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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325744 |
| Date | January 2006 |
| Contributors | Chan, Man Ling., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 114 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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