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Chan Man Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-114). / Abstracts in English and Chinese. / Table of Contents / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- The importance of rice --- p.4 / Chapter 2.2 --- The prevalence of hunger and malnutrition --- p.4 / Chapter 2.3 --- Limitation of essential amino acids in crop plants --- p.5 / Chapter 2.4 --- Lysine biosynthesis and catabolism --- p.7 / Chapter 2.5 --- Lysine biosynthetic pathway --- p.7 / Chapter 2.5.1 --- The biosynthesis of aspartate --- p.7 / Chapter 2.5.2 --- The aspartate family pathway --- p.9 / Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.11 / Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.13 / Chapter 2.5.2.3 --- Threonine Synthase (TS) and other enzymes --- p.15 / Chapter 2.6 --- The lysine catabolic pathway --- p.16 / Chapter 2.6.1 --- "LKR-SDHproteins, mRNAs and genes" --- p.18 / Chapter 2.6.2 --- Regulation of lysine catabolic pathway --- p.21 / Chapter 2.6.2.1 --- Regulation at biochemical level --- p.21 / Chapter 2.6.2.2 --- Regulation through linkage between LKR and SDH --- p.22 / Chapter 2.6.2.3 --- Regulation through LKR/SDH gene expression --- p.24 / Chapter 2.6.2.4 --- Implication of regulatory mechanism of saccharopine pathway --- p.26 / Chapter 2.7 --- Overall regulation of lysine content in plants --- p.27 / Chapter 2.8 --- Increasing lysine content in plants --- p.28 / Chapter 2.8.1 --- "Breeding, selection and naturally occurring mutants" --- p.28 / Chapter 2.8.2 --- Induced biochemical mutants --- p.29 / Chapter 2.8.3 --- Transgenic plants --- p.31 / Chapter 2.8.4 --- Insight into the way of lysine accumulation --- p.35 / Chapter 2.9 --- Gene silencing in plant --- p.36 / Chapter 2.9.1 --- Mechanism of antisense RNA and RNAi --- p.36 / Chapter 2.9.2 --- Application of antisense technology to produce transgenic plants --- p.39 / Chapter 2.10 --- Hypothesis --- p.41 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.43 / Chapter 3.1 --- Chemicals --- p.43 / Chapter 3.2 --- Bacterial strains --- p.43 / Chapter 3.3 --- Chimeric gene construction for rice transformation --- p.43 / Chapter 3.3.1 --- Plasmids and genetic materials --- p.43 / Chapter 3.3.2 --- Construction of chimeric genes with seed-specific promoters --- p.46 / Chapter 3.3.3 --- Construction of chimeric gene with 35S promoter --- p.51 / Chapter 3.3.4 --- Construction of antisense and RNAi constructs --- p.53 / Chapter 3.3.5 --- "Construction of chimeric genes expressing AK, DHPS and RNAi synchronously" --- p.58 / Chapter 3.3.6 --- Confirmation of sequence fidelity of chimeric genes --- p.59 / Chapter 3.4 --- Rice transformation --- p.59 / Chapter 3.4.1 --- Plant materials --- p.59 / Chapter 3.4.2 --- Preparation of Agrobacterium --- p.59 / Chapter 3.4.3 --- Agrobacterium-mediated rice transformation --- p.60 / Chapter 3.4.3.1 --- Callus induction from mature seed embryos --- p.60 / Chapter 3.4.3.2 --- Callus induction from immature seed embryos --- p.60 / Chapter 3.4.3.3 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.60 / Chapter 3.5 --- Analysis of transgenic expression --- p.62 / Chapter 3.5.1 --- Genomic DNA extraction --- p.62 / Chapter 3.5.2 --- Total RNA extraction --- p.62 / Chapter 3.5.3 --- Synthesis of DIG-labeled DNA probe / Chapter 3.5.4 --- Southern blot analysis --- p.65 / Chapter 3.5.5 --- Northern blot analysis --- p.65 / Chapter 3.5.6 --- Extraction of immature seed protein --- p.65 / Chapter 3.5.7 --- Tricine SDS-PAGE --- p.66 / Chapter 3.5.8 --- Western blot analysis --- p.66 / Chapter 3.6 --- Free amino acid analysis --- p.67 / Chapter CHAPTER 4. --- RESULTS --- p.68 / Chapter 4.1 --- Construction of chimeric genes --- p.68 / Chapter 4.2 --- Rice transformation --- p.70 / Chapter 4.3 --- Detection of target genes in transgenic rice lines --- p.72 / Chapter 4.3.1 --- PCR of Genomic DNA --- p.72 / Chapter 4.3.2 --- Southern blot analysis --- p.75 / Chapter 4.4 --- Northern blot analysis --- p.77 / Chapter 4.5 --- "Western blot analysis ofAK, DHPS and LKR protein" --- p.80 / Chapter 4.6 --- Free amino acid analysis --- p.82 / Chapter 4.6.1 --- Free lysine content --- p.82 / Chapter 4.6.2 --- Changes of other amino acids --- p.84 / Chapter CHAPTER 5. --- DISCUSSION --- p.93 / Chapter 5.1 --- Rice transformation and transgene expression --- p.93 / Chapter 5.2 --- Co-expression of E. coli feedback-insensitive AK and DHPS --- p.94 / Chapter 5.3 --- Enhancing free Lys through down-regulation of LKR --- p.95 / Chapter 5.4 --- Co-expression of AK and DHPS together with down-regulation of LKR --- p.96 / Chapter 5.5 --- Free amino acid changes in different genotypes --- p.97 / Chapter 5.6 --- Future perspectives --- p.98 / Chapter CHAPTER 6. --- CONCLUSION --- p.100 / REFERENCES --- p.102

by Wai Ching Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-159). / Abstracts in English and Chinese. / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / List of Abbreviations --- p.viii / Table of contents --- p.x / List of Tables --- p.xiv / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.4 / Chapter 2.1 --- Plant lectins --- p.4 / Chapter 2.1.1 --- Introduction --- p.4 / Chapter 2.1.2 --- Definition and subdivision of plant lectins --- p.4 / Chapter 2.2 --- Monocot mannose-binding lectins --- p.6 / Chapter 2.2.1 --- Occurrence and carbohydrate binding specificity --- p.6 / Chapter 2.2.2 --- Molecular structure and amino acid sequence --- p.7 / Chapter 2.2.3 --- "Molecular cloning, biosynthesis and post-translational modification" --- p.10 / Chapter 2.2.4 --- Mannose-binding lectins of Family Liliaceae --- p.11 / Chapter 2.2.4.1 --- Tulipa gesneriana lectins (TGL) --- p.12 / Chapter 2.2.4.2 --- Aloe arborescens lectins (AAL) --- p.13 / Chapter 2.2.4.3 --- Polygonatum multiflorum agglutinin (PMA) and lectin-related protein --- p.14 / Chapter 2.3 --- Polygonatum odoratum lectins (POL) --- p.15 / Chapter 2.3.1 --- Isolation and purification of POL from Yu Chu --- p.15 / Chapter 2.3.2 --- Agglutinating activity and anti-viral activities of POL --- p.17 / Chapter 2.3.3 --- Bacterial expression of POL in Escherichia coli --- p.18 / Chapter 2.4 --- Plant-based production of recombinant proteins --- p.20 / Chapter 2.4.1 --- Advantages of using plants as expression system --- p.20 / Chapter 2.4.2 --- Plant-derived recombinant proteins --- p.22 / Chapter 2.5 --- Expression of heterologous proteins in rice --- p.24 / Chapter 2.5.1 --- The facts of rice --- p.24 / Chapter 2.5.2 --- Rice storage proteins --- p.25 / Chapter 2.5.2 --- Expression of lysine-rich protein (LRP)/glutelin fusion proteinin rice seeds --- p.28 / Chapter 2.5.3 --- Expression of Galanthus nivalis agglutinin in rice --- p.29 / Chapter 2.6 --- Protein trafficking in plants --- p.30 / Chapter 2.6.1 --- Golgi-dependent pathways --- p.30 / Chapter 2.6.2 --- Golgi-independent pathway --- p.32 / Chapter 2.6.3 --- Expression of protein targeting determinants in tobacco plants and suspension cells --- p.33 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Chemcials --- p.35 / Chapter 3.3 --- Bacterial strains --- p.35 / Chapter 3.4 --- Cloning of POL cDNA --- p.36 / Chapter 3.4.1 --- Plant materials --- p.36 / Chapter 3.4.2 --- RNA extraction --- p.36 / Chapter 3.4.3 --- RT-PCR amplification of POL cDNA --- p.36 / Chapter 3.4.4 --- 5'RACE and 3'RACE --- p.38 / Chapter 3.4.5 --- Sequencing of POL cDNA --- p.39 / Chapter 3.5 --- Analysis of POL protein --- p.40 / Chapter 3.5.1 --- Protein extraction and Tricine-SDS PAGE --- p.40 / Chapter 3.5.2 --- Western blot analysis --- p.41 / Chapter 3.6 --- Chimeric gene construction --- p.42 / Chapter 3.6.1 --- Construction of the Cauliflower mosaic virus (CaMV)35S promoter/POL constructs --- p.44 / Chapter 3.6.2 --- Construction of the glutelin-1 promoter/POL constructs --- p.48 / Chapter 3.6.3 --- Sequence fidelity of chimeric genes --- p.55 / Chapter 3.7 --- Expression of transgenes in rice --- p.55 / Chapter 3.7.1 --- Plant materials --- p.55 / Chapter 3.7.2 --- Agrobacterium transformation --- p.55 / Chapter 3.7.3 --- Callus induction --- p.56 / Chapter 3.7.4 --- Agrobacterium culture and rice transformation --- p.56 / Chapter 3.7.5 --- Selection and regeneration of rice callus --- p.56 / Chapter 3.7.6 --- Isolation of genomic DNA --- p.58 / Chapter 3.7.7 --- Southern blot analysis --- p.58 / Chapter 3.7.8 --- Extraction of leaf total RNA --- p.59 / Chapter 3.7.9 --- Extraction of seed total RNA --- p.59 / Chapter 3.7.10 --- Northern blot analysis --- p.60 / Chapter 3.7.11 --- Protein extraction and Tricine SDS-PAGE --- p.60 / Chapter 3.7.12 --- Western blot analysis --- p.61 / Chapter 3.8 --- Cytopathic effect (CPE) reduction assay --- p.61 / Chapter 3.8.1 --- Protein extraction --- p.61 / Chapter 3.8.2 --- CPE reduction assay --- p.62 / Chapter 3.9 --- Confocal immunofluorescence --- p.63 / Chapter 3.9.1 --- Preparation of sections --- p.63 / Chapter 3.9.2 --- Labelling of fluorescence probes --- p.63 / Chapter 3.9.3 --- Image collection --- p.64 / Chapter Chapter 4 --- Results --- p.65 / Chapter 4.1 --- Cloning of POL cDNA from Yu Chu --- p.65 / Chapter 4.1.1 --- RNA extraction and partial POL cDNA amplification --- p.65 / Chapter 4.1.2 --- 5'RACE and 3'RACE --- p.67 / Chapter 4.1.3 --- Sequencing of POL cDNA --- p.68 / Chapter 4.1.4 --- Sequences comparison of POL and Liliaceae lectins --- p.75 / Chapter 4.2 --- Occurence of POL protein in Yu Chu plant --- p.77 / Chapter 4.3 --- Constitutional expression of POL in rice --- p.79 / Chapter 4.3.1 --- Construction of Cauliflower mosaic virus 35S promoter constructs --- p.80 / Chapter 4.3.2 --- Southern blot analysis --- p.82 / Chapter 4.3.3 --- Northern blot analysis --- p.84 / Chapter 4.3.4 --- Western blot analysis --- p.85 / Chapter 4.3.5 --- Western blot analysis of 35S/POL T1 plant --- p.87 / Chapter 4.4 --- Seed-specific expression of POL in rice --- p.88 / Chapter 4.4.1 --- Construction of the glutelin-1 promoter constructs --- p.89 / Chapter 4.4.2 --- Southern blot analysis --- p.92 / Chapter 4.4.3 --- Northern blot analysis --- p.96 / Chapter 4.4.4 --- Western blot analysis --- p.101 / Chapter 4.4.5 --- Western blot analysis of POL-BP-8O and POL-α-TIP T1 transgenic plants --- p.117 / Chapter 4.5 --- Cytopathic effect (CPE) reduction assay --- p.122 / Chapter 4.6 --- Confocal immunofluorescence studies --- p.125 / Chapter Chapter 5 --- Discussion --- p.134 / Chapter 5.1 --- Cloning of POL cDNA --- p.134 / Chapter 5.2 --- Analysis of constitutional expression of POL in rice --- p.136 / Chapter 5.3 --- Analysis of seed-specific expression of POL in rice --- p.138 / Chapter 5.4 --- Localization of POL in POL-BP-8O and POL-α-TIP transgenic rice seeds --- p.146 / Chapter 5.5 --- Cytopathic effect (CPE) reduction assay --- p.148 / Chapter 5.6 --- Future prospects --- p.151 / Chapter Chapter 6 --- Conclusion --- p.153