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

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

Long, Xiaohang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 146-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Leung, Hoi Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-128). / Abstracts in English and Chinese. / THESIS/ASSESSMENT COMMITTEE --- p.i / STATEMENT FROM AUTHOR --- p.ii / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.xi / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xviii / LIST OF ABBREVIATIONS --- p.xix / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- Nutritional quality of rice --- p.4 / Chapter 2.1.1 --- Classification of seed proteins --- p.4 / Chapter 2.1.2 --- Amino acid composition of rice proteins --- p.5 / Chapter 2.1.3 --- Other nutritional components of rice --- p.6 / Chapter 2.2 --- Rice seed storage proteins --- p.7 / Chapter 2.2.1 --- Properties and classification of seed storage proteins --- p.7 / Chapter 2.2.2 --- Composition and stucture --- p.9 / Chapter 2.2.2.1 --- Glutelin --- p.9 / Chapter 2.2.2.2 --- Prolamin --- p.10 / Chapter 2.2.2.3 --- Albumin and globulin --- p.12 / Chapter 2.2.3 --- "Synthsis, assembly and deposition of rice seed storage proteins" --- p.13 / Chapter 2.2.3.1 --- Storage protein folding and assembly in the ER --- p.14 / Chapter 2.2.3.2 --- Storage protein transport and protein body formation --- p.16 / Chapter 2.2.3.3 --- Protein bodies and their distribution in endosperm --- p.18 / Chapter 2.3 --- Transgenic approaches to improve the nutritional quality of rice seed proteins --- p.19 / Chapter 2.3.1 --- General introduction --- p.19 / Chapter 2.3.2 --- Attempts to improve the nutritional quality of seed proteins --- p.20 / Chapter 2.3.3 --- Rice grain quality improvement by genetic engineering --- p.22 / Chapter 2.3.3.1 --- Increase in the lysine content of rice endosperm --- p.22 / Chapter 2.2.3.2 --- Other examples of rice nutritional quality improvement --- p.25 / Chapter 2.3.4 --- Expression of recombinant protein in transgenic plants --- p.26 / Chapter 2.3.5 --- Effects of recombinant proteins on the high-lysine rice --- p.27 / Chapter 2.4 --- Proteomics --- p.28 / Chapter 2.4.1 --- General overview --- p.28 / Chapter 2.4.1.1 --- Two-dimensional polyacrylamide gel electrophoresis for proteome analysis --- p.29 / Chapter 2.4.1.2 --- Protein visualization --- p.32 / Chapter 2.4.1.3 --- Computer-aided image analysis --- p.34 / Chapter 2.4.1.4 --- Mass spectrometry-based methods for protein identification --- p.35 / Chapter 2.4.1.5 --- Database search --- p.36 / Chapter 2.4.1.6 --- Protein sequence database --- p.37 / Chapter 2.4.2 --- Plant proteomics --- p.40 / Chapter 2.4.2.1 --- Rice proteomics --- p.41 / Chapter 2.4.2.2 --- Comparative proteomics --- p.43 / Chapter 2.5 --- Hypothesis and objectives --- p.45 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.47 / Chapter 3.1 --- Materials --- p.47 / Chapter 3.1.1 --- Chemicals and commercial kits --- p.47 / Chapter 3.1.2 --- Instruments --- p.47 / Chapter 3.1.3 --- Softwares --- p.48 / Chapter 3.1.4 --- Plant materials --- p.48 / Chapter 3.2 --- Methods --- p.49 / Chapter 3.2.1 --- Collection of developing rice seeds --- p.49 / Chapter 3.2.2 --- Extraction of rice seed proteins --- p.51 / Chapter 3.2.2.1 --- Extraction of total protein --- p.51 / Chapter 3.2.3.2 --- Extraction of four fractions of rice seed proteins --- p.51 / Chapter 3.2.3 --- 2D gel electrophoresis --- p.53 / Chapter 3.2.3.1 --- Protein precipitation and quantification --- p.53 / Chapter 3.2.3.2 --- Isoelectric focusing (IEF) --- p.54 / Chapter 3.2.3.3 --- IPG strips equilibration --- p.54 / Chapter 3.2.3.4 --- Second-dimension SDS-PAGE --- p.55 / Chapter 3.2.3.5 --- Silver staining of 2D gel --- p.55 / Chapter 3.2.3.6 --- Image and data analysis --- p.56 / Chapter 3.2.4 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.56 / Chapter 3.2.4.1 --- Sample destaining --- p.56 / Chapter 3.2.4.2 --- In-gel digestion with trypsin --- p.57 / Chapter 3.2.4.3 --- Desalination of the digested sample with Zip Tip --- p.58 / Chapter 3.2.4.4 --- Protein identification by mass spectrometry and database searching --- p.58 / Chapter 3.2.5 --- Detection of LRP fusion protein in 2D PAGE --- p.59 / Chapter 3.2.5.1 --- 2D gel electrophoresis --- p.59 / Chapter 3.2.5.2 --- Western blotting using anti-LRP antibody --- p.60 / Chapter 3.2.6 --- Antiserum production --- p.61 / Chapter 3.2.6.1 --- Purification of glutelin and prolamin proteins --- p.61 / Chapter 3.2.6.2 --- Immunization of rabbits and mice --- p.62 / Chapter 3.2.6.3 --- Testing of antibody specificity --- p.62 / Chapter 3.2.7 --- Transmission electron microscopy (TEM) --- p.63 / Chapter 3.2.7.1 --- Sample fixation and section preparation --- p.63 / Chapter 3.2.7.2 --- TEM observation --- p.64 / Chapter 3.2.7.3 --- Immunocytochemical observation --- p.64 / Chapter CHAPTER 4. --- RESULTS --- p.66 / Chapter 4.1 --- Proteomic analysis of high-lysine rice --- p.66 / Chapter 4.1.1 --- Extraction of proteins --- p.66 / Chapter 4.1.2 --- The proteomic profiles of different storage proteins in developing high-lysine rice seeds --- p.67 / Chapter 4.1.3 --- Quantitative analysis of protein spots --- p.76 / Chapter 4.1.4 --- Proteomic analysis of salt-soluble proteins --- p.79 / Chapter 4.1.5 --- Proteomic analysis of alcohol-soluble proteins --- p.81 / Chapter 4.1.6 --- Proteomic analysis of salt-soluble proteins --- p.82 / Chapter 4.1.7 --- Proteomic analysis of water-soluble proteins --- p.89 / Chapter 4.1.8 --- Comparison of changes in expression patterns of specific proteins in the high lysine rice --- p.89 / Chapter 4.2 --- Antibody production --- p.92 / Chapter 4.2.1 --- The production of anti-prolamin and anti-glutelin antibodies --- p.92 / Chapter 4.2.2 --- The specificity of anti-prolamin and anti-glutelin antibodies --- p.93 / Chapter 4.3 --- Transmission electron microscopy observation of rice protein bodies --- p.95 / Chapter 4.3.1 --- Morphology of protein bodies in high-lysine rice --- p.95 / Chapter 4.3.2 --- Subcellular localization of storage proteins and LRP --- p.98 / Chapter CHAPTER 5. --- DISCUSSION --- p.100 / Chapter 5.1 --- Protein profiling of LRP fusion protein and its effects on the expression of other proteins --- p.100 / Chapter 5.2 --- Over-expression of glutelin and its effects on the expression of other proteins --- p.102 / Chapter 5.3 --- Formation of malformed protein bodies and deposition of storage proteins --- p.103 / Chapter 5.4 --- Relationship between changes in protein expression and the Unfolded Protein Response --- p.105 / Chapter 5.5 --- Effects of transgenes on rice grain quality --- p.108 / Chapter 5.6 --- Allergenic effects of transgenic rice --- p.109 / Chapter 5.7 --- Future perspectives --- p.110 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.112 / REFERENCES --- p.114

by Yuan Dingyang. / "September 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 206-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Yu, Chun Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 115-124). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / LIST OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xx / LIST OF ABBREVIATIONS --- p.xxi / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.20 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.22 / Chapter 2.1 --- Major storage proteins in rice --- p.22 / Chapter 2.1.1 --- Structure and composition of glutelin --- p.22 / Chapter 2.1.2 --- Structure and composition of prolamin --- p.22 / Chapter 2.2 --- Biosynthesis pathway --- p.23 / Chapter 2.2.1 --- "The Biosynthesis, processing & compartmentalization of glutelin" --- p.23 / Chapter 2.2.1.1 --- Endoplasmic reticulum as the site of protein folding and compartmentalization --- p.23 / Chapter 2.2.1.2 --- COP-coated vesicles for protien trafficking between ER and Golgi --- p.25 / Chapter 2.2.1.3 --- Glutelin trafficking beyond ER --- p.26 / Chapter 2.2.1.3.1 --- Golgi as the site of post-translational modification of glutelin / Chapter 2.2.1.3.1.1 --- """Sorting for entry"" and ""sorting by retention"" models: mechanism of dense vesicle formation" --- p.26 / Chapter 2.2.1.3.1.2 --- "“Classical ligand-receptor"" and ""aggregation-mediated"" as the model describing protein sorting in Golgi" --- p.27 / Chapter 2.2.1.3.2 --- Pathway bypassing Golgi apparatus --- p.30 / Chapter 2.2.1.4 --- Prevacuolar compartment and protein body --- p.30 / Chapter 2.2.2 --- "The Biosynthesis, processing and compartmentalization of prolamin" --- p.31 / Chapter 2.3 --- Protein processing enzymes --- p.31 / Chapter 2.3.1 --- Luminal chaperone binding protein (BiP) --- p.31 / Chapter 2.3.2 --- Protein disulfide isomerase (PDI) --- p.33 / Chapter 2.4 --- ER quality control： unfolded protein response --- p.34 / Chapter 2.4.1 --- The importance of quality control in ER --- p.34 / Chapter 2.4.2 --- The target of ER quality control: misfolded protein --- p.35 / Chapter 2.4.3 --- Unfolded protein response --- p.36 / Chapter 2.4.3.1 --- IRE1 --- p.37 / Chapter 2.4.3.2 --- PERK --- p.37 / Chapter 2.4.3.3 --- ATF6 --- p.38 / Chapter 2.4.3.4 --- BiP as the master regulator of three transducers --- p.38 / Chapter 2.5 --- The cause of chalkiness --- p.41 / Chapter 2.5.1 --- "The relationship between ER stress, unfolded protein response and chalkiness" --- p.42 / Chapter 2.6 --- Organelle separation： sucrose density gradient centrifugation --- p.43 / Chapter 2.6.1 --- General introduction --- p.43 / Chapter 2.6.2 --- Plant organelle separation --- p.43 / Chapter 2.6.3 --- Organelle marker enzyme as a mean to elucidate the homogeneity of isolated organelle fraction --- p.44 / Chapter 2.7 --- Rice grain quality improvement by genetic engineering --- p.45 / Chapter 2.7.1 --- Increase in lysine content of rice endosperm --- p.45 / Chapter 2.7.2 --- Physiological and phenotypic changes in GT and LRP-fusion lines --- p.46 / Chapter 2.8 --- Hypotheses and objectives --- p.48 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.49 / Chapter 3.1 --- Materials --- p.49 / Chapter 3.1.1 --- Chemicals and commercial kits --- p.49 / Chapter 3.1.2 --- Instruments --- p.49 / Chapter 3.1.3 --- Plant materials --- p.49 / Chapter 3.1.3.1 --- Glutelin-enriched line (GT) --- p.50 / Chapter 3.1.3.2 --- Gtl-LRP-fusion line (LRP fusion) --- p.50 / Chapter 3.2 --- RNA extraction and northern-blot analysis --- p.50 / Chapter 3.2.1 --- Seed harvesting and RNA extraction --- p.50 / Chapter 3.2.2 --- Northern-blot analysis --- p.51 / Chapter 3.3 --- SDS-PAGE and western-blot analysis --- p.52 / Chapter 3.3.1 --- Seed harvesting and protein extraction --- p.52 / Chapter 3.3.2 --- SDS-PAGE and western-blot analysis s --- p.52 / Chapter 3.4 --- Purification of cellular organelles by SDG centrifugation --- p.53 / Chapter 3.4.1 --- Purification of ER by SDG centrifugation --- p.53 / Chapter 3.4.2 --- Purification of protein body by SDG centrifugation --- p.54 / Chapter 3.4.3 --- Protein body isolation by pepsin treatment --- p.54 / Chapter 3.5 --- Electron-microscopic observation --- p.55 / Chapter 3.5.1 --- Sample preparation for immuno-localization analysis --- p.55 / Chapter 3.5.1.1 --- Sample preparation --- p.55 / Chapter 3.5.1.2 --- Immunocytochemical observation --- p.55 / Chapter 3.5.2 --- Sample preparation for structural analysis --- p.56 / Chapter 3.6 --- Antibodies --- p.56 / Chapter 3.6.1 --- KLH conjugation of synthetic peptide --- p.57 / Chapter 3.6.2 --- Immunization of rabbits --- p.57 / Chapter 3.6.3 --- Antibody purification by affinity column --- p.57 / Chapter 3.6.3.1 --- Preparation of column for coupling --- p.57 / Chapter 3.6.3.2 --- Affinity purification of antibody by prepared column --- p.58 / Chapter 3.6.4 --- Testing of antibody specificity --- p.58 / Chapter CHAPTER 4. --- RESULTS --- p.60 / Chapter 4.1 --- Pro-glutelin accumulation in GT and LRP fusion transgenic lines --- p.60 / Chapter 4.2 --- General morphology and glutelin localization in rice seed --- p.61 / Chapter 4.3 --- "Studies on glutelin, BiP and pdi expression profiles of GT, LRP fusion lines and wild type rice" --- p.63 / Chapter 4.3.1 --- Comparison of the protein and RNA profiles of BiP between wild type and FH transgenic rice lines --- p.64 / Chapter 4.3.2 --- Comparison of the protein and RNA profiles of PDI between wild type and FH transgenic rice lines --- p.66 / Chapter 4.3.3 --- "Comparison of the RNA and protein profiles of BiP between wild type, GH and GL transgenic rice lines" --- p.68 / Chapter 4.3.4 --- "Comparison of the RNA and protein expression profiles of PDI between wild type, GH and GL transgenic lines" --- p.70 / Chapter 4.3.5 --- Summary of RNA and protein level comparison of different transgenic lines with wild type --- p.72 / Chapter 4.4 --- Electron microscopic studies of morphological changes in GLUTELIN OVER-EXPRESSED AND GT1-LRP-FUSION TRANSGENIC LINES AND WILD type rice --- p.73 / Chapter 4.5 --- Isolation of ER-enriched fractions by sucrose density gradient centrifugation --- p.76 / Chapter 4.5.1 --- Cross-contamination assessment by organelle specific marker proteins --- p.77 / Chapter 4.5.2 --- Identification of ER enriched fractions of different transgenic lines --- p.78 / Chapter 4.5.3 --- Studies on ER enriched fraction --- p.85 / Chapter 4.6 --- Isolation and studies on PB enriched fractions of different transgenic lines --- p.91 / Chapter 4.7 --- TEM studies on immuno-localization of ER chaperones (BlP and pdI) in immature rice seeds of different transgenic lines --- p.94 / Chapter CHAPTER 5. --- DISCUSSIONS --- p.101 / Chapter 5.1 --- Distortion of glutelin processing and translocation pathway --- p.101 / Chapter 5.1.1 --- The relationship between proglutelin localization and novel protein body in Gt1-LRP-fusion lines --- p.101 / Chapter 5.1.2 --- The presence of BiP and PDI in novel protein body in Gt1-LR-fusion lines --- p.103 / Chapter 5.1.2.1 --- Glutelin translocation pathway bypassing Golgi --- p.105 / Chapter 5.1.2.2 --- Glutelin translocation pathway through Golgi --- p.105 / Chapter 5.1.2.3 --- Gt1-LRP-fusion protein and proglutelin are trapped in ER --- p.107 / Chapter 5.2 --- "The relationship between novel protein body formation, ER stress, unfolded protein response and chalkiness" --- p.108 / Chapter 5.2.1 --- Relationship between novel protein body formation and unfolded protein response --- p.108 / Chapter 5.2.2 --- Repressing the expression of other storage proteins: consequence of unfold protein response or protein nutrients regulation --- p.109 / Chapter 5.2.3 --- Relationship between novel protein body formation and chalkiness --- p.110 / Chapter 5.3 --- The causes of ER dilation --- p.110 / Chapter 5.4 --- The relationship between different physiological changes in transgenic glutelin lines --- p.111 / Chapter 5.5 --- Future perspectives --- p.112 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.114 / REFERENCES --- p.115 / APPENDIX --- p.125

Yang, Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-132). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter Chatper 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.5 / Chapter 2.1 --- Facts on food allergy --- p.5 / Chapter 2.1.1 --- Food allergy and its prevalence --- p.5 / Chapter 2.1.2 --- Pathogenesis of food allergy --- p.6 / Chapter 2.1.3 --- Clineal disorders caused and diagnosis of food allergy --- p.8 / Chapter 2.2 --- Allergenicity assessment of genetically engineered food --- p.13 / Chapter 2.2.1 --- The structural and sequence homology of proteins as a criterion for food allergenicity assessment --- p.14 / Chapter 2.2.2 --- Digestion stability as a criterion for food allergenicity assessment --- p.15 / Chapter 2.2.3 --- Animal models for Food Allergenicity Assessment --- p.21 / Chapter 2.3 --- The importance of rice and its nutritional facts --- p.27 / Chapter 2.3.1 --- The importance of rice --- p.27 / Chapter 2.3.2 --- Rice nutritional facts and its relationship with malnutrition --- p.28 / Chapter 2.4 --- Food allergenicity research in rice --- p.30 / Chapter 2.5 --- Glutelin overexpression transgenic rice GT3 --- p.33 / Chapter 2.6 --- Recent and future perspectives for treatment of food allergy --- p.36 / Chapter Chapter 3. --- Materials and Methods --- p.39 / Chapter 3.1 --- Rice Seed Protein Extraction --- p.39 / Chapter 3.1.1 --- Rice varieties for protein extraction --- p.39 / Chapter 3.1.2 --- Protein extraction from rice seeds --- p.39 / Chapter 3.1.3 --- Fractionation of major rice seed storage proteins --- p.40 / Chapter 3.1.4. --- Protein quantification --- p.41 / Chapter 3.1.5 --- Tricine SDS-PAGE --- p.42 / Chapter 3.2 --- Simulated Gastric Digestibility Assay --- p.43 / Chapter 3.2.1 --- Assay System --- p.43 / Chapter 3.2.2 --- Preparation of Simulated Gastric Fluid --- p.43 / Chapter 3.2.3 --- Assay Procedures --- p.44 / Chapter 3.2.4 --- Results Interpretation --- p.44 / Chapter 3.3 --- Construction of Mouse Models --- p.45 / Chapter 3.3.1 --- Mouse strain and reagents used --- p.45 / Chapter 3.3.2 --- Mouse Model I --- p.46 / Chapter 3.3.3 --- Mouse Model II --- p.50 / Chapter 3.3.4 --- Mouse Model III --- p.51 / Chapter 3.4 --- Bioinformatic Analysis of Glutelin Sequence --- p.52 / Chapter 3.5 --- Epitope Mapping of Glutelin --- p.55 / Chapter 3.5.1 --- Bioinformatic Analysis --- p.55 / Chapter 3.5.2 --- Direct and Competitive ELISA --- p.56 / Chapter 3.5.3 --- Western Blot Analysis --- p.57 / Chapter 3.5.4 --- IgE-binding assay --- p.58 / Chapter Chapter 4. --- Results and Discussion --- p.60 / Chapter 4.1 --- Rice Seed Protein Extraction --- p.60 / Chapter 4.1.1 --- Rice Protein Extraction --- p.60 / Chapter 4.1.2 --- Extraction of rice major seed storage protein fractions --- p.62 / Chapter 4.2 --- Simulated Gastric Digestibility Assay --- p.64 / Chapter 4.2.1 --- Pepsin Digestibility of total protein from GT3 and WT rice seeds --- p.64 / Chapter 4.2.2 --- Pepsin Digestibility of major storage protein fractions in GT3 and WT rice --- p.68 / Chapter 4.2.3 --- Summary of Pepsin Digestibility Assay --- p.74 / Chapter 4.3 --- Mouse Model I --- p.75 / Chapter 4.3.1 --- Protein-specific IgE levels --- p.75 / Chapter 4.3.2 --- Protein-specific IgG1 and IgG2a levels --- p.77 / Chapter 4.3.3 --- Allergic Response Test --- p.79 / Chapter 4.3.4 --- Summary from Mouse Model I --- p.81 / Chapter 4.4 --- Mouse Model II --- p.83 / Chapter 4.4.1 --- Proteins specific IgE levels --- p.84 / Chapter 4.4.2 --- Proteins specific IgG1 and IgG2a levels --- p.85 / Chapter 4.4.3 --- Allergic Response Test --- p.87 / Chapter 4.4.4 --- Summary from Mouse Model II --- p.88 / Chapter 4.5 --- Mouse Model III --- p.90 / Chapter 4.5.1 --- Protein-specific IgE levels --- p.90 / Chapter 4.5.2 --- Proteins specific IgG1 and IgG2a levels --- p.91 / Chapter 4.5.3 --- Allergic Response Test --- p.93 / Chapter 4.5.4 --- Summary from Mouse Model III --- p.93 / Chapter 4.6 --- Potential allergenicity of rice glutelin by bioinformatics and epitope mapping --- p.94 / Chapter 4.6.1 --- Bioinformatic analysis --- p.94 / Chapter 4.6.2 --- ELISA analysis of synthesized epitopes --- p.97 / Chapter 4.6.3 --- Western Blot Analysis --- p.99 / Chapter 4.6.4 --- IgE-binding assay --- p.103 / Chapter Chapter 5. --- Conclusion and Future Perspectives --- p.109 / References --- p.111

Ho, Wing Ho. / On t.p. "beta" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 157-183). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xxiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter 2.1 --- Vitamin A --- p.4 / Chapter 2.1.1 --- Genral and properties --- p.4 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Dietary source of vitamin A --- p.12 / Chapter 2.1.3.1 --- Plant-derived provitamin A and animal-derived vitamin A --- p.12 / Chapter 2.1.3.2 --- Dependence on the plant-derived provitamin A by the poor --- p.14 / Chapter 2.1.3.2.1 --- Plant-derived provitamin A --- p.14 / Chapter 2.1.3.2.1.1 --- General and properties --- p.14 / Chapter 2.1.3.2.1.2 --- Biosynthesis of provitamin A in plants --- p.17 / Chapter 2.1.3.2.1.2.1 --- Assembly of C40 backbone … --- p.17 / Chapter 2.1.3.2.1.2.2 --- Desaturation and cyclization --- p.26 / Chapter 2.1.3.2.1.2.3 --- Oxygenation --- p.29 / Chapter 2.1.3.2.1.2.4 --- Carotenogenic enzymes --- p.31 / Chapter 2.1.4 --- Metabolism of dietary vitamin A and provitamin A in human system --- p.35 / Chapter 2.1.4.1 --- Digestion and absorption --- p.35 / Chapter 2.1.4.2 --- Biocon version --- p.37 / Chapter 2.1.4.2.1 --- "Beta, beta '-carotene 15, 15'-monooxygenase (BCMO)" --- p.40 / Chapter 2.1.4.3 --- "Transport, uptake and storage" --- p.43 / Chapter 2.1.4.4 --- Provision or excretion --- p.46 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.48 / Chapter 2.2.1 --- Green revolution --- p.48 / Chapter 2.2.2 --- Rice as the major staple food for feeding the poor --- p.49 / Chapter 2.2.3 --- Provitamin A content in processed rice seeds --- p.49 / Chapter 2.2.4 --- Symptoms of VAD --- p.51 / Chapter 2.2.5 --- Global prevalence of VAD --- p.53 / Chapter 2.3 --- Previous efforts for dealing with the deficiency --- p.55 / Chapter 2.3.1 --- The key for dealing with the deficiency --- p.55 / Chapter 2.3.2 --- Selective plant breeding --- p.55 / Chapter 2.3.3 --- Supplementation and post-harvesting fortification --- p.56 / Chapter 2.3.4 --- Bio-fortification by genetic engineering --- p.57 / Chapter 2.3.4.1 --- Advantages of genetic engineering --- p.57 / Chapter 2.3.4.1.1 --- Genetic engineering of non-cereal crops --- p.58 / Chapter 2.3.4.1.2 --- Genetic engineering of cereal crops --- p.62 / Chapter 2.3.4.1.2.1 --- Golden Rice 1 --- p.62 / Chapter 2.3.4.1.2.2 --- Golden Rice 2 --- p.64 / Chapter 2.4 --- Motivation for striking forward --- p.67 / Chapter 2.4.1 --- Recommended Dietary Amount of vitamin A --- p.67 / Chapter 2.4.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.68 / Chapter 2.4.2.1 --- Bioavailability --- p.68 / Chapter 2.4.2.2 --- Bioconvertibility --- p.69 / Chapter 2.4.2.3 --- Health and nutritional status --- p.71 / Chapter 2.4.3 --- Further improvement for dealing with the deficiency --- p.73 / Chapter 2.5 --- Hypothesis --- p.75 / Chapter Chapter 3 --- Materials and Methods --- p.78 / Chapter 3.1 --- Chemicals --- p.78 / Chapter 3.2 --- Bacterial strains --- p.78 / Chapter 3.3 --- Transient expression of BCMOs in plant system --- p.79 / Chapter 3.3.1 --- Choice of BCMOs --- p.79 / Chapter 3.3.2 --- Plasmids and genetic material --- p.79 / Chapter 3.3.3 --- Construction of chimeric genes for transient expression --- p.82 / Chapter 3.3.4 --- Microprojectile bombardment and GUS assay --- p.83 / Chapter 3.4 --- Construction of chimeric genes for rice co-transformation --- p.84 / Chapter 3.4.1 --- Choice of carotenogenic genes --- p.84 / Chapter 3.4.2 --- Choice of promoters --- p.84 / Chapter 3.4.3 --- Necessities and choice of transit peptide (TP) --- p.85 / Chapter 3.4.4 --- Arrangement of chimeric gene-cassettes --- p.86 / Chapter 3.4.5 --- Plasmids and genetic materials --- p.87 / Chapter 3.4.6 --- Construction of chimeric gene expressing PSY and PDS coordinately --- p.87 / Chapter 3.4.7 --- "Construction of chimeric gene expressing PSY, PDS and TP equipped CHBCMO coordinately" --- p.92 / Chapter 3.4.8 --- "Construction of chimeric gene expressing PSY, PDS and TP equipped ZEBCMO coordinately" --- p.98 / Chapter 3.4.9 --- Construction of chimeric gene expressing ZDS and LYCB coordinately --- p.103 / Chapter 3.4.10 --- Confirmation of sequence fidelity --- p.108 / Chapter 3.5 --- Rice co-transformation --- p.109 / Chapter 3.5.1 --- Plant materials --- p.109 / Chapter 3.5.2 --- Preparation of Agrobacterium tumefaciens --- p.109 / Chapter 3.5.3 --- Agrobacterium mediated co-transformation --- p.110 / Chapter 3.5.3.1 --- Callus induction from mature rice seeds --- p.110 / Chapter 3.5.3.2 --- Callus induction from immature rice seeds --- p.110 / Chapter 3.5.3.3 --- "Co-cultivation, selection and regeneration" --- p.111 / Chapter 3.6 --- Detection of transgene expression --- p.112 / Chapter 3.6.1 --- Detection at DNA level --- p.112 / Chapter 3.6.1.1 --- Genomic DNA extraction --- p.112 / Chapter 3.6.1.2 --- PCR screening --- p.112 / Chapter 3.6.1.3 --- Synthesis of DIG-labeled DNA probes --- p.114 / Chapter 3.6.1.4 --- Southern blot analysis --- p.115 / Chapter 3.6.2 --- Detection at RNA level --- p.116 / Chapter 3.6.2.1 --- Total RNA extraction --- p.116 / Chapter 3.6.2.2 --- Northern blot analysis --- p.116 / Chapter 3.6.3 --- Detection at product level --- p.117 / Chapter 3.6.3.1 --- Phenotypic identification --- p.117 / Chapter 3.6.3.2 --- HPLC analysis --- p.117 / Chapter 3.6.3.2.1 --- Extraction of total carotenoids and retinoids --- p.117 / Chapter 3.6.3.2.2 --- HPLC identification --- p.118 / Chapter 3.6.3.2.3 --- HPLC quantification --- p.118 / Chapter Chapter 4 --- Results --- p.119 / Chapter 4.1 --- Transient expression of BCMOs in plant system --- p.119 / Chapter 4.1.1 --- Construction of chimeric genes for transient expression --- p.119 / Chapter 4.1.2 --- Microprojectile bombardment and GUS assay --- p.120 / Chapter 4.2 --- Construction of chimeric genes for rice co-transformation --- p.121 / Chapter 4.3 --- Rice co-transformation --- p.123 / Chapter 4.3.1 --- Callus induction from mature and immature rice seeds --- p.123 / Chapter 4.3.2 --- "Co-cultivation, selection and regeneration" --- p.124 / Chapter 4.4 --- Detection of transgene expression --- p.126 / Chapter 4.4.1 --- Detection at DNA level --- p.126 / Chapter 4.4.1.1 --- PCR screening --- p.126 / Chapter 4.4.1.2 --- Southern blot analysis --- p.129 / Chapter 4.4.2 --- Detection at RNA level --- p.133 / Chapter 4.4.2.1 --- Northern blot analysis --- p.133 / Chapter 4.4.3 --- Detection at product level --- p.135 / Chapter 4.4.3.1 --- Phenotypic identification --- p.135 / Chapter 4.4.3.2 --- HPLC identification --- p.137 / Chapter 4.4.3.3 --- HPLC quantification --- p.147 / Chapter Chapter 5 --- Discussion --- p.150 / Chapter Chapter 6 --- Conclusion --- p.156 / References --- p.157

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