Leung Chun-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 113-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgment --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of abbreviations --- p.xiv / List of figures --- p.xvii / List of tables --- p.xix / Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Artificial sweeteners --- p.3 / Chapter 1.2.1 --- SACCHARIN --- p.3 / Chapter 1.2.2 --- cyclamate --- p.4 / Chapter 1.2.3 --- Aspartame --- p.4 / Chapter 1.2.4 --- acesulfame-k --- p.5 / Chapter 1.2.5 --- SUCRALOSE --- p.5 / Chapter 1.3 --- natural sweet plant proteins --- p.7 / Chapter 1.3.1 --- THAUMATIN --- p.7 / Chapter 1.3.2 --- MONELLIN --- p.10 / Chapter 1.3.3 --- CURCULIN --- p.11 / Chapter 1.3.4 --- PENTADIN AND BRAZZEIN --- p.11 / Chapter 1.3.5 --- MIRACULIN --- p.12 / Chapter 1.3.6 --- MABINLIN --- p.12 / Chapter 1.4 --- Genetic Engineering of Sweet Plant Protein --- p.19 / Chapter 1.4.1 --- biotechnological studies on thaumatin --- p.20 / Chapter 1.4.1.1 --- Protein modification and sweetness --- p.20 / Chapter 1.4.1.2 --- Transgenic expression in microbes --- p.21 / Chapter 1.4.1.3 --- Transgenic expression in higher plants --- p.23 / Chapter 1.4.2 --- BIOTECHNOLOGICAL STUDIES ON MONELLIN --- p.24 / Chapter 1.4.2.1 --- Gene modification and transgenic expression in microbes --- p.24 / Chapter 1.4.2.2 --- Transgenic expression in plants --- p.25 / Chapter 1.4.3 --- TRANSGENIC EXPRESSION OF MABINLIN IN PLANTS --- p.26 / Chapter 1.5 --- phaseolin and its regulatory sequences --- p.27 / Chapter 1.6 --- ARABIDOPSIS --- p.29 / Chapter 1.6.1 --- ARABIDOPSIS THALIANA as a model plant --- p.29 / Chapter 1.6.2 --- Transformation methods --- p.29 / Chapter 1.6.2.1 --- Direct DNA uptake --- p.30 / Chapter 1.6.2.2 --- Agrobacterium-mediated transformation --- p.31 / Chapter 1.6.2.3 --- In planta transformation --- p.31 / Chapter 2 --- GENKRAL INTRODUTION AND HYPOTHESIS --- p.22 / Chapter 2.1 --- General Introduction --- p.33 / Chapter 2.2 --- Hypothesis --- p.34 / Chapter 3 --- MOLECULAR STUDIES ON SWEET PROTEIN MARINLIN : THERMAL STABILITY --- p.28 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Materials --- p.40 / Chapter 3.2.1 --- laboratory wares --- p.40 / Chapter 3.2.2 --- Equipments --- p.40 / Chapter 3.2.3 --- Chemicals --- p.40 / Chapter 3.2.4 --- commerical kits --- p.41 / Chapter 3.2.5 --- DNA primers --- p.42 / Chapter 3.2.6 --- DNA plasmids --- p.43 / Chapter 3.2.7 --- bacterial strains --- p.43 / Chapter 3.2.8 --- Plant materials --- p.44 / Chapter 3.2.9 --- Protein and Antibody --- p.44 / Chapter 3.3 --- Methods --- p.45 / Chapter 3.3.1 --- Transformation of Arabidopsis with mbliii and mbli genes --- p.45 / Chapter 3.3.1.1 --- Construction of mutant MBLIII and MBLI genes containing single codon mutation by megaprimer PCR --- p.45 / Chapter 3.3.1.2 --- Cloning of PCR-amplified MBLIII and MBLI cDNAs into vector pD3-8 --- p.48 / Chapter 3.3.1.3 --- In vitro site-directed mutagensis (for the construction of MBLIII and MBLI cDNAs containing single codon mutation) --- p.49 / Chapter 3.3.1.4 --- Cloning of the wild-type and mutated MBLIII and MBLI cDNA into vector pTZ / phas --- p.53 / Chapter 3.3.1.5 --- Confirmation of sequence fidelity and mutated codon in MBLIII and MBLI cDNA by DNA sequencing --- p.53 / Chapter 3.3.1.6 --- Transfer of wild-type MBLIII and MBLI cDNA flanked by phaseolin regulatory sequence into Agrobacterium binary vector --- p.55 / Chapter 3.3.1.7 --- Transformation of Agrobacterium with pBI / phas / MBLIII and pBI / phas / MBLI chimeric gene constructs --- p.57 / Chapter 3.3.1.8 --- Vacuum infiltration transformation of A rabidopsis --- p.58 / Chapter 3.3.1.9 --- Screening of homozygous transgenic Arabidopsis --- p.59 / Chapter 3.3.2 --- Expression analysis of MBLIII transgene --- p.61 / Chapter 3.3.2.1 --- GUS assay of transgenic plants --- p.61 / Chapter 3.3.2.2 --- Genomic DNA isolation from transgenic plants --- p.61 / Chapter 3.3.2.3 --- PCR amplification of transgene --- p.62 / Chapter 3.3.2.4 --- Total RNA isolation from transgenic Arabidopsis --- p.63 / Chapter 3.3.2.5 --- RT-PCR of total RNA from transgenic Arabidopsis --- p.64 / Chapter 3.3.2.6 --- Verification of the presence of mutagenic site and the fidelity of RNA transcript from transgenic Arabidopsis --- p.65 / Chapter 3.3.2.7 --- Protein extraction and tricine SDS-PAGE of putative transgenic protein from Arabidopsis --- p.65 / Chapter 3.3.2.8 --- N-terminal amino acid sequencing --- p.66 / Chapter 3.3.2.9 --- Isoelectric precipitation of MBL --- p.67 / Chapter 3.3.2.10 --- Production of polyclonal antibody against purified MBL --- p.67 / Chapter 3.3.2.11 --- Western-blotting and immunodectection of Arabidopsis protein by anti-MBL polyclonal antibody --- p.69 / Chapter 3.4 --- results & discussion --- p.71 / Chapter 3.4.1 --- Site-specific mutations of Arginine residue in mbliii cdna and glutamine in mbli cdna --- p.71 / Chapter 3.4.1.1 --- Megaprimer PCR --- p.71 / Chapter 3.4.1.2 --- Cloning into the seed-specific expression vector pD38 --- p.74 / Chapter 3.4.1.3 --- In vitro site-directed mutagenesis --- p.76 / Chapter 3.4.2 --- Construction of plant expression vectors containing chimeric MBLIII and MBLI --- p.80 / Chapter 3.4.2.1 --- Cloning of MBLIII and MBLI cDNAs into the seed-specific expression vector pTZ / phas --- p.80 / Chapter 3.4.2.2 --- Cloning into the plant expression vector pBI121 --- p.83 / Chapter 3.4.3 --- Generation of homozygous transgenic Arabidopsis --- p.84 / Chapter 3.4.3.1 --- Screening of transgenic R1 Arabidopsis --- p.84 / Chapter 3.4.3.2 --- Screening of transgenic R2 plants --- p.86 / Chapter 3.4.3.3 --- Screening of homozygous R3 transgenic plants --- p.88 / Chapter 3.4.4 --- Detection of MBLIII transgene in Arabidopsis --- p.89 / Chapter 3.4.4.1 --- Gus Assay --- p.89 / Chapter 3.4.4.2 --- Detection of transgene integration --- p.90 / Chapter 3.4.5 --- DETECTION of MBLIII TRANSCRIPT IN TRANSGENIC arabidopsis --- p.92 / Chapter 3.4.5.1 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.92 / Chapter 3.4.5.2 --- Verification of the presence of the mutant codon and sequence fidelity of the RT-PCR product --- p.94 / Chapter 3.4.6 --- DETECTION OF MBL III PROTEIN IN TRANSGENIC arabidopsis --- p.97 / Chapter 3.4.6.1 --- Expression of MBL protein --- p.97 / Chapter 3.4.6.2 --- Isoelectric precipitation --- p.101 / Chapter 3.4.6.3 --- Assay of titers and quality of primary polyclonal antibody against purified MBL protein --- p.103 / Chapter 3.4.6.4 --- Western blot / Immunodetection --- p.106 / Chapter 4 --- GENERAL DISCUSSION --- p.109 / Conclusion --- p.112 / References --- p.113

Fung Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- An overview of the secretory pathway in eukaryotic cells --- p.2 / Chapter 1.2 --- The secretory pathway in plants --- p.4 / Chapter 1.2.1 --- Plant cells contain two functionally distinct vacuoles --- p.4 / Chapter 1.2.2 --- Three vesicular pathways to two vacuole --- p.6 / Chapter 1.2.3 --- Transport vesicles in the three vesicular pathways --- p.9 / Chapter 1.2.4 --- Vacuolar sorting determinants (VSDs) --- p.10 / Chapter 1.2.5 --- Vacuolar sorting receptors (VSRs) --- p.12 / Chapter 1.3 --- The PSVs in mature seeds --- p.15 / Chapter 1.3.1 --- Biogenesis of PSV --- p.15 / Chapter 1.3.2 --- The two chimeric integral membrane reporters --- p.16 / Chapter 1.3.3 --- Subcellular localization of the two chimeric integral membrane reporters in PSVs of mature tobacco seeds --- p.17 / Chapter 1.4 --- Project objectives --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Construction of the YFP-BP-80 and the YFP- a -TIP reporters --- p.21 / Chapter 2.1.1 --- The pYFP-BP-80-K construct --- p.21 / Chapter 2.1.2 --- The pYFP- a -TIP-K construct --- p.22 / Chapter 2.2 --- Construction of GFP-RMR reporter --- p.23 / Chapter 2.2.1 --- Cloning of pGFP-RMR --- p.23 / Chapter 2.2.2 --- Cloning of pGFP-RMR-K --- p.23 / Chapter 2.3 --- Construction of pGONST1-YFP construct --- p.26 / Chapter 2.3.1 --- The pGONSTl-YFP construct --- p.26 / Chapter 2.4 --- Transformation of Agrobacterium by electroporation --- p.27 / Chapter 2.5 --- Tobacco transformation and selection --- p.28 / Chapter 2.5.1 --- Plant materials --- p.28 / Chapter 2.5.2 --- Tobacco transformation --- p.28 / Chapter 2.6 --- Screening of transgenic tobacco plants expressing YFP fusion proteins --- p.30 / Chapter 2.6.1 --- Kanamycin screening --- p.30 / Chapter 2.6.2 --- Extraction of genomic DNA from leaves --- p.30 / Chapter 2.6.3 --- PCR of genomic DNA --- p.31 / Chapter 2.7 --- Southern blot analysis of genomic DNA --- p.32 / Chapter 2.8 --- Western blot analysis of transgenic tobacco plants --- p.33 / Chapter 2.8.1 --- Extraction of total protein from tobacco leaves or seeds --- p.33 / Chapter 2.8.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.9 --- Confocal immunofluorescence studies --- p.35 / Chapter 2.9.1 --- Preparation of sections --- p.35 / Chapter 2.9.2 --- Single labeling --- p.35 / Chapter 2.9.3 --- Double labeling with one polyclonal and one monoclonal antibodies --- p.36 / Chapter 2.9.4 --- Double labeling with two polyclonal antibodies --- p.36 / Chapter 2.9.5 --- Collection of images --- p.37 / Chapter 2.10 --- Chemicals --- p.38 / Chapter 2.11 --- Primers --- p.38 / Chapter 2.12 --- Bacterial strain --- p.38 / Chapter 2.13 --- Antibodies --- p.39 / Chapter 2.14 --- Growing condition of transgenic plants and determining the developmental stage of tobacco flowers --- p.39 / Chapter Chapter 3 --- Results --- p.41 / Chapter 3.1 --- Generation of transgenic tobacco plants --- p.42 / Chapter 3.2 --- PCR screening of transgenic tobacco plants --- p.46 / Chapter 3.3 --- Southern blot analysis --- p.48 / Chapter 3.4 --- Detection of the YFP fusion proteins in transgenic tobacco plants by western blot analysis --- p.50 / Chapter 3.4.1 --- Detection of the YFP fusion proteins in leaves --- p.50 / Chapter 3.4.2 --- Western blot analysis of vegetative tissues --- p.57 / Chapter 3.4.3 --- Western blot analysis of mature seeds --- p.59 / Chapter 3.5 --- Confocal immunofluorescence studies --- p.61 / Chapter 3.5.1 --- Detection of YFP signals in root tip cells --- p.61 / Chapter 3.5.2 --- Detection of YFP signals in developing seeds --- p.65 / Chapter 3.5.3 --- Subcellular localization of the YFP fusion proteins in mature seeds --- p.67 / Chapter Chapter 4 --- Discussion --- p.72 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.77 / Chapter 5.1 --- Summary --- p.78 / Chapter 5.1.1 --- Generation of transgenic tobacco plants expressing the YFP fusion proteins --- p.78 / Chapter 5.1.2 --- Full-length fusion proteins and cleaved soluble YFP were detected in vegetative tissues --- p.79 / Chapter 5.1.3 --- Only cleaved soluble YFP was detected in mature seeds --- p.79 / Chapter 5.1.4 --- The two fusion proteins might localized in different compartments in developing seeds --- p.79 / Chapter 5.1.5 --- Both fusion proteins were localized within the PSVs of mature seeds --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / References --- p.83

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

Chiao Ying Ann. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 127-140). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VII / Abbreviations --- p.IX / Abbreviation of chemicals --- p.XI / Table of Contents --- p.XII / List of figures and tables --- p.XVIII / Chapter Chapter 1. --- Literature review / Chapter 1.1 --- Significances of manipulation of nitrogen sink-source relationship --- p.1 / Chapter 1.2 --- Nitrogen sink-source relationship in plants --- p.2 / Chapter 1.3 --- Aspartate family amino acid metabolism --- p.5 / Chapter 1.3.1 --- Asparagine metabolism --- p.9 / Chapter 1.3.1.1 --- "Asparagine synthetase (AS, EC 6.3.5.4)" --- p.9 / Chapter 1.3.1.2 --- "Asparaginase (ANS, EC 3.5.1.1)" --- p.10 / Chapter 1.3.2 --- Metabolism of aspartate-derived essential amino acids --- p.10 / Chapter 1.3.2.1 --- "Aspartate kinase (AK, EC 2.7.2.4)" --- p.10 / Chapter 1.3.2.2 --- "Homoserine dehydrogenase (HSD, EC 1.1.1.3)" --- p.12 / Chapter 1.3.2.3 --- "Dihydrodipicolinate synthase (DHPS, EC 4.2.1.52)" --- p.13 / Chapter 1.3.2.4 --- "Lysine a-ketoglutarate reductase (LKR, EC 1.5.1.7)" --- p.14 / Chapter 1.3.2.5 --- "Threonine synthase (TS, EC 4.2.3.1)" --- p.15 / Chapter 1.3.2.6 --- Cystathionine γ-synthase (CGS，EC 2.5.1.48) --- p.16 / Chapter 1.3.2.7 --- Threonine deaminase (TD，EC 4.3.1.19) --- p.17 / Chapter 1.4 --- Previous attempts to manipulate seed protein quantity and quality --- p.18 / Chapter 1.4.1 --- Enhancement of amino acids transported from source to sink --- p.18 / Chapter 1.4.2 --- Redirection of metabolic pathways to increase target amino acids --- p.19 / Chapter 1.4.2.1 --- Production of aspartate by Aspartate Aminotransferase (AAT) --- p.24 / Chapter 1.4.2.2 --- Deregulation of AK to increase the common substrate for all essential aspartate family amino acids --- p.25 / Chapter 1.4.2.3 --- Inhibition of TS and enhancement of CGS to increase Met biosynthesis --- p.25 / Chapter 1.4.2.3.1 --- Inhibition of TS --- p.26 / Chapter 1.4.2.3.2 --- Enhancement of CGS --- p.26 / Chapter 1.4.2.4 --- Deregulation of DHPS and reduction of lysine catabolism to increase lysine content --- p.27 / Chapter 1.4.2.4.1 --- Deregulation of DHPS --- p.28 / Chapter 1.4.2.4.2 --- Reduction of Lys catabolism --- p.29 / Chapter 1.4.2.3.3 --- Deregulation of DHPS and reduction of LKR --- p.29 / Chapter 1.4.3 --- Expression of seed storage proteins to entrap the free amino acids --- p.30 / Chapter 1.5 --- Expression of multiple transgenes in plants --- p.34 / Chapter 1.5.1 --- Significance of multiple genes manipulation in seed quality improvement --- p.34 / Chapter 1.5.2 --- Difficulties in introduction of multiple genes into plant genomes --- p.34 / Chapter 1.5.3 --- Recent advances in introduction of multiple genes into plant genome --- p.35 / Chapter 1.6 --- Global nitrogen regulators in plants --- p.36 / Chapter 1.6.1 --- Global regulation of nitrogen metabolism --- p.36 / Chapter 1.6.2 --- General amino acid control by GCN system --- p.38 / Chapter 1.6.3 --- General amino acid control in plants --- p.39 / Chapter 1.6.4 --- GCN system in plants --- p.41 / Chapter 1.7 --- Hypothesis and specific objectives of this study --- p.42 / Chapter Chapter 2 --- Materials and methods --- p.46 / Chapter 2.1 --- Materials --- p.46 / Chapter 2.1.1 --- "Vectors, bacterial strains and plants" --- p.46 / Chapter 2.1.2 --- Chemicals and reagents used --- p.49 / Chapter 2.1.3 --- "Buffer, solution, gel and medium" --- p.49 / Chapter 2.1.4 --- Commercial kits used --- p.49 / Chapter 2.1.5 --- Equipments and facilities used --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Molecular techniques --- p.50 / Chapter 2.2.1.1 --- DNA gel electrophoresis --- p.59 / Chapter 2.2.1.2 --- PCR technique --- p.50 / Chapter 2.2.1.3 --- Restriction digestion --- p.50 / Chapter 2.2.1.4 --- Ligation (for sticky-end ligation) --- p.51 / Chapter 2.2.1.5 --- DNA purification --- p.51 / Chapter 2.2.1.6 --- DNA sequencing --- p.51 / Chapter 2.2.1.7 --- Transformation of competent E. coli cells --- p.52 / Chapter 2.2.1.8 --- Preparation of plasmid from bacterial cells --- p.53 / Chapter 2.2.1.9 --- Transformation of competent Agrobacterium tumefaciens cells --- p.53 / Chapter 2.2.1.10 --- DNA extraction from plant tissue (Small-scale) --- p.54 / Chapter 2.2.1.11 --- RNA extraction from plant tissue --- p.55 / Chapter 2.2.2 --- Growth conditions of A. thaliana --- p.55 / Chapter 2.2.2.1 --- Surface sterilization of A. thaliana seeds --- p.55 / Chapter 2.2.2.2 --- Growing A. thaliana --- p.55 / Chapter 2.2.3 --- Characterization of transgenic A. thaliana with altered sink-source relationship --- p.57 / Chapter 2.2.3.1. --- Determination of amino acid contents in seeds --- p.57 / Chapter 2.2.3.2. --- Expression study of developing siliques of transgenic lines --- p.58 / Chapter 2.2.3.2.1 --- Tagging siliques of different developmental stages --- p.58 / Chapter 2.2.3.2.2 --- Extraction of silique RNA --- p.58 / Chapter 2.2.3.2.3 --- cDNA synthesis --- p.58 / Chapter 2.2.3.2.4 --- Real-time PCR --- p.59 / Chapter 2.2.4 --- Characterization of transgenic A. thaliana overexpressing GCN2 --- p.60 / Chapter 2.2.4.1 --- Gene expression study of vegetative tissues by real-time PCR --- p.60 / Chapter 2.2.4.2 --- Gene expression study of developing siliques by real-time PCR --- p.61 / Chapter 2.2.5 --- Making transgenic A. thaliana --- p.61 / Chapter 2.2.5.1 --- Cloning of multigene construct --- p.61 / Chapter 2.2.5.1.1 --- Subcloning of target genes into donor vectors --- p.61 / Chapter 2.2.5.1.1.1 --- Cloning of LRP into donor vector VS --- p.61 / Chapter 2.2.5.1.1.2 --- Cloning of dapA into donor vector SV --- p.64 / Chapter 2.2.5.1.1.3 --- Cloning of ansB into donor vector VS --- p.67 / Chapter 2.2.5.1.1.4 --- Cloning of antisense LKR fragment into donor vector SV --- p.70 / Chapter 2.2.5.1.2 --- Preparation of phosphorylated linkers --- p.73 / Chapter 2.2.5.1.3 --- Introduction of target genes to acceptor vector --- p.73 / Chapter 2.2.5.2 --- Agrobacterium-mediated transformation of A. thaliana via Vacuum infiltration --- p.78 / Chapter 2.2.5.3 --- Screening of transformants --- p.79 / Chapter Chapter 3. --- Results --- p.80 / Chapter 3.1 --- Characterization of transgenic lines with altered sink-source relationship --- p.80 / Chapter 3.1.1 --- Amino acid analysis of mature seeds of transgenic lines --- p.80 / Chapter 3.1.1.1 --- Aspartate family amino acids levels remain steady in seeds of transgenic plants --- p.83 / Chapter 3.1.1.2 --- Increase in seed Met content in Met-rich protein expressing transgenic plants --- p.85 / Chapter 3.1.1.3 --- Increase in seed Lys content in phas-dapA/phas-LRP transgenic plants --- p.87 / Chapter 3.1.2 --- Gene expression study of transgenic line --- p.89 / Chapter 3.1.2.1 --- Down-regulation of akthr1 and akthr2 in transgenic plants with altered N sink-source relationship --- p.89 / Chapter 3.1.2.2 --- Down regulation of GCN2 in transgenic plants with altered N sink-source relationship --- p.90 / Chapter 3.1.2.4 --- Expression study of other genes in aspartate family pathway --- p.90 / Chapter 3.2 --- Characterization of GCN2 overexpressing line --- p.93 / Chapter 3.2.1 --- Gene expression study of seedlings of GCN2 overexpressing plants --- p.93 / Chapter 3.2.1.1 --- Increased GCN2 expression by azaserine treatment --- p.93 / Chapter 3.2.1.2 --- Increased akthrl and akthr2 expression in GCN2 overexpressing plants --- p.96 / Chapter 3.2.1.3 --- Expression study of other genes in aspartate family pathway --- p.96 / Chapter 3.2.2 --- Gene expression study of GCN2 overexpressing plants during seed development --- p.98 / Chapter 3.3 --- Construction of transgenic plants by multigene assembly system --- p.100 / Chapter 3.3.1 --- Successful construction of recombinant plasmid carrying four target genes --- p.100 / Chapter 3.3.2 --- Transformation of A. thaliana with multigene vector --- p.103 / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- Characterization of transgenic plants with altered sink-source relationship of aspartate family amino acid metabolism --- p.104 / Chapter 4.1.1 --- Total content of aspartate family amino acids remains steady in transgenic lines --- p.105 / Chapter 4.1.2 --- Methionine content increases in phas-PN2S and phas-MetL transgenic plants --- p.106 / Chapter 4.1.3 --- Relative lysine content increases in phas-dapA/phas-LRP transgenic plants --- p.107 / Chapter 4.1.4 --- Coordinated regulation of gene expressions of akthrl and akthr2 with GCN2 expression in transgenic plants with altered sink-source relationship --- p.109 / Chapter 4.2 --- GCN system in plants --- p.110 / Chapter 4.2.1 --- Transcriptional regulation of GCN2 in A. thaliana --- p.110 / Chapter 4.2.2 --- Regulation of amino acid biosynthesis by GCN system --- p.111 / Chapter 4.2.2.1 --- Regulation of akthrl and akthr2 by GCN2 --- p.111 / Chapter 4.2.2.2 --- GCN4 homolog in plants? --- p.112 / Chapter 4.2.2.3 --- Regulation of amino acid metabolism by GCN system --- p.113 / Chapter 4.3 --- Generation of transgenic plants with a combination of altered sink- source relationship --- p.114 / Chapter Chapter 5. --- Conclusion and Future Prospective --- p.116 / Appendix I: The major chemicals and reagents used in this research --- p.118 / "Appendix II: Major buffers, solutions and mediums used in this research" --- p.120 / Appendix III: Commercial kits used in this research --- p.125 / Appendix IV: Major equipment and facilities used in this research --- p.126 / References --- p.127