Yu, Wai Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-101). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / 摘要 --- p.vi / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- The importance of rice --- p.3 / Chapter 2.2 --- Limitation of essential amino acids in rice --- p.4 / Chapter 2.3 --- Lysine biosynthetic pathway --- p.6 / Chapter 2.3.1 --- The biosynthesis of aspartate --- p.6 / Chapter 2.3.2 --- Aspartate family pathway --- p.3 / Chapter 2.3.2.1 --- Aspartate kinase (AK) --- p.10 / Chapter 2.3.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.12 / Chapter 2.3.2.3 --- Other enzymes --- p.14 / Chapter 2.4 --- Regulation of lysine content in plant --- p.15 / Chapter 2.5 --- Enhancement of lysine content in plants --- p.16 / Chapter 2.5.1 --- "Breeding, selection and naturally occuring muatnts" --- p.17 / Chapter 2.5.2 --- Induced biochemical mutants --- p.18 / Chapter 2.5.3 --- Transgenic plants --- p.19 / Chapter 2.6 --- Hypothesis --- p.24 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.25 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Chemicals --- p.25 / Chapter 3.3 --- Bacterial strains --- p.25 / Chapter 3.4 --- Cloning of AK and DHPS cDNAs --- p.25 / Chapter 3.4.1 --- Plant materials --- p.25 / Chapter 3.4.2 --- RNA extraction --- p.26 / Chapter 3.4.3 --- RT-PCR amplification of AK and DHPS cDNAs --- p.26 / Chapter 3.4.4 --- Sequence modification of AK and DHPS cDNAs --- p.27 / Chapter 3.4.5 --- DNA sequencing of AK and DHPS cDNAs --- p.32 / Chapter 3.5 --- Chimeric gene construction for rice transformation --- p.32 / Chapter 3.5.1 --- Plasmid and genetic material --- p.32 / Chapter 3.5.2 --- Construction of chimeric genes with seed-specific promoter --- p.35 / Chapter 3.5.3 --- Sequence fidelity of chimeric genes --- p.37 / Chapter 3.6 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.37 / Chapter 3.7 --- Rice transformation --- p.38 / Chapter 3.7.1 --- Plant materials --- p.38 / Chapter 3.7.2 --- Preparation of agrobacterium --- p.33 / Chapter 3.7.3 --- Agrobacterium-mediated rice transformation --- p.39 / Chapter 3.7.3.1 --- Callus induction from mature rice seed embryos --- p.39 / Chapter 7.3.2 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.39 / Chapter 3.8 --- Analysis of transgenic expression --- p.41 / Chapter 3.8.1 --- Genomic DNA extraction --- p.41 / Chapter 3.8.2 --- Total RNA extraction --- p.41 / Chapter 3.8.3 --- Synthesis of DIG-labeled DNA probe --- p.42 / Chapter 3.8.4 --- Southern blot analysis --- p.43 / Chapter 3.8.5 --- Northern blot analysis --- p.43 / Chapter 3.8.6 --- Extraction of rice seed protein --- p.43 / Chapter 3.8.7 --- Tricine SDS-PAGE --- p.44 / Chapter 3.8.8 --- Raising AK and DHPS antibody --- p.44 / Chapter 3.8.9 --- Western blot analysis --- p.46 / Chapter 3.9 --- Free amino acid analysis --- p.46 / Chapter CHAPTER 4. --- RESULTS --- p.48 / Chapter 4.1 --- Cloning of AK and DHPS cDNAs from rice --- p.48 / Chapter 4.1.1 --- RNA extraction and cDNAs amplification --- p.43 / Chapter 4.1.2 --- Sequencing of AK and DHPS cDNAs --- p.50 / Chapter 4.2 --- Sequence modification of AK and DHPS cDNAs --- p.50 / Chapter 4.3 --- Construction of chimeric genes --- p.50 / Chapter 4.4 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.56 / Chapter 4.5 --- Rice transformation --- p.58 / Chapter 4.6 --- Detection of target genes in transgenic rice lines --- p.60 / Chapter 4.6.1 --- PCR of genomic DNA --- p.60 / Chapter 4.6.2 --- Southern blot analysis --- p.63 / Chapter 4.7 --- Northern blot analysis --- p.65 / Chapter 4.8 --- Western blot analysis of AK and DHPS proteins --- p.66 / Chapter 4.9 --- Free amino acid analysis --- p.68 / Chapter 4.9.1 --- Free lysine content --- p.68 / Chapter 4.9.2 --- Changes in other amino acids --- p.69 / Chapter CHAPTER 5. --- DISCUSSION --- p.82 / Chapter 5.1 --- Cloning and modification of AK and DHPS cDNAs --- p.82 / Chapter 5.2 --- Seed-specific expression of modified AK and DHPS in rice --- p.82 / Chapter 5.3 --- Free amino acid changes in transgenic rice lines --- p.83 / Chapter 5.4 --- Future perspectives --- p.85 / Chapter CHAPTER 6. --- CONCLUSION --- p.86 / REFERENCES --- p.87 / APPENDIX --- p.102
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327062 |
| Date | January 2011 |
| Contributors | Yu, Wai Han., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 116 leaves : ill. (chiefly 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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