Thesis committee --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vii / List of figures --- p.x / List of tables --- p.xi / Chapter Chapter 1: --- General introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.3 / Chapter 2.1 --- Nutritional quality of plant proteins --- p.3 / Chapter 2.2 --- Using traditional plant breeding method to enhance amino acid quality of plant proteins --- p.3 / Chapter 2.3 --- Molecular strategies to enhance amino acid quality of plant proteins --- p.4 / Chapter 2.3.1 --- Heterologous gene expression --- p.5 / Chapter 2.3.2 --- Protein sequence modification --- p.8 / Chapter 2.3.3 --- Modification of biosynthesis pathway --- p.10 / Chapter 2.3.4 --- Synthetic gene expression --- p.11 / Chapter 2.3.5 --- Homologous gene overexpression --- p.13 / Chapter 2.4 --- Arabidopsis --- p.14 / Chapter 2.4.1 --- Arabidopsis as a model plant --- p.14 / Chapter 2.4.2 --- Transformation methods --- p.14 / Chapter 2.4.2.1 --- Direct DNA uptake --- p.15 / Chapter 2.4.2.2 --- Agrobacterium-mediated transformation --- p.15 / Chapter 2.5 --- Winged Bean Lysine-Rich protein --- p.17 / Chapter 2.5.1 --- Identification of winged bean polypeptides rich in lysine --- p.17 / Chapter 2.5.2 --- Cloning of the lysine-rich protein gene --- p.17 / Chapter 2.5.3 --- Further characterization of the WBLRP gene --- p.18 / Chapter 2.6 --- Phaseolin --- p.19 / Chapter Chapter 3: --- Expression of LRP in transgenic Arabidopsis --- p.20 / Chapter 3.1 --- Introduction --- p.20 / Chapter 3.2 --- Materials and methods --- p.21 / Chapter 3.2.1 --- Targeting LRP to cytosol --- p.21 / Chapter 3.2.1.1 --- Chemicals --- p.21 / Chapter 3.2.1.2 --- Plant materials --- p.21 / Chapter 3.2.1.3 --- Bacterial strains --- p.22 / Chapter 3.2.1.4 --- Construction of chimeric LRP gene (pBILRP-1) --- p.22 / Chapter 3.2.1.4.1 --- PCR amplification of LRP --- p.22 / Chapter 3.2.1.4.2 --- Cloning of PCR-amplified LRP into vector pD3-8 --- p.26 / Chapter 3.2.1.4.3 --- Cloning of recombinant plasmid pLRP-1 into binary vector --- p.26 / Chapter 3.2.1.5 --- Transformation of Agrobacterium with pBILRP-1 --- p.27 / Chapter 3.2.1.6 --- Vacuum infiltration transformation of Arabidopsis --- p.28 / Chapter 3.2.1.7 --- Selection of transgenic plants --- p.29 / Chapter 3.2.1.8 --- GUS assay --- p.30 / Chapter 3.2.1.9 --- DNA isolation --- p.31 / Chapter 3.2.1.10 --- PCR amplification and detection of transgenes --- p.31 / Chapter 3.2.1.11 --- Southern blot hybridization --- p.31 / Chapter 3.2.1.12 --- RNA isolation --- p.32 / Chapter 3.2.1.13 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.32 / Chapter 3.2.1.14 --- Protein extraction and SDS-PAGE --- p.33 / Chapter 3.2.1.15 --- Protein sequencing --- p.33 / Chapter 3.2.1.16 --- Amino acid analysis --- p.34 / Chapter 3.2.2 --- Targeting LRP to protein bodies --- p.35 / Chapter 3.2.2.1 --- Chemicals --- p.35 / Chapter 3.2.2.2 --- Plant materials --- p.35 / Chapter 3.2.2.3 --- Bacterial strains --- p.35 / Chapter 3.2.2.4 --- Construction of chimeric LRP gene (pBILRP-2) --- p.35 / Chapter 3.2.2.4.1 --- Site-directed mutagenesis --- p.36 / Chapter 3.2.2.4.2 --- Cloning of the mutated phaseolin fragment into pBluescript --- p.36 / Chapter 3.2.2.4.3 --- PCR amplification of LRP --- p.39 / Chapter 3.2.2.4.4 --- Insertion of LRP into plasmid pBK/phas* --- p.39 / Chapter 3.2.2.4.5 --- Insertion of plasmid pLRP-2 into Agrobacterium binary vector --- p.41 / Chapter 3.2.2.5 --- Transformation of Agrobacterium with pBILRP-2 --- p.41 / Chapter 3.2.2.6 --- Vacuum infiltration transformation of Arabidopsis --- p.41 / Chapter 3.2.2.7 --- Selection of transgenic plants --- p.41 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- Targeting LRP to protein bodies --- p.42 / Chapter 3.3.1.1 --- Morphology of transgenic Arabidopsis --- p.42 / Chapter 3.3.1.2 --- Selection of transgenic plants --- p.42 / Chapter 3.3.2 --- Targeting LRP to cytosol --- p.46 / Chapter 3.3.2.1 --- Morphology of transgenic Arabidopsis --- p.46 / Chapter 3.3.2.2 --- Selection of transgenic plants --- p.46 / Chapter 3.3.2.3 --- Detection of GUS activity --- p.49 / Chapter 3.3.2.4 --- Integration of LRP transgene into Arabidopsis genome --- p.54 / Chapter 3.3.2.5 --- LRP transcript in transgenic Arabidopsis --- p.58 / Chapter 3.3.2.6 --- Stable accumulation of LRP in transgenic Arabidopsis --- p.61 / Chapter 3.3.2.7 --- Amino acid analysis of seed protein --- p.64 / Chapter Chapter 4: --- General discussion --- p.67 / Conclusion --- p.70 / References --- p.71

by Lee Juon Kiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-152). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xvii / List of graphs --- p.xviii / List of abbreviations --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.4 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.4 / Chapter 2.1.1 --- Physiological roles --- p.4 / Chapter 2.1.2 --- Molecular properties --- p.8 / Chapter 2.1.3 --- Biochemical properties --- p.9 / Chapter 2.1.4 --- Comparison to G-CSF of other specie --- p.10 / Chapter 2.1.5 --- Clinical application --- p.11 / Chapter 2.1.6 --- Economic value --- p.13 / Chapter 2.2 --- Expression systems producing recombinant hG-CSF --- p.15 / Chapter 2.2.1 --- Bacteria --- p.15 / Chapter 2.2.2 --- Yeasts --- p.17 / Chapter 2.2.3 --- Animal cell lines --- p.18 / Chapter 2.2.4 --- Transgenic animals --- p.19 / Chapter 2.2.5 --- Transgenic plants --- p.20 / Chapter 2.3 --- Plant as bioreactors --- p.21 / Chapter 2.3.1 --- Characteristics of using plant as bioreactors --- p.22 / Chapter 2.3.2 --- Transgenic plants producing hematopoietic growth factors --- p.24 / Chapter 2.3.2.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.24 / Chapter 2.3.2.2 --- Erythropoietin (Epo) --- p.26 / Chapter 2.3.3 --- Arabidopsis and tobacco as model plants --- p.27 / Chapter 2.3.3.1 --- Arabidopsis --- p.28 / Chapter 2.3.3.2 --- Tobacco --- p.28 / Chapter 2.3.4 --- Phaseolin and its regulatory sequences --- p.29 / Chapter 2.4 --- Plant transformation methods --- p.31 / Chapter 2.4.1 --- Agrobacterium-mediated transformation --- p.31 / Chapter 2.4.1.1 --- Tissue culture methods --- p.31 / Chapter 2.4.1.2 --- Non-tissue culture (In planta) methods --- p.32 / Chapter 2.4.2 --- Direct DNA uptake transformation --- p.33 / Chapter 2.4.2.1 --- Chemical methods --- p.33 / Chapter 2.4.2.2 --- Electrical methods --- p.34 / Chapter 2.4.2.3 --- Physical methods --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Chemicals --- p.37 / Chapter 3.3 --- Bacterial strains --- p.37 / Chapter 3.4 --- Chimeric gene construction --- p.37 / Chapter 3.4.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.41 / Chapter 3.4.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.44 / Chapter 3.4.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.47 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.50 / Chapter 3.4.5 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.51 / Chapter 3.5 --- Expression in Arabidopsis --- p.52 / Chapter 3.5.1 --- Agrobacterium GV3101/pMP90 transformation --- p.52 / Chapter 3.5.2 --- Arabidopsis transformation --- p.53 / Chapter 3.5.2.1 --- Plant materials --- p.53 / Chapter 3.5.2.2 --- Vacuum infiltration --- p.54 / Chapter 3.5.3 --- Screening of successful R1 transformants --- p.55 / Chapter 3.5.4 --- Screening of hemizygous and homozygous transgenic Arabidopsis --- p.56 / Chapter 3.5.5 --- GUS assay --- p.57 / Chapter 3.5.6 --- Genomic DNA extraction --- p.57 / Chapter 3.5.7 --- Southern blot analysis --- p.58 / Chapter 3.5.8 --- Total RNA extraction from developing siliques --- p.59 / Chapter 3.5.9 --- Northern blot analysis --- p.60 / Chapter 3.5.10 --- Protein extraction and Tricine SDS-PAGE --- p.61 / Chapter 3.5.11 --- Western blot analysis --- p.62 / Chapter 3.5.12 --- Functional analysis --- p.63 / Chapter 3.5.12.1 --- Culture ofNFS-60 cells --- p.64 / Chapter 3.5.12.2 --- MTT assay --- p.65 / Chapter 3.6 --- Expression in tobacco --- p.67 / Chapter 3.6.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.67 / Chapter 3.6.2 --- Tobacco transformation --- p.68 / Chapter 3.6.2.1 --- Plant materials --- p.68 / Chapter 3.6.2.2 --- Tobacco transformation using leaf-disc technique --- p.68 / Chapter 3.6.3 --- Regeneration of transgenic tobacco --- p.69 / Chapter 3.6.4 --- GUS assay --- p.70 / Chapter 3.6.5 --- Genomic DNA extraction --- p.70 / Chapter 3.6.6 --- Southern blot analysis --- p.70 / Chapter 3.6.7 --- Total RNA extraction from immature seeds --- p.70 / Chapter 3.6.8 --- Northern blot analysis --- p.71 / Chapter 3.6.9 --- Protein extraction and Tricine SDS-PAGE --- p.71 / Chapter 3.6.10 --- Western blot analysis --- p.71 / Chapter 3.6.11 --- Functional analysis --- p.71 / Chapter 3.6.11.1 --- Culture of NFS-60 cells --- p.72 / Chapter 3.6.11.2 --- MTT assay --- p.72 / Chapter Chapter 4: --- Results --- p.73 / Chapter 4.1 --- Chimeric gene construction --- p.73 / Chapter 4.1.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.73 / Chapter 4.1.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.75 / Chapter 4.1.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.77 / Chapter 4.1.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.79 / Chapter 4.2 --- Expression in Arabidopsis --- p.81 / Chapter 4.2.1 --- Agrobacterium GV3101/pMP90 transformation --- p.81 / Chapter 4.2.2 --- Arabidopsis transformation and screening of R1 transformants --- p.83 / Chapter 4.2.3 --- Screening of hemizygous transgenic R1 Arabidopsis --- p.84 / Chapter 4.2.4 --- Screening of homozygous transgenic R2 Arabidopsis --- p.86 / Chapter 4.2.5 --- GUS assay --- p.88 / Chapter 4.2.6 --- Genomic DNA extraction --- p.89 / Chapter 4.2.7 --- Southern blot analysis --- p.91 / Chapter 4.2.8 --- Total RNA extraction from developing siliques --- p.93 / Chapter 4.2.9 --- Northern blot analysis --- p.94 / Chapter 4.2.10 --- Protein extraction and Tricine SDS-PAGE --- p.96 / Chapter 4.2.11 --- Western blot analysis --- p.99 / Chapter 4.2.12 --- Functional analysis --- p.103 / Chapter 4.3 --- Expression in tobacco --- p.108 / Chapter 4.3.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.108 / Chapter 4.3.2 --- Tobacco transformation and regeneration of transformants --- p.109 / Chapter 4.3.3 --- GUS assay --- p.111 / Chapter 4.3.4 --- Genomic DNA extraction --- p.112 / Chapter 4.3.5 --- Southern blot analysis --- p.114 / Chapter 4.3.6 --- Total RNA extraction from immature seeds --- p.116 / Chapter 4.3.7 --- Northern blot analysis --- p.116 / Chapter 4.3.8 --- Protein extraction and Tricine SDS-PAGE --- p.118 / Chapter 4.3.9 --- Western blot analysis --- p.120 / Chapter 4.3.10 --- Functional analysis --- p.123 / Chapter Chapter 5: --- Discussion --- p.126 / Chapter 5.1 --- Introduction --- p.126 / Chapter 5.2 --- Successful in producing biologically active rhG-CSF from transgenic plants --- p.128 / Chapter 5.2.1 --- Production level --- p.129 / Chapter 5.2.2 --- O-glycosylation --- p.130 / Chapter 5.2.3 --- Phaseolin signal peptide --- p.131 / Chapter 5.2.4 --- Functional analysis --- p.131 / Chapter 5.3 --- Comparison of the productivity of other expression systems producing rhG-CSF --- p.132 / Chapter 5.4 --- Comparison of the productivity of plants producing different human proteins --- p.135 / Chapter 5.5 --- Future perspectives --- p.137 / Chapter Chapter 6: --- Conclusion --- p.138 / References --- p.139

Chi-shing Lo. / Thesis submitted in: November 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 119-138). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.v / Abstract --- p.vii / Abstract (Chinese version) --- p.ix / Table of contents --- p.xi / List of Figure --- p.xvii / List of Table --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- BIOLOGY OF TOXOPLASMA GONDII --- p.1 / Chapter 1.1.1 --- Life cycle of Toxoplasma gondii --- p.2 / Chapter (a) --- Tachyzoite --- p.3 / Chapter (b) --- Bradyzoite --- p.3 / Chapter 1.1.2 --- Genetics of Toxoplasma gondii --- p.4 / Chapter (a) --- Population genetics --- p.4 / Chapter (b) --- Molecular genetics --- p.5 / Chapter (c) --- Genome analysis --- p.7 / Chapter 1.1.3 --- Invasion --- p.8 / Chapter 1.1.4 --- Surface of Toxoplasma gondii --- p.9 / Chapter (a) --- Tachyzoite surface --- p.9 / Chapter (b) --- Bradyzoite surface --- p.11 / Chapter (c) --- Sporozite surface --- p.11 / Chapter (d) --- Glycoprotein antigens --- p.12 / Chapter 1.2 --- TREATMENT OF TOXOPLASMOSIS --- p.13 / Chapter 1.2.1 --- Chemotherapy --- p.13 / Chapter (a) --- Drug against metabolism and protein synthesis on nuclear genome --- p.13 / Chapter (b) --- Drug against other organelles --- p.14 / Chapter (c) --- Drug resistance --- p.15 / Chapter 1.2.2 --- Toxoplasma vaccine --- p.16 / Chapter (a) --- Mutant strains of Toxoplasma gondii as vaccine --- p.17 / Chapter (b) --- Subunit vaccine --- p.19 / Chapter (c) --- P30 as subunit vaccine --- p.20 / Chapter 1.3 --- AIM OF THE STUDY --- p.22 / Chapter Chapter 2 --- : Expression of P30 in Escherichia coli --- p.23 / Chapter 2.1 --- INTRODUCTION --- p.23 / Chapter 2.1.1 --- Why Escherichia coli? --- p.23 / Chapter 2.1.2 --- protein folding --- p.24 / Chapter 2.1.3 --- T7-based gene expression system --- p.25 / Chapter (a) --- Biology of T7 RNA polymerase --- p.26 / Chapter (b) --- pET translational vector --- p.26 / Chapter (c) --- Hislidine-tagged protein --- p.27 / Chapter (d) --- Host strain for expression --- p.28 / Chapter 2.2 --- MATERIALS --- p.29 / Chapter 2.2.1 --- Bactcrial strains --- p.29 / Chapter 2.2.2 --- Mouse strain --- p.29 / Chapter 2.2.3 --- Chemicals --- p.29 / Chapter 2.2.4 --- Nucleic acids --- p.30 / Chapter 2.2.5 --- Kit and reagents --- p.31 / Chapter 2.2.6 --- Antibodies --- p.31 / Chapter 2.2.7 --- Solutions --- p.32 / Chapter 2.2.8 --- Enzymes --- p.33 / Chapter 2.2.9 --- Sequencing primers --- p.33 / Chapter 2.3 --- METHODS --- p.34 / Chapter 2.3.1 --- Modification of P30 gene --- p.34 / Chapter (a) --- Preparation of recombinant plasmids，pBV220-ASP30PI and pBV220- SP30hisAPI --- p.36 / Chapter (b) --- Digestion of pBV220-ASP30PI and pBV220-SP30hisAPI with DraII and EcoRI --- p.37 / Chapter (c) --- Purification of DNA fragments from agarose gel --- p.37 / Chapter (d) --- Ligation of fragments of pBV220-ΔSP30PI and pBV220-SP30hisAPI --- p.38 / Chapter (e) --- Preparation of DH5α competent cells --- p.38 / Chapter (f) --- Transformation of recombinant pBV220-ΔSP30hisAPI --- p.38 / Chapter (g) --- Plasmid preparation of putative pBV220-ΔSP30API --- p.39 / Chapter (h) --- Plasmid preparation of pET-ΔSP30API --- p.39 / Chapter (i) --- Cycle sequencing reaction on putative plasmid pET-ASP30API --- p.40 / Chapter 2.3.2 --- Expression and Purification of his-tag P30 --- p.41 / Chapter (a) --- Expression profile of his-tag P30 production by IPTG induction --- p.41 / Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.41 / Chapter (c) --- Purification of his-tag P30 --- p.43 / Chapter (d) --- Bradford Protein Microassay (Bio-Rad) --- p.43 / Chapter 2.3.3 --- Characterization of his-tag P30 --- p.44 / Chapter (a) --- Western blot of induced bacterial lysate by monoclonal anti-his-tag antibody --- p.44 / Chapter (b) --- Western blot of his-tag with seropositive sera of mice，rabbit and human --- p.46 / Chapter (c) --- Enterokinase digestion of his-tag P30 --- p.46 / Chapter (d) --- N'terminal amino acid sequencing of pure and enterokinase-cut his-tag --- p.47 / Chapter (e) --- Western blot of T. gondii lysate with antiserum against his-tag P30 --- p.47 / Chapter 2.4 --- RESULTS --- p.49 / Chapter 2.4.1 --- Modification of P30 gene --- p.49 / Chapter 2.4.2 --- "Expression, purification and characteriziation of his-tag P30 in bacteria" --- p.54 / Chapter 2.5 --- DISCUSSIONS --- p.64 / Chapter 2.5.1 --- Modification of P30 gene --- p.64 / Chapter 2.5.2 --- Expression and purification of his-tag P30 --- p.66 / Chapter 2.5.3 --- Characterization of his-tag P30 --- p.67 / Chapter Chapter 3 --- : Expression of P30 in Arabidopsis thalina --- p.69 / Chapter 3.1 --- INTRODUCTION --- p.69 / Chapter 3.1.1 --- Why Arabidopsis thalina? --- p.69 / Chapter 3.1.2 --- In planta transformation --- p.70 / Chapter 3.1.3 --- Transgenic plants as vacine production systems --- p.72 / Chapter (a) --- Stable expression of E. coli heat-liable enterotoxin B subunit and cholera-toxin B subunit --- p.73 / Chapter (b) --- Stable expression of Hepatitis B surface antigen (HBsAg) --- p.74 / Chapter (c) --- Stable expression of Norwalk virus capsid protein --- p.75 / Chapter (d) --- Transient expression by tobacco mosaic virus --- p.75 / Chapter (e) --- Transient expression by Cowpea mosaic virus capsid protein fusion --- p.76 / Chapter 3.2 --- MATERIALS --- p.77 / Chapter 3.2.1 --- Bacterial strains --- p.77 / Chapter 3.2.2 --- Arabidopsis strains --- p.77 / Chapter 3.2.3 --- Chemicals --- p.77 / Chapter 3.2.4 --- Nucleic acids --- p.78 / Chapter 3.2.5 --- Kit and reagents --- p.78 / Chapter 3.2.6 --- Solutions --- p.79 / Chapter 3.2.7 --- Enzymes and buffers --- p.81 / Chapter 3.2.8 --- PCR and Sequencing primers --- p.81 / Chapter 3.3 --- METHODS --- p.82 / Chapter 3.3.1 --- Construction of V7-ASP30API --- p.82 / Chapter 3.3.2 --- Agrobacterium-mediated transformation of Arabidopsis by vacuum infiltration --- p.83 / Chapter (a) --- Preparation of electro-competent Agrobacterium --- p.83 / Chapter (b) --- Transformation of electro-competent Agrobacterium with V7- ASP30API --- p.84 / Chapter (c) --- Plasmid preparation of V7-ASP30API from transformed Agrobacterium --- p.84 / Chapter (d) --- Vacuum infiltration --- p.85 / Chapter 3.3.3 --- Screening of homozygous transgenic plants --- p.86 / Chapter 3.3.4 --- Detecton of transgene P30 in genomic DNA of transgenic plants --- p.87 / Chapter (a) --- Preparation of DIG-labelled probe --- p.87 / Chapter (b) --- Estimation the yield of DIG-labelled probe --- p.88 / Chapter (c) --- Extraction of genomic DNA from transgenic plants --- p.88 / Chapter (d) --- Restriction digestion of genomic DNA with EcoRI and HindIII --- p.89 / Chapter (e) --- DNA transfer from gel to nylon membrane --- p.89 / Chapter (f) --- Detection of hybridized DIG-labelled probe on membrane/ blot --- p.90 / Chapter (g) --- PCR on genomic DNA of transgenic plants with specific primers --- p.91 / Chapter 3.3.5 --- Analysis of transgene RNA expression in transgenic plants --- p.91 / Chapter (a) --- Extraction of total RNA from plants --- p.91 / Chapter (b) --- Northern blot on RNA of F2 transgenic plants --- p.92 / Chapter (c) --- RT-PCR on RNA of F3 transgenic plants --- p.93 / Chapter 3.3.6 --- Detection of his-tag P30 protein in F3 transgenic plants --- p.93 / Chapter 3.4 --- RESULTS --- p.95 / Chapter 3.4.1 --- Construction of V7-ASP30API --- p.95 / Chapter 3.4.2 --- Screening of homozygous transgenic plants --- p.99 / Chapter 3.4.3 --- Molecular analysis of transgene P30 in transgenic plants --- p.101 / Chapter 3.5 --- DISCUSSIONS --- p.108 / Chapter 3.5.1 --- Construction and optimization of expression construct --- p.108 / Chapter 3.5.2 --- Screening and selection of homozyous transgenic plants --- p.109 / Chapter 3.5.3 --- Analysis of transgenic plants --- p.110 / Chapter Chapter 4 : --- General Discussions --- p.112 / Chapter 4.1 --- Significances of studying Toxoplasma gondii --- p.112 / Chapter 4.2 --- Expression of recombinant P30 in prokaryotic systems --- p.113 / Chapter 4.2 --- Expression of recombinant P30 in eukaryotic systems --- p.115 / Reference --- p.119

by Ng Wang Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Table of Contents --- p.xv / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global picture --- p.3 / Chapter 2.1.2 --- Malaria mechanics --- p.4 / Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4 / Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5 / Chapter 2.2.1 --- Antimalarial drugs --- p.5 / Chapter 2.2.2 --- Drug resistance --- p.6 / Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7 / Chapter 2.3.1 --- Malarial vaccine developments --- p.7 / Chapter 2.3.2 --- Transmission blocking vaccines --- p.7 / Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9 / Chapter 2.3.4 --- Blood stage vaccines --- p.10 / Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11 / Chapter 2.5 --- Plants as bioreactors --- p.12 / Chapter 2.5.1 --- Products of transgenic plants --- p.13 / Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14 / Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15 / Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15 / Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16 / Chapter 2.7.1 --- Arabidopsis --- p.16 / Chapter 2.7.2 --- Tobacco --- p.17 / Chapter 2.8 --- Transformation methods --- p.17 / Chapter 2.8.1 --- Direct DNA uptake --- p.17 / Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17 / Chapter 2.8.1.2 --- Biolistic transformation --- p.18 / Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18 / Chapter 2.8.2.1 --- Leaf-disc technique --- p.18 / Chapter 2.8.2.2 --- In planta transformation --- p.19 / Chapter 2.9 --- Phaseolin --- p.20 / Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21 / Chapter 2.11 --- Aims of study and hypotheses --- p.22 / Chapter Chapter 3: --- Materials and Methods --- p.24 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Chemicals --- p.24 / Chapter 3.3 --- Expression in tobacco system --- p.24 / Chapter 3.3.1 --- Plant materials --- p.24 / Chapter 3.3.2 --- Bacterial strains --- p.25 / Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25 / Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26 / Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30 / Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30 / Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33 / Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34 / Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35 / Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36 / Chapter 3.3.8 --- GUS assay --- p.37 / Chapter 3.3.9 --- Genomic DNA isolation --- p.37 / Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38 / Chapter 3.3.11 --- Southern blot analysis --- p.38 / Chapter 3.3.12 --- Total seeds RNA isolation --- p.39 / Chapter 3.3.13 --- RT-PCR --- p.39 / Chapter 3.3.14 --- Northern blot analysis --- p.40 / Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40 / Chapter 3.3.16 --- Western blot analysis --- p.41 / Chapter 3.4 --- Expression in Arabidopsis system --- p.42 / Chapter 3.4.1 --- Plant materials --- p.42 / Chapter 3.4.2 --- Bacterial strains --- p.42 / Chapter 3.4.3 --- Chimeric gene construction --- p.42 / Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43 / Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45 / Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45 / Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49 / Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49 / Chapter 3.4.7 --- Arabidopsis Transformation --- p.49 / Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50 / Chapter 3.4.9 --- Selection of successful transformants --- p.51 / Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51 / Chapter 3.4.11 --- GUS assay --- p.52 / Chapter 3.4.12 --- Genomic DNA isolation --- p.52 / Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52 / Chapter 3.4.14 --- Southern Blot analysis --- p.52 / Chapter 3.4.15 --- Total siliques RNA isolation --- p.53 / Chapter 3.4.16 --- RT-PCR --- p.53 / Chapter 3.4.17 --- Northern blot analysis --- p.53 / Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54 / Chapter 3.4.18 --- Western blot analysis --- p.54 / Chapter 3.5 --- In vitro transcription and translation --- p.54 / Chapter 3.5.1 --- In vitro transcription --- p.54 / Chapter 3.5.2 --- In vitro translation --- p.55 / Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56 / Chapter 3.6.1 --- Chimeric gene constructs --- p.56 / Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61 / Chapter Chapter 4: --- Results --- p.63 / Chapter 4.1 --- Tobacco system --- p.63 / Chapter 4.1.1 --- Chimeric gene constructs --- p.63 / Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65 / Chapter 4.1.3 --- GUS activity assay --- p.67 / Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68 / Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68 / Chapter 4.1.4.2 --- Southern blot analysis --- p.70 / Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72 / Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72 / Chapter 4.1.5.2 --- Northern blot analysis --- p.75 / Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76 / Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78 / Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78 / Chapter 4.1.7.2 --- Western blot analysis --- p.78 / Chapter 4.2 --- Arabidopsis system --- p.83 / Chapter 4.2.1 --- Chimeric gene constructs --- p.83 / Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85 / Chapter 4.2.3 --- Selection of transgenic plants --- p.87 / Chapter 4.2.4 --- Assay of GUS activity --- p.91 / Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92 / Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92 / Chapter 4.2.5.2 --- Southern blot analysis --- p.96 / Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99 / Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99 / Chapter 4.2.6.2 --- Northern blot analysis --- p.106 / Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107 / Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108 / Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108 / Chapter 4.2.8.2 --- Western blot analysis --- p.108 / Chapter 4.3 --- In vitro transcription and translation --- p.112 / Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115 / Chapter Chapter 5: --- Discussion and Future perspectives --- p.117 / Chapter 5.1 --- Failure in detecting transgene expression --- p.117 / Chapter 5.2 --- Poor transgene expression --- p.120 / Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120 / Chapter 5.2.2 --- AT-richness --- p.121 / Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123 / Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125 / Chapter 5.2.5 --- Codon usage --- p.126 / Chapter 5.3 --- Future perspectives --- p.127 / Chapter Chapter 6: --- Conclusion --- p.129 / References --- p.131

[Truncated abstract] Mitochondria play an essential role in diverse metabolic pathways in plants. Their primary roles are the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons from reduced NAD+ to oxygen via the electron transport chain. Plant mitochondria also contain nonphosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX), type II NAD(P)H dehydrogenases (NDHs) and uncoupling proteins (UCPs). Each of these components bypasses energy conservation by either circumventing the formation or utilization of the electrochemical proton gradient, and each is encoded by a small gene family in Arabidopsis. It is proposed that the alterative pathways are likely to be involved in balancing cellular redox and energy status and in minimizing the production of ROS generated by over-reduction of basal respiratory chain components. Furthermore the alternative respiratory pathways are thought to play a role in plant responses to stress. In this study a transcriptomic approach was taken to investigate the role of the alternative respiratory pathways in Arabidopsis, with a focus on elucidating the role and regulation of the AOX gene family. Analysis of the expression of the five AOX genes in Arabidopsis over development and in a range of tissues revealed a unique spatiotemporal expression pattern for each gene. Expression profiling using quantitative RT-PCR, MPSS and microarrays detected an abundance of the AOX1a transcript throughout the plant and over development. The expression patterns of other AOX genes provide insight into their putative roles, AOX1b was expressed predominantly in the flower, AOX1d was particularly abundant in senescing leaves and AOX2 expression was only observed in the seed.