by Chan, Yuk Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 166-175). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / 摘要 --- p.vii / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xiv / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Vitamin A --- p.5 / Chapter 2.1.1 --- General and properties --- p.5 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Deficiency symptoms --- p.9 / Chapter 2.1.4 --- Dietary source of vitamin A --- p.10 / Chapter 2.1.5 --- Metabolism of dietary vitamin A and provitamin A in human --- p.12 / Chapter 2.1.5.1 --- Digestion and absorption --- p.12 / Chapter 2.1.5.2 --- Bioconversion --- p.12 / Chapter 2.1.5.2.1 --- "β, β-carotene-15,15'-monooxygenase (BCMO)" --- p.13 / Chapter 2.1.5.3 --- "Transport, uptake and storage" --- p.15 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.19 / Chapter 2.2.1 --- Present situation --- p.19 / Chapter 2.2.2 --- Global efforts in dealing with VAD --- p.21 / Chapter 2.2.2.1 --- Vitamin A supplementation --- p.21 / Chapter 2.2.2.2 --- Food fortification --- p.22 / Chapter 2.2.2.3 --- Biofortification --- p.23 / Chapter 2.2.2.3.1 --- Conventional selective breeding --- p.23 / Chapter 2.2.2.3.2 --- Biosynthesis of provitamin A in plants --- p.25 / Chapter 2.2.2.3.3 --- Carotenoids enhancement in major plants and food crops --- p.31 / Chapter 2.3 --- Inherent problems of the present carotenoid enhancement --- p.34 / Chapter 2.3.1 --- Recommended Dietary Amount of vitamin A --- p.34 / Chapter 2.3.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.35 / Chapter 2.3.2.1 --- Bioavailability --- p.36 / Chapter 2.3.2.2 --- Bioconvertibility --- p.38 / Chapter 2.3.2.3 --- Health and nutrition status --- p.39 / Chapter 2.4 --- Previous study in our lab --- p.41 / Chapter 2.4.1 --- Overexpression of rice PSY1 --- p.41 / Chapter 2.4.2 --- Introduction of carotenoid genes and BCMOs into rice --- p.44 / Chapter 2.5 --- Overview of the project --- p.50 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.52 / Chapter 3.1 --- Chemicals --- p.52 / Chapter 3.2 --- Bacterial strains in regular cloning --- p.52 / Chapter 3.3 --- BCMO genes and carotenogenic genes --- p.53 / Chapter 3.4 --- Expression of BCMOs in bacterial system --- p.54 / Chapter 3.4.1 --- lac promoter system --- p.54 / Chapter 3.4.2 --- pBAD-TOPO® system --- p.56 / Chapter 3.5 --- Construction of gene cassettes for plant transformation --- p.58 / Chapter 3.5.1 --- Gene cassettes for carrot transformation --- p.58 / Chapter 3.5.1.1 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by CaMV 35S promoter --- p.58 / Chapter 3.5.1.2 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by lycopene-β-cyclase promoter --- p.63 / Chapter 3.5.2 --- Gene cassettes for Arabidopsis transformation --- p.67 / Chapter 3.5.2.1 --- Construction of gene cassettes expressing Dcpsy --- p.67 / Chapter 3.5.2.2 --- Construction of gene cassettes expressing mbcmos --- p.69 / Chapter 3.5.3 --- Gene cassettes for Rice transformation --- p.72 / Chapter 3.5.3.1 --- Construction of gene cassettes expressing mbcmos --- p.72 / Chapter 3.5.3.2 --- Construction of gene cassettes expressing Ospsyl and mbcmos --- p.74 / Chapter 3.5.4 --- Confirmation of sequence fidelity --- p.76 / Chapter 3.6 --- Carrot transformation --- p.76 / Chapter 3.6.1 --- Plant materials --- p.76 / Chapter 3.6.2 --- Preparation of Agrobacterium --- p.76 / Chapter 3.6.3 --- Agrobacterium mediated transformation --- p.77 / Chapter 3.6.3.1 --- Seed germination --- p.78 / Chapter 3.6.3.2 --- Co-cultivation with hypocotyls --- p.78 / Chapter 3.6.3.3 --- Callus induction and selection --- p.78 / Chapter 3.6.3.4 --- Liquid cell culture preparation and embryogenesis induction --- p.79 / Chapter 3.6.3.5 --- Regeneration --- p.80 / Chapter 3.7 --- Arabidopsis Transformation --- p.80 / Chapter 3.7.1 --- Plant materials --- p.80 / Chapter 3.7.2 --- Preparation of Agrobacterium --- p.81 / Chapter 3.7.3 --- Agrobacterium mediated transformation --- p.81 / Chapter 3.7.3.1 --- Co-cultivation --- p.81 / Chapter 3.7.3.2 --- Selection --- p.82 / Chapter 3.8 --- Rice transformation --- p.83 / Chapter 3.8.1 --- Plant materials --- p.83 / Chapter 3.8.2 --- Preparation of Agrobacterium --- p.83 / Chapter 3.8.3 --- Agrobacterium mediated transformation --- p.83 / Chapter 3.8.3.1 --- Callus induction from mature rice seeds --- p.84 / Chapter 3.8.3.2 --- Co-cultivation and selection --- p.84 / Chapter 3.9 --- Detection of transgene expression --- p.86 / Chapter 3.9.1 --- Detection at DNA level --- p.86 / Chapter 3.9.1.1 --- Genomic DNA extraction --- p.86 / Chapter 3.9.1.2 --- PCR screening --- p.86 / Chapter 3.9.1.3 --- Synthesis of DIG-labelled DNA probes --- p.86 / Chapter 3.9.1.4 --- Southern blot analysis --- p.87 / Chapter 3.9.2 --- Detection at RNA level --- p.88 / Chapter 3.9.2.1 --- Total RNA extraction --- p.88 / Chapter 3.9.2.2 --- Northern blot analysis --- p.89 / Chapter 3.9.2.3 --- RT-PCR --- p.89 / Chapter 3.9.3 --- Detection at protein level --- p.89 / Chapter 3.9.3.1 --- Antibody production --- p.89 / Chapter 3.9.3.1.1 --- B.CMO protein induction in pET30a-bacterial system --- p.90 / Chapter 3.9.3.1.2 --- Immunization of rabbit and serum collection --- p.93 / Chapter 3.9.3.2 --- Protein extraction and Tricine SDS-PAGE --- p.93 / Chapter 3.9.3.3 --- Western blot analysis --- p.94 / Chapter 3.9.4 --- Detection at final product level --- p.95 / Chapter 3.9.4.1 --- UPLC analysis --- p.95 / Chapter 3.9.4.1.1 --- Extraction of total carotenoids and retinoids --- p.95 / Chapter 3.9.4.1.2 --- UPLC identification --- p.96 / Chapter CHAPTER 4. --- RESULTS --- p.97 / Chapter 4.1 --- Modified bcmo genes --- p.97 / Chapter 4.2 --- Expression of BCMOs in bacterial system --- p.102 / Chapter 4.2.1 --- lac promoter system --- p.104 / Chapter 4.2.2 --- pBAD-TOPO® system --- p.106 / Chapter 4.2.3 --- UPLC detection --- p.108 / Chapter 4.3 --- Carrot transformation --- p.110 / Chapter 4.3.1 --- Construction of gene cassettes for carrot transformation --- p.110 / Chapter 4.3.2 --- Seed germination and co-cultivation --- p.112 / Chapter 4.3.3 --- Callus induction and selection --- p.113 / Chapter 4.3.4 --- Embryogenesis induction and regeneration --- p.113 / Chapter 4.3.5 --- Callus induction in the dark --- p.115 / Chapter 4.3.6 --- Detection of native BCMO --- p.116 / Chapter 4.3.6.1 --- Genomic PCR screening of 35Spro - zebcmo transgenic lines --- p.116 / Chapter 4.3.6.2 --- Southern blot analysis of 35Spro - zebcmo transgenic lines --- p.117 / Chapter 4.3.6.3 --- RT-PCR of 35Spro - zebcmo transgenic lines --- p.118 / Chapter 4.3.6.4 --- Detection at protein level --- p.119 / Chapter 4.3.6.4.1 --- Antibody production --- p.119 / Chapter 4.3.6.5 --- Western blot analysis of 35Spro - zebcmo transgenic lines --- p.123 / Chapter 4.3.6.6 --- Genomic PCR screening of later transgenic lines --- p.123 / Chapter 4.3.6.7 --- Western blot analysis of later transgenic lines --- p.125 / Chapter 4.3.6.8 --- UPLC analysis of later transgenic lines --- p.127 / Chapter 4.3.7 --- Detection of modified BCMO --- p.130 / Chapter 4.3.7.1 --- Genomic PCR screening --- p.130 / Chapter 4.3.7.2 --- Northern blot analysis --- p.132 / Chapter 4.3.7.3 --- Western blot analysis --- p.134 / Chapter 4.3.8 --- UPLC analysis --- p.136 / Chapter 4.4 --- Arabidopsis transformation --- p.138 / Chapter 4.4.1 --- Construction of gene cassettes for Arabidopsis transformation --- p.138 / Chapter 4.4.2 --- Selection --- p.139 / Chapter 4.4.3 --- Genmoic PCR screening of Arabidopsis transformants --- p.140 / Chapter 4.4.4 --- UPLC analysis for Arabidopsis transformants --- p.142 / Chapter 4.5 --- Rice transformation --- p.144 / Chapter 4.5.1 --- Construction of gene cassettes for rice transformation --- p.144 / Chapter 4.5.2 --- "Callus induction from mature rice seeds, co-cultivation and selection" --- p.146 / Chapter 4.5.3 --- Genomic PCR screening of Rice transformants --- p.147 / Chapter 4.5.4 --- UPLC analysis of rice transformants --- p.149 / Chapter CHAPTER 5. --- DISCUSSION --- p.151 / Chapter 5.1 --- Bacterial expression of BCMO --- p.151 / Chapter 5.2 --- Analysis of BCMO in plants --- p.153 / Chapter 5.2.1 --- Carrot --- p.154 / Chapter 5.2.1.1 --- Expression of BCMO in carrot transformants --- p.154 / Chapter 5.2.1.2 --- UPLC analysis of carrot transformants --- p.155 / Chapter 5.2.2 --- Arabidopsis --- p.156 / Chapter 5.2.3 --- Rice --- p.158 / Chapter 5.3 --- Proposed explanation for the failure of retinal production --- p.159 / Chapter 5.3.1 --- Retinal sequestration --- p.160 / Chapter 5.3.2 --- Localization of BCMO --- p.161 / Chapter 5.4 --- Future prospects --- p.163 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.165 / REFERENCES --- p.166 / APPENDICES --- p.176