Engineering lysine metabolic pathway in rice.

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 ContentsACKNOWLEDGEMENTS --- p.iiiABSTRACT --- p.ivTABLE OF CONTENTS --- p.viiLIST OF FIGURES --- p.xiLIST OF TABLES --- p.xiiiLIST OF ABBREVIATIONS --- p.xivChapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4Chapter 2.1 --- The importance of rice --- p.4Chapter 2.2 --- The prevalence of hunger and malnutrition --- p.4Chapter 2.3 --- Limitation of essential amino acids in crop plants --- p.5Chapter 2.4 --- Lysine biosynthesis and catabolism --- p.7Chapter 2.5 --- Lysine biosynthetic pathway --- p.7Chapter 2.5.1 --- The biosynthesis of aspartate --- p.7Chapter 2.5.2 --- The aspartate family pathway --- p.9Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.11Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.13Chapter 2.5.2.3 --- Threonine Synthase (TS) and other enzymes --- p.15Chapter 2.6 --- The lysine catabolic pathway --- p.16Chapter 2.6.1 --- "LKR-SDHproteins, mRNAs and genes" --- p.18Chapter 2.6.2 --- Regulation of lysine catabolic pathway --- p.21Chapter 2.6.2.1 --- Regulation at biochemical level --- p.21Chapter 2.6.2.2 --- Regulation through linkage between LKR and SDH --- p.22Chapter 2.6.2.3 --- Regulation through LKR/SDH gene expression --- p.24Chapter 2.6.2.4 --- Implication of regulatory mechanism of saccharopine pathway --- p.26Chapter 2.7 --- Overall regulation of lysine content in plants --- p.27Chapter 2.8 --- Increasing lysine content in plants --- p.28Chapter 2.8.1 --- "Breeding, selection and naturally occurring mutants" --- p.28Chapter 2.8.2 --- Induced biochemical mutants --- p.29Chapter 2.8.3 --- Transgenic plants --- p.31Chapter 2.8.4 --- Insight into the way of lysine accumulation --- p.35Chapter 2.9 --- Gene silencing in plant --- p.36Chapter 2.9.1 --- Mechanism of antisense RNA and RNAi --- p.36Chapter 2.9.2 --- Application of antisense technology to produce transgenic plants --- p.39Chapter 2.10 --- Hypothesis --- p.41Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.43Chapter 3.1 --- Chemicals --- p.43Chapter 3.2 --- Bacterial strains --- p.43Chapter 3.3 --- Chimeric gene construction for rice transformation --- p.43Chapter 3.3.1 --- Plasmids and genetic materials --- p.43Chapter 3.3.2 --- Construction of chimeric genes with seed-specific promoters --- p.46Chapter 3.3.3 --- Construction of chimeric gene with 35S promoter --- p.51Chapter 3.3.4 --- Construction of antisense and RNAi constructs --- p.53Chapter 3.3.5 --- "Construction of chimeric genes expressing AK, DHPS and RNAi synchronously" --- p.58Chapter 3.3.6 --- Confirmation of sequence fidelity of chimeric genes --- p.59Chapter 3.4 --- Rice transformation --- p.59Chapter 3.4.1 --- Plant materials --- p.59Chapter 3.4.2 --- Preparation of Agrobacterium --- p.59Chapter 3.4.3 --- Agrobacterium-mediated rice transformation --- p.60Chapter 3.4.3.1 --- Callus induction from mature seed embryos --- p.60Chapter 3.4.3.2 --- Callus induction from immature seed embryos --- p.60Chapter 3.4.3.3 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.60Chapter 3.5 --- Analysis of transgenic expression --- p.62Chapter 3.5.1 --- Genomic DNA extraction --- p.62Chapter 3.5.2 --- Total RNA extraction --- p.62Chapter 3.5.3 --- Synthesis of DIG-labeled DNA probeChapter 3.5.4 --- Southern blot analysis --- p.65Chapter 3.5.5 --- Northern blot analysis --- p.65Chapter 3.5.6 --- Extraction of immature seed protein --- p.65Chapter 3.5.7 --- Tricine SDS-PAGE --- p.66Chapter 3.5.8 --- Western blot analysis --- p.66Chapter 3.6 --- Free amino acid analysis --- p.67Chapter CHAPTER 4. --- RESULTS --- p.68Chapter 4.1 --- Construction of chimeric genes --- p.68Chapter 4.2 --- Rice transformation --- p.70Chapter 4.3 --- Detection of target genes in transgenic rice lines --- p.72Chapter 4.3.1 --- PCR of Genomic DNA --- p.72Chapter 4.3.2 --- Southern blot analysis --- p.75Chapter 4.4 --- Northern blot analysis --- p.77Chapter 4.5 --- "Western blot analysis ofAK, DHPS and LKR protein" --- p.80Chapter 4.6 --- Free amino acid analysis --- p.82Chapter 4.6.1 --- Free lysine content --- p.82Chapter 4.6.2 --- Changes of other amino acids --- p.84Chapter CHAPTER 5. --- DISCUSSION --- p.93Chapter 5.1 --- Rice transformation and transgene expression --- p.93Chapter 5.2 --- Co-expression of E. coli feedback-insensitive AK and DHPS --- p.94Chapter 5.3 --- Enhancing free Lys through down-regulation of LKR --- p.95Chapter 5.4 --- Co-expression of AK and DHPS together with down-regulation of LKR --- p.96Chapter 5.5 --- Free amino acid changes in different genotypes --- p.97Chapter 5.6 --- Future perspectives --- p.98Chapter CHAPTER 6. --- CONCLUSION --- p.100REFERENCES --- p.10