Promoter analysis and endosperm-specific expression of rice phytoene synthase genes (psy1 and psy2) in rice.

Leung, Chiu Yi.Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.Includes bibliographical references (leaves 219-235).Abstracts in English and Chinese.ACKNOWLEDGEMENTS --- p.iiiABSTRACT --- p.v摘要 --- p.viiTABLE OF CONTENTS --- p.ixLIST OF FIGURES --- p.xivLIST OF TABLES --- p.xviiiLIST OF ABBREVIATIONS --- p.xixChapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.5Chapter 2.1 --- Introduction to carotenoids --- p.5Chapter 2.1.1 --- Structures and general chemical properties --- p.6Chapter 2.1.2 --- Dietary source of carotenoids --- p.8Chapter 2.2 --- Biosynthesis of carotenoids in plants --- p.8Chapter 2.2.1 --- Formation of isopretenyl diphosphate (IPP) --- p.12Chapter 2.2.2 --- "Formation of C40 backbone, phytoene" --- p.13Chapter 2.2.3 --- Desaturation reactions --- p.16Chapter 2.2.4 --- Isomerization --- p.18Chapter 2.2.5 --- Cyclization --- p.18Chapter 2.2.6 --- Xanthophylls synthesis --- p.19Chapter 2.2.6.1 --- Hydroxylation --- p.19Chapter 2.2.6.2 --- Epoxidation and de-epoxidation --- p.19Chapter 2.2.6.3 --- Neoxanthin formation --- p.20Chapter 2.2.7 --- Carotenoids catabolism by cleavage enzymes --- p.20Chapter 2.2.8 --- Carotenoids sequestration --- p.20Chapter 2.2.9 --- Regulations of Carotenogenesis in plant --- p.21Chapter 2.3 --- Roles of carotenoids in plants --- p.24Chapter 2.3.1 --- Precursor of abscisic acid (ABA) production --- p.24Chapter 2.3.2 --- Photomorphogenesis: Prolamella body formation --- p.27Chapter 2.3.3 --- "Light-harvesting, energy transfer and photoprotection" --- p.30Chapter 2.4 --- Importance of carotenoids to human --- p.34Chapter 2.4.1 --- Provitamin A activity and conversion --- p.34Chapter 2.4.2 --- Roles of vitamin A and carotenoids in diseases prevention --- p.36Chapter 2.4.2.1 --- Visual cycle and related diseases --- p.36Chapter 2.4.2.2 --- Cardiovascular diseases --- p.37Chapter 2.4.2.3 --- Cancer --- p.38Chapter 2.4.3 --- Roles of vitamin A in gene regulation --- p.39Chapter 2.4.4 --- Bioavailability and daily intake recommendation --- p.39Chapter 2.5 --- Vitamin A deficiency (VAD) --- p.42Chapter 2.5.1 --- Clinicopathological features --- p.44Chapter 2.5.1.1 --- Visual problems --- p.44Chapter 2.5.1.2 --- Infection --- p.45Chapter 2.6 --- Global efforts in combating VAD --- p.45Chapter 2.6.1 --- Dietary diversification --- p.46Chapter 2.6.2 --- Supplementation --- p.48Chapter 2.6.3 --- Pro-vitamin A enriched crops by genetical engineering --- p.49Chapter 2.6.3.1 --- Tomato --- p.49Chapter 2.6.3.2 --- Potato --- p.50Chapter 2.6.3.3 --- Canola --- p.50Chapter 2.6.3.4 --- The Golden Rice (GR) project --- p.51Chapter 2.6.3.4.1 --- The 1st generation (GR1) --- p.52Chapter 2.6.3.4.2 --- The 2nd generation (GR2) --- p.54Chapter 2.7 --- Rice phytoene synthase as a GR candidate enzyme --- p.55Chapter 2.7.1 --- General properties of phytoene synthase in higher plant --- p.55Chapter 2.7.1.1 --- Gene duplication and structure --- p.55Chapter 2.7.1.2 --- Membrane association and cation requirement --- p.57Chapter 2.7.1.3 --- Expression pattern and tissue-specificity --- p.58Chapter 2.7.2 --- "Rice phytoene synthases: Ospsy1, Ospsy2 and Ospsy3" --- p.60Chapter 2.7.2.1 --- Gene duplication and structure --- p.60Chapter 2.7.2.2 --- Protein structures and phylogenetic analysis --- p.60Chapter 2.7.2.3 --- Expression pattern --- p.62Chapter 2.7.2.4 --- Light- and stress-induced expression --- p.63Chapter 2.7.3 --- Rice phytoene synthases activity in rice seeds --- p.64Chapter 2.7.3.1 --- Previous study in rice seed carotenogenic capacity --- p.64Chapter 2.8 --- Seed-specific rice promoters --- p.66Chapter 2.8.1 --- Previous studies on seed-specific expression in rice --- p.66Chapter 2.8.1.1 --- Endosperm-specific cis-acting regulatory elements --- p.67Chapter 2.8.1.2 --- Requirements to confer endosperm-specific expression in rice --- p.69Chapter 2.9 --- Project overview and hypothesis --- p.71Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.73Chapter 3.1 --- Chemicals --- p.73Chapter 3.2 --- Vectors and bacterial strains in regular cloning --- p.73Chapter 3.3 --- Plant materials --- p.74Chapter 3.4 --- Construction of gene cassettes for plant transformation --- p.74Chapter 3.4.1 --- Construction of gene cassettes for Ospsyl and Ospsy2 study --- p.74Chapter 3.4.1.1 --- Cloning of Ospsyl and Ospsy2 from rice --- p.76Chapter 4.1.1.1 --- Total RNA extraction --- p.75Chapter 3.4.1.1.2 --- Amplification of cDNA of Ospsyl by RT-PCR --- p.77Chapter 3.4.1.1.3 --- Amplification of cDNA of Ospsy2 by PCR --- p.80Chapter 3.4.1.2 --- Cloning of constitutive CaMV35S promoter --- p.82Chapter 3.4.1.2.1 --- Preparation of pBI121 vector --- p.82Chapter 3.4.1.2.2 --- Amplification of CaMV35S promoter by PCR --- p.82Chapter 3.4.1.3 --- Cloning of endosperm-specific rice glutelin-1 (Gt-1) promoter --- p.84Chapter 3. 4.1.3.1 --- Genomic DNA extraction --- p.84Chapter 3.4.1.3.2 --- Amplification of Gt-1 promoter by PCR --- p.84Chapter 3.4.1.4 --- Construction of gene cassettes for Ospsyl and Ospsy2 driven by CaMV35S and rice Gt-1 promoter --- p.87Chapter 3.4.2 --- Construction of gene cassettes for promoter analysis --- p.90Chapter 3.4.2.1 --- Cloning of full length and fragments of Ospsyl promoter --- p.92Chapter 3.4.2.1.1 --- Genomic DNA extraction --- p.95Chapter 3.4.2.1.2 --- Amplification of full length and fragments of Ospsy1 promoter --- p.95Chapter 3.4.2.2 --- Cloning of CaMV35S minimal promoter --- p.96Chapter 3.4.2.2.1 --- Amplification of CaMV35S minimal promoter --- p.97Chapter 3.4.2.3 --- Cloning of rice Gt-1 promoter --- p.97Chapter 3.4.2.3.1 --- Genomic DNA extraction --- p.97Chapter 3.4.2.3.2 --- Amplification of Gt-1 promoter --- p.97Chapter 3.4.2.4 --- Annealing of linker --- p.93Chapter 3.4.3.5 --- Making of rice GCN4 multimers --- p.98Chapter 3.4.2.6 --- Construction of gene cassettes for promoter analysis --- p.99Chapter 3.4.2.7 --- Construction of gene cassettes for Gt-1 promoter analysis --- p.104Chapter 3.4.3 --- Confirmation of sequence fidelity --- p.106Chapter 3.5 --- Rice transformation --- p.107Chapter 3.5.1 --- Plant materials --- p.107Chapter 3.5.2 --- Preparation of Agrobacterium --- p.107Chapter 3.5.3 --- Agrobacterium mediated transformation --- p.108Chapter 3.5.4 --- Callus induction from mature rice seeds --- p.109Chapter 3.5.6 --- Co-cultivation and selection --- p.109Chapter 3.5.7 --- Pre-regeneration and regeneration of transgenic rice --- p.110Chapter 3.5.8 --- Plantation of transgenic rice --- p.110Chapter 3.6 --- Detection of transgene expression --- p.112Chapter 3.6.1 --- Detection at DNA level --- p.112Chapter 3.6.1.1 --- Genomic DNA extraction --- p.112Chapter 3.6.1.2 --- PCR screening --- p.122Chapter 3.6.1.3 --- Synthesis of DIG-labeled DNA probes --- p.116Chapter 3.6.1.4 --- Southern blot analysis --- p.118Chapter 3.6.2 --- Detection at RNA level --- p.119Chapter 3.6.2.1 --- Total RNA extraction --- p.119Chapter 3.6.2.2 --- Northern blot analysis --- p.119Chapter 3.6.3 --- Detection at protein level --- p.119Chapter 3.6.3.1 --- Antibody production --- p.119Chapter 3.6.3.1 --- Ospsyl and Ospsy2 induction in pET system --- p.120Chapter 3.63.1.2 --- Immunization of rabbit and serum collection --- p.123Chapter 3.6.3.2 --- Total protein extraction from plant materials --- p.124Chapter 3.6.3.2.1 --- Protein extraction from rice calli and leaves --- p.124Chapter 3.6.3.2.2 --- Protein extraction from immature and mature rice seeds --- p.124Chapter 3.6.3.3 --- Tricine SDS-PAGE --- p.125Chapter 3.6.3.4 --- Western blot analysis --- p.125Chapter 3.6.4 --- Detection at metabolite level --- p.126Chapter 3.6.4.1 --- Isoprenoids extraction from plant materials --- p.126Chapter 3.6.4.2 --- UPLC analysis for isoprenoid identification --- p.127Chapter 3.6.5 --- Detection of promoter activity --- p.128Chapter 3.6.5.1 --- Histochemical staining of GUS activity --- p.128Chapter 3.6.5.1.1 --- Histochemical staining --- p.128Chapter 3.6.5.1.2 --- Plant tissue fixation for microscopic observation --- p.128Chapter 3.6.5.2 --- GUS activity assay --- p.129Chapter 3.6.5.2.1 --- Protein extraction and quantitation with Bio-Rad protein assay --- p.129Chapter 3.6.5.2.2 --- G US activity assay --- p.130Chapter CHAPTER 4. --- RESULTS --- p.131Chapter 4.1 --- Tissue-specificity and endosperm specific expression of rice psy1 and Psy2 --- p.131Chapter 4.1.1 --- Construction of gene cassettes for study in Ospsy1 and Ospsy2 --- p.133Chapter 4.1.2 --- Rice transformation --- p.135Chapter 4.1.3 --- Transgene detection --- p.137Chapter 4.1.3.1 --- Genomic DNA PCR screening --- p.137Chapter 4.1.3.2 --- Southern blot analysis --- p.139Chapter 4.1.3.2.1 --- Southern blot analysis on transgenic rice calli --- p.141Chapter 4.1.3.2.2 --- Southern blot analysis on regenerated rice --- p.143Chapter 4.1.4 --- Detection of transgene expression --- p.149Chapter 4.1.4.1 --- Northern blot analysis on immature transgenic seed --- p.149Chapter 4.1.4.2 --- Western blot analysis on transgenic rice tissues --- p.152Chapter 4.1.4.2.1 --- Antibody production --- p.152Chapter 4.1.4.2.2 --- Western blot analysis of transgenic rice calli --- p.155Chapter 4.1.4.2.3 --- Western blot analysis of transgenic rice leaves --- p.157Chapter 4.1.4.2.4 --- Western blot analysis of immature transgenic rice seeds --- p.160Chapter 4.1.5 --- Detection of OsPSYs activity at metabolite level --- p.163Chapter 4.1.5.1 --- UPLC analysis on transgenic rice tissues --- p.163Chapter 4.1.5.1.1 --- Carotenoid profiling of transgenic rice calli --- p.163Chapter 4.1.5.1.2 --- Carotenoid profiling of transgenic rice leaves --- p.168Chapter "4.1.5.1,3" --- Carotenoid profiling of mature transgenic rice seeds --- p.172Chapter 4.2 --- Promoter analysis of modified rice psy1 promoter --- p.176Chapter 4.2.1 --- Construction of gene cassettes for promoter analysis --- p.178Chapter 4.2.2 --- Rice transformation --- p.180Chapter 4.2.3 --- Transgene detection --- p.180Chapter 4.2.3.1 --- Genomic DNA PCR screening --- p.180Chapter 4.2.3.2 --- Southern blot analysis --- p.185Chapter 4.2.3.2.1 --- Southern blot analysis of regenerated rice --- p.186Chapter 4.2.4 --- Detection of promoter activity --- p.196Chapter 4.2.4.1 --- Promoter activity in transgenic rice leaves --- p.196Chapter 4.2.4.1.1 --- Histochemical staining of GUS --- p.196Chapter 4.2.4.1.2 --- G US activity assay --- p.199Chapter 4.2.4.2 --- Promoter activity in transgenic immature seeds --- p.202Chapter 4.2.4.2.1 --- Histochemical staining of GUS --- p.202Chapter 4.2.4.2.2 --- GUS activity assay --- p.206Chapter CHAPTER 5. --- DISCUSSIONS --- p.209Chapter 5.1 --- Tissue-specificity and endosperm specific expression of rice psyl and psy2 --- p.209Chapter 5.1.1 --- OsPSYl and OsPSY2 activities in rice calli --- p.209Chapter 5.1.2 --- OsPSYl and OsPSY2 activities in rice leaves --- p.210Chapter 5.1.3 --- OsPSYl and OsPSY2 activities in rice seeds --- p.211Chapter 5.2 --- Analysis of modified rice psyl promoter --- p.213Chapter 5.3 --- Future prospects of Golden Rice --- p.214Chapter CHAPTER 6. --- CONCLUSIONS --- p.217REFERENCES --- p.21