published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Duan Meijuan. / Thesis submitted in: August 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 170-194). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Chinese abstract --- p.vi / List of Tables --- p.viii / List of Figures --- p.ix / List of Abbreviations --- p.xiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.3 / Chapter 2.1 --- Hybrid rice: Genetics and breeding --- p.3 / Chapter 2.1.1 --- Classification in rice --- p.5 / Chapter 2.1.2 --- Heterosis in rice --- p.6 / Chapter 2.1.2.1 --- Performance of heterosis in rice --- p.6 / Chapter 2.1.2.2. --- Genetic mechanism of heterosis in rice --- p.7 / Chapter 2.1.3 --- Utilization of heterosis in rice --- p.9 / Chapter 2.2 --- Grain quality in rice --- p.11 / Chapter 2.2.1 --- Composition of rice grain quality --- p.11 / Chapter 2.2.1.1 --- Appearance quality --- p.11 / Chapter 2.2.1.2 --- Milling quality --- p.11 / Chapter 2.2.1.3 --- Nutritional quality --- p.12 / Chapter 2.2.1.4 --- Cooking and eating quality --- p.17 / Chapter 2.2.2 --- Genetic and breeding for high nutritional quality rice --- p.17 / Chapter 2.2.3 --- "Structural, physiological and biochemical changes during rice seed development" --- p.18 / Chapter 2.3 --- Molecular biological characteristics of rice seed storage protein --- p.20 / Chapter 2.3.1 --- "Property, classification and structure" --- p.20 / Chapter 2.3.1.1 --- Property and classification --- p.20 / Chapter 2.3.1.2 --- Composition structure --- p.20 / Chapter 2.3.1.2.1 --- Glutelin --- p.21 / Chapter 2.3.1.2.2 --- Prolamin --- p.22 / Chapter 2.3.1.2.3 --- Globulin and Albumin --- p.23 / Chapter 2.3.2 --- "Structure, expression and regulation of genes encoding rice seed storage protein genes" --- p.24 / Chapter 2.3.2.1 --- Structure --- p.24 / Chapter 2.3.2.1.1 --- Glutelin gene family --- p.24 / Chapter 2.3.2.1.2 --- Prolamin gene family --- p.26 / Chapter 2.3.2.1.3 --- Albumin and globulin gene family --- p.27 / Chapter 2.3.2.2 --- Expression of storage proteins in rice seed development --- p.28 / Chapter 2.3.2.3 --- Regulation of expression of seed storage protein genes --- p.29 / Chapter 2.3.2.3.1 --- Regulation at transcriptional level --- p.29 / Chapter 2.3.2.3.2 --- Regulation at post-transcriptional level --- p.31 / Chapter 2.3.2.3.3 --- Regulation at translational level --- p.31 / Chapter 2.3.3 --- "Synthesis, processing and deposition of rice seed storage proteins" --- p.32 / Chapter 2.4 --- Molecular characteristics of starch in rice grain --- p.34 / Chapter 2.4.1 --- Property of rice starch --- p.34 / Chapter 2.4.2 --- Starch biosynthesis in rice --- p.37 / Chapter 2.4.3 --- Enzymes involved in starch biosynthesis in rice --- p.39 / Chapter 2.4.3.1 --- ADP-glucose pyrophosphorylase (AGPase) --- p.39 / Chapter 2.4.3.2 --- Soluble starch synthase (SSS) --- p.41 / Chapter 2.4.3.3 --- Granular-bound starch synthase (GBSS) --- p.42 / Chapter 2.4.3.4 --- Starch branching enzyme (SBE) --- p.44 / Chapter 2.4.3.5 --- Starch debranching enzyme (SDE) --- p.46 / Chapter 2.5 --- Aspartate family amino acid biosynthetic pathway in rice --- p.48 / Chapter 2.5.1 --- Introduction --- p.48 / Chapter 2.5.2 --- Two key regulatory enzymes involved in lysine synthesis pathway --- p.50 / Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.50 / Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.51 / Chapter 2.5.2.3. --- Regulation of lysine and the other end products of AK pathway --- p.52 / Chapter 2.6 --- Proteomics in plants --- p.53 / Chapter 2.7 --- Approaches for grain quality improvement in rice --- p.56 / Chapter 2.7.1 --- Improvement of nutrition quality --- p.56 / Chapter 2.7.2 --- Improvement of eating and cooking quality --- p.57 / Chapter 2.8 --- Objectives of my project --- p.58 / Chapter Chapter 3. --- Materials and Methods --- p.60 / Chapter 3.1 --- Materials --- p.60 / Chapter 3.1.1 --- Chemicals --- p.60 / Chapter 3.1.2 --- Apparatus and commercial kits --- p.60 / Chapter 3.1.3 --- Plant materials --- p.61 / Chapter 3.1.4 --- DNA sequencing --- p.61 / Chapter 3.1.5 --- Software --- p.61 / Chapter 3.2 --- Methods --- p.62 / Chapter 3.2.1 --- Search for protein and DNA sequences of all genes --- p.62 / Chapter 3.2.1.1 --- Genes encoding rice glutelin family --- p.62 / Chapter 3.2.1.2 --- Genes encoding rice prolamin family --- p.63 / Chapter 3.2.1.3 --- Genes encoding rice albumin family --- p.63 / Chapter 3.2.1.4 --- Genes encoding rice globulin family --- p.64 / Chapter 3.2.1.5 --- Genes encoding rice starch synthesis enzymes --- p.64 / Chapter 3.2.2 --- Alignment of homologous DNA sequence between family member genes --- p.65 / Chapter 3.2.2.1 --- Seeds storage protein gene families of rice seeds --- p.65 / Chapter 3.2.2.2 --- Rice starch synthase gene families --- p.67 / Chapter 3.2.3 --- Primer design --- p.69 / Chapter 3.2.4 --- Collection of developing hybrid rice seeds --- p.71 / Chapter 3.2.5 --- Total RNA extraction --- p.75 / Chapter 3.2.6 --- Quantification of total RNA and determination of internal control --- p.75 / Chapter 3.2.7 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.77 / Chapter 3.2.8 --- Northern blot analysis --- p.78 / Chapter 3.2.9 --- DNA sequencing --- p.79 / Chapter 3.2.10 --- Protein extraction --- p.80 / Chapter 3.2.10.1 --- Extraction of four kinds of storage proteins --- p.80 / Chapter 3.2.10.2 --- Extraction of the Wx protein --- p.81 / Chapter 3.2.11 --- Tricine SDS-PAGE --- p.82 / Chapter 3.2.12 --- "Determination of crude protein and amylose content in P64S,9311 and F1 hybrid" --- p.83 / Chapter 3.2.12.1 --- Determination of crude protein --- p.83 / Chapter 3.2.12.2 --- Determination of amylose content --- p.84 / Chapter 3.2.13 --- Two-dimension gel electrophoresis --- p.85 / Chapter 3.2.13.1 --- Clean up of protein sample for 2-D gel --- p.85 / Chapter 3.2.13.2 --- Quantification of protein samples --- p.86 / Chapter 3.2.13.3 --- First-dimension IEF (isoelectric focusing) --- p.86 / Chapter 3.2.13.4 --- IPG strips equilibration --- p.87 / Chapter 3.2.13.5 --- Second-dimension SDS PAGE --- p.87 / Chapter 3.2.13.6 --- Silver staining of 2-D gel --- p.88 / Chapter 3.2.14 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.88 / Chapter 3.2.14.1 --- Sample destaining --- p.88 / Chapter 3.2.14.2 --- In-gel digestion with trypsin enzyme --- p.89 / Chapter 3.2.14.3 --- Desalination of the digested sample with Zip Tip --- p.90 / Chapter 3.2.14.4 --- Mass spectrometry --- p.90 / Chapter Chapter 4. --- Results --- p.91 / Chapter 4.1 --- Quantification of the total RNA from developing seeds at different developingstages --- p.91 / Chapter 4.2 --- Determination of internal control --- p.92 / Chapter 4.3 --- DNA sequence analysis --- p.95 / Chapter 4.4 --- Profiling the expression of genes encoding rice seed storage proteins --- p.97 / Chapter 4.4.1 --- The glutelin genes --- p.97 / Chapter 4.4.1.1 --- The Gtl (GluA-2) gene --- p.100 / Chapter 4.4.1.2 --- The Gt2 (GluA-1) gene --- p.100 / Chapter 4.4.1.3 --- The Gt3 (GluA-3) gene --- p.101 / Chapter 4.4.1.4 --- Comparison of the expression profiles of GluA subfamily genes --- p.101 / Chapter 4.4.1.5 --- The GluB-1 gene --- p.101 / Chapter 4.4.1.6 --- The GluB-2 gene --- p.102 / Chapter 4.4.1.7 --- The GluB-4 gene --- p.102 / Chapter 4.4.1.8 --- Comparing of the expression profiles of GluB subfamily genes --- p.102 / Chapter 4.4.1.9 --- Profiling the expression of glutilin family genes in developing hybrid rice seeds --- p.103 / Chapter 4.4.1.10 --- Profiling glutelin gene expression in developing seeds at protein level --- p.103 / Chapter 4.4.2 --- Profiling the expression of genes encoding prolamin familyin developing hybrid rice seeds --- p.105 / Chapter 4.4.2.1 --- The 10-kDa prolamin gene --- p.105 / Chapter 4.4.2.2 --- The RP5 gene --- p.108 / Chapter 4.4.2.3 --- The RP6 gene --- p.108 / Chapter 4.4.2.4 --- The Prol 7 gene --- p.109 / Chapter 4.4.2.5 --- The Prol 14 gene --- p.109 / Chapter 4.4.2.6 --- The Prol 17 gene --- p.109 / Chapter 4.4.2.7 --- Expression profiles of prolamin family genes --- p.110 / Chapter 4.4.2.8 --- Expression profiles of prolamin genes in developing hybrid rice seeds at protein level --- p.111 / Chapter 4.4.3 --- Profiling the expression of genes encoding globulin familyin developing hybrid rice seed --- p.113 / Chapter 4.4.3.1 --- The 26-kDa globulin (alpha-globulin) gene --- p.113 / Chapter 4.4.3.2 --- The globulin 1 gene --- p.113 / Chapter 4.4.3.3 --- The globulin 2 gene --- p.115 / Chapter 4.4.3.4 --- The Low molecular weight (LMW) globulin gene --- p.115 / Chapter 4.4.3.5 --- Profiling the expression of the globulin family genes --- p.115 / Chapter 4.4.3.6 --- Expression profiles of globulin proteins in developing hybrid rice seeds at protein level --- p.117 / Chapter 4.4.4 --- Profiling the expression of genes encoding rice albumin familyin developing hybrid rice seeds --- p.118 / Chapter 4.4.4.1 --- The RA5 gene --- p.118 / Chapter 4.4.4.2 --- The RA 14 gene --- p.119 / Chapter 4.4.4.3 --- The RA 17 gene --- p.119 / Chapter 4.4.4.4 --- Profiling the expression of the albumin family genes --- p.121 / Chapter 4.4.4.5 --- Albumin gene expression in developing hybrid rice seeds at protein level --- p.121 / Chapter 4.4.5 --- Comparison of expression pattern of all genes encoding rice seed storage proteins in developing hybrid rice seeds --- p.123 / Chapter 4.4.6 --- Profiling the total proteins in developing hybrid rice seeds --- p.126 / Chapter 4.5 --- Profiling the expression of genes encoding rice starch synthasesin developing hybrid rice seeds --- p.127 / Chapter 4.5.1 --- Rice ADP-glucose pyrophosphorylase (AGPase) genes --- p.127 / Chapter 4.5.1.1 --- The AGPase large subunit gene --- p.127 / Chapter 4.5.1.2 --- The AGPase small subunit gene --- p.127 / Chapter 4.5.2 --- "The Wx (Granule bound starch synthase, GBSS) gene" --- p.129 / Chapter 4.5.3 --- Genes encoding rice SSS (Soluble starch synthase) family --- p.132 / Chapter 4.5.3.1 --- The SSS1 gene --- p.132 / Chapter 4.5.3.2 --- The SSS II-1 gene --- p.132 / Chapter 4.5.3.3 --- The SSS II-2 gene --- p.132 / Chapter 4.5.3.4 --- The SSS II-3 gene --- p.135 / Chapter 4.5.3.5 --- The SSS III-2 gene --- p.135 / Chapter 4.5.3.6 --- The SSS IV-1 gene --- p.135 / Chapter 4.5.3.7 --- The SSS IV-2 gene --- p.135 / Chapter 4.5.3.8 --- Profiling the expression of SSS family genes --- p.136 / Chapter 4.5.4 --- Genes encoding rice starch branching enzyme (SBE) family --- p.138 / Chapter 4.5.4.1 --- The SBE-1 gene --- p.138 / Chapter 4.5.4.2 --- The SBE-3 gene --- p.138 / Chapter 4.5.4.3 --- The SBE-4 gene --- p.138 / Chapter 4.5.4.4 --- Profiling the expression of SBE family genes --- p.140 / Chapter 4.5.5 --- Genes encoding rice starch debranching enzyme (SDE) family --- p.141 / Chapter 4.5.5.1 --- The isoamylase gene --- p.141 / Chapter 4.5.5.2 --- The pullulanase gene --- p.141 / Chapter 4.5.5.3 --- Difference between isoamylose and pullulanase --- p.141 / Chapter 4.5.6 --- Comparison of the expression patterns of genes encoding the enzymes involved in starch synthesis in developing hybrid rice seeds --- p.143 / Chapter 4.6 --- Profiling the expression of genes encoding aspartate family amino acid biosynthetic pathway in rice in developing hybrid rice seeds --- p.145 / Chapter 4.6.1 --- Rice AK (aspartate kinase) gene --- p.145 / Chapter 4.6.2 --- The DHPS gene --- p.145 / Chapter 4.7 --- Two-dimension gel electrophoresis and MALDI-ToF seed proteins analysis of rice --- p.147 / Chapter Chapter 5. --- Discussion --- p.152 / Chapter 5.1 --- Super hybrid rice as experimental material and its significance --- p.152 / Chapter 5.2 --- RT-PCR and northern blotting as methods to profile gene expression --- p.153 / Chapter 5.3 --- Regulation of genes related to nutritional quality in rice --- p.155 / Chapter 5.3.1 --- Storage protein genes --- p.155 / Chapter 5.3.2 --- Lysine synthesis enzymes --- p.158 / Chapter 5.4 --- Regulation of genes related to cooking and eating quality in rice --- p.159 / Chapter 5.5 --- Heredity of genes expression in F1 hybrid --- p.161 / Chapter 5.6 --- Application of 2-D gel electrophoresis --- p.162 / Chapter 5.7 --- Future perspectives --- p.163 / Chapter Chapter 6. --- Conclusion --- p.164 / References --- p.170

Zhang Junjun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-160). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement from Author --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xvi / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Literature Review --- p.2 / Chapter 1.2.1 --- Rice as a Model Monocot Cereal --- p.2 / Chapter 1.2.1.1 --- Genome Size --- p.3 / Chapter 1.2.1.2 --- Synteny --- p.3 / Chapter 1.2.1.3 --- Well-mapped Genome --- p.4 / Chapter 1.2.1.4 --- Amenable to Transformation --- p.4 / Chapter 1.2.2 --- Rice Genome Sequencing --- p.4 / Chapter 1.2.3 --- Rice Functional Genomics --- p.6 / Chapter 1.2.3.1 --- International Collaboration on Rice Functional Genomics --- p.6 / Chapter 1.2.3.2 --- Recent Progress on Rice Functional Genomics in China --- p.7 / Chapter 1.2.4 --- Large-scale Expressed Sequenced Tag (EST) Analysis --- p.8 / Chapter 1.2.4.1 --- Introduction --- p.8 / Chapter 1.2.4.2 --- Large-scale EST Analysis vs. SAGE and MPSS: Advantages and Shortcomings --- p.12 / Chapter 1.2.4.3 --- Large-scale Rice EST analysis --- p.14 / Chapter 1.2.4.4 --- Seed Specific Large-scale EST Analysis --- p.16 / Chapter 1.2.4.5 --- Bioinformatics Tools Involved --- p.17 / Chapter 1.2.5 --- Profiling Gene Expression using cDNA Microarray --- p.20 / Chapter 1.2.5.1 --- Introduction --- p.20 / Chapter 1.2.5.2 --- Advantages of cDNA Microarray in Gene Expression Profiling --- p.22 / Chapter 1.2.5.3 --- Profiling Gene Expression by cDNA Microarrays --- p.24 / Chapter 1.2.5.4 --- Profiling Seed-specific Gene Expression --- p.26 / Chapter 1.2.6 --- Overview of Current Information on the Biosynthesis and Gene Regulation of Major Components during Rice Seed Formation --- p.30 / Chapter 1.2.6.1 --- Starch --- p.30 / Chapter 1.2.6.2 --- Storage Proteins --- p.38 / Chapter 1.2.6.3 --- Lipids --- p.42 / Chapter Chapter 2 --- Materials and Methods --- p.44 / Chapter 2.1 --- Materials --- p.44 / Chapter 2.1.1 --- Chemicals --- p.44 / Chapter 2.1.2 --- Facilities and Instruments --- p.45 / Chapter 2.1.3 --- Commercial Kits --- p.46 / Chapter 2.1.4 --- Oligonucleotides --- p.47 / Chapter 2.1.5 --- Plant Materials --- p.50 / Chapter 2.1.6 --- Bacterial Strains --- p.52 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- mRNA Isolation --- p.52 / Chapter 2.2.2 --- Construction of cDNA Library --- p.53 / Chapter 2.2.2.1 --- Construction of cDNA Library in Phage --- p.53 / Chapter 2.2.2.2 --- Construction of cDNA Library in E.coli --- p.56 / Chapter 2.2.3 --- Screening of cDNA Library --- p.62 / Chapter 2.2.3.1 --- In situ Hybridization --- p.62 / Chapter 2.2.3.2 --- Probe Synthesis --- p.63 / Chapter 2.2.3.3 --- Hybridization and Detection --- p.64 / Chapter 2.2.4 --- Single-pass Sequencing of cDNAs --- p.65 / Chapter 2.2.5 --- Sequence Analysis --- p.67 / Chapter 2.2.6 --- BLAST Search --- p.71 / Chapter 2.2.7 --- Contig Analysis --- p.71 / Chapter 2.2.8 --- Database Management --- p.72 / Chapter 2.2.9 --- Selection Criteria for ESTs in Different Pathways --- p.72 / Chapter 2.2.10 --- Construction of Super Hybrid Rice Seed cDNA Microarray --- p.75 / Chapter 2.2.11 --- "Probe Synthesis, Microarray Hybridization and Detection" --- p.75 / Chapter 2.2.12 --- Quality Test of External Control --- p.78 / Chapter 2.2.13 --- Scanning of Microarray --- p.79 / Chapter 2.2.14 --- Analysis of Microarray Data --- p.79 / Chapter 2.2.15 --- Normalization --- p.79 / Chapter 2.2.16 --- Northern Blot Analysis --- p.80 / Chapter Chapter 3 --- Results and Discussion --- p.82 / Chapter 3.1 --- cDNA Libraries --- p.82 / Chapter 3.1.1 --- cDNA Library in Phage --- p.82 / Chapter 3.1.2 --- cDNA Library in E.coli --- p.84 / Chapter 3.2 --- Statistic Data of Screening and Single-pass Sequencing --- p.87 / Chapter 3.3 --- Genes Expressing in Developing Rice Seeds --- p.89 / Chapter 3.4 --- Novel ESTs in the Seed EST Set --- p.94 / Chapter 3.5 --- Unique Genes in the EST Set from the Phage cDNA Library --- p.94 / Chapter 3.6 --- Mapping ESTs onto Rice Genome --- p.95 / Chapter 3.7 --- The Most Abundant Transcripts Reflected by EST Redundancy --- p.98 / Chapter 3.8 --- ESTs and Representive Genes in Different Pathways --- p.102 / Chapter 3.9 --- ESTs Involved in Carbon Flows --- p.106 / Chapter 3.10 --- Microarray Configuration and Contents --- p.109 / Chapter 3.11 --- Quantification of Total RNA from Seeds at 5 Developing Stages --- p.110 / Chapter 3.12 --- Quality Test of External Control --- p.111 / Chapter 3.13 --- Scanning of Array Hybridization --- p.113 / Chapter 3.14 --- Reproducibility of Technical Duplicates --- p.116 / Chapter 3.15 --- Summary of Gene Expression Profiles in Developing Super Hybrid Rice Seeds --- p.117 / Chapter 3.16 --- Expression Profiles of Major Storage Compounds in Rice Seeds --- p.119 / Chapter 3.16.1 --- Expression profiles of storage protein genes --- p.119 / Chapter 3.16.2 --- Expression Profiles of Starch Synthesis Related Genes --- p.122 / Chapter 3.16.3 --- Expression Profiles of Lipid Synthesis Related Genes --- p.124 / Chapter 3.16.4 --- General Expression Patterns of Major Storage Compounds --- p.126 / Chapter 3.18 --- General Discussion --- p.131 / Chapter 3.18.1 --- Cautions when Interpreting the EST Data --- p.131 / Chapter 3.18.2 --- Comparison of EST Data Sets of Developing Seeds between Arabidopsis and Rice --- p.131 / Chapter 3.18.3 --- Comparison of cDNA Library Construction by Two Methods --- p.135 / Chapter 3.18.4 --- Comparison of Different Normalization Methods --- p.138 / Chapter 3.18.5 --- Comparison between Microarray and Northern Data --- p.140 / Chapter Chapter 4 --- Conclusion --- p.142 / Chapter Chapter 5 --- Future Perspectives --- p.145 / Reference Lists --- p.147

着丝粒和端粒是真核细胞染色体的重要组成部分，他们都是由DNA和蛋白质组成的复合体。研究发现水稻的着丝粒DNA含有大量CentO卫星重复序列，而端粒DNA由富含鸟嘌呤的重复序列组成。他们的蛋白质成份在着丝粒和端粒发挥其功能的过程中起到非常重要的作用，然而对这些蛋白的了解却很少。该项研究通过使用affinity pull down技术和其他蛋白质组学方法捕捉到了一系列DNA特异性结合的蛋白；其中有86个与CentO序列结合和135个与水稻端粒重复序列结合的蛋白质被捕捉到。通过使用体外蛋白质与DNA结合验证方法，其中的一个蛋白Os02g0288200被证实了具有特异性结合着丝粒DNA的功能。同时，发现了4个水稻端粒结合蛋白。这些结果显示了affinity pull down技术能有效的应用于分离DNA特异结合蛋白，特别是着丝粒和端粒DNA结合蛋白的研究中。此外，在CenH3体外功能研究中，我们发现水稻内源CenH3蛋白对不同DNA序列的结合能力不同；与水稻rDNA序列相比，CenH3对着丝粒特异的DNA序列的结合能力更强。在研究DNA甲基化对CenH3与DNA结合能力的实验中，我们同时发现水稻CenH3蛋白对甲基化的CentO序列比未修饰的CentO序列的结合能力弱。这个结果同着丝粒功能区DNA次甲基化相吻合。 / Centromeres and telomeres are both DNA/protein complex and essential functional components of eukaryotic chromosomes. Previous researches have shown that rice centromeres and telomeres are occupied by CentO satellite repeat and G-rich telomere repeats, respectively. However, the protein components are not fully understood. DNA binding proteins associated with centromeric or telomeric DNA components will be most likely important for the understanding of centromere and telomere structure and functions. To capture DNA specific binding proteins, affinity pull down technique was applied in this research to isolate rice centromeric and telomeric DNA binding proteins. 86 proteins and 135 proteins were pulled down from CentO column and telomere repeat column respectively. One putative CentO binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this CentO binding protein, and proven to be responsible for the specific binding to CentO sequence in vitro. Four proteins identified as telomere binding proteins in this research were studied by different groups and reported previously. These results demonstrate that the DNA affinity pull down technique is powerful in the isolation of sequence specific binding proteins and may be applicable in future studies of centromere and telomere proteins. In addition, the binding affinity of CenH3 to various forms of DNAs was analyzed by in vitro studies. The results show that rice endogenous CenH3 binds stronger to rice centromeric DNA sequences than rDNA sequence control, and prefers unmethylated CentO DNA sequence to methylated form. This phenomenon may provide explanation of the hypomethylation of centromeric DNAs in active centromeres. / Detailed summary in vernacular field only. / He, Qi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 50-55). / Abstracts also in Chinese. / List of Figures --- p.iii / List of Tables --- p.iv / List of Abbreviations --- p.v / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.ix / Chapter Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter Chapter 2 --- IDENTIFICATION OF CENTROMERE AND TELOMERE DNA BINDING PROTEINS IN RICE --- p.10 / Chapter Chapter 3 --- IN VITRO STUDIES OF CENH3 BINDING TO CENTRIMERIC DNA AND ITS METHYLATED FORM --- p.36 / Chapter Chapter 4 --- CONCLUSIONS AND PERSPECTIVES --- p.48 / References --- p.50

Cheung, Wai Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 66-76). / Abstracts in English and Chinese. / List of Figures --- p.v / List of Tables --- p.vi / List of Abbreviations --- p.vii / Acknowledgements --- p.ix / Abstract --- p.xi / 摘要 --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Centromere and Kinetochore --- p.1 / Chapter 1.2 --- The Kinetochore Subunits: Centromeric Nucleosomes --- p.2 / Chapter 1.3 --- CenH3: The Centromere Specific Histone --- p.5 / Chapter 1.4 --- The Centromeric DNA: Tandem Repeats and Retrotransposons --- p.8 / Chapter 1.5 --- The Genetic and Epigenetic Nature of the Centromeres --- p.9 / Chapter 1.6 --- Point Centromeres and Regional Centromeres --- p.10 / Chapter 1.7 --- Holocentric Chromosomes --- p.11 / Chapter 1.8 --- Hypothesis --- p.13 / Chapter Chapter 2 --- Materials and methods --- p.15 / Chapter 2.1 --- Chemicals --- p.15 / Chapter 2.2 --- Bacterial strains in routine cloning --- p.15 / Chapter 2.3 --- Plant materials in cloning and transformation --- p.15 / Chapter 2.4 --- Construction of LnCENH3-GFP and CeHCP3-DsRED chimeric gene cassettes for rice transformation --- p.15 / Chapter 2.5 --- Cloning of CENH3 gene of Luzula spp --- p.22 / Chapter 2.6 --- Construction of full length OsCENH3 RNAi and 150bp OsCENH3 RNAi constructs for rice transformation --- p.22 / Chapter 2.7 --- Agrobacterium-mediated transformation of rice (Oryza sativa L.japonica. cv. Nipponbare) --- p.24 / Chapter 2.8 --- Gene gun transformation of rice (Oryza sativa L.japonica. cv. Nipponbare) by Biolistic PDS-1000/He´ёØ System (Bio-rad) --- p.26 / Chapter 2.9 --- Detection of transgenes expression --- p.28 / Chapter 2.10 --- Nuclear protein extraction --- p.29 / Chapter 2.11 --- Protein-DNA Binding Assay --- p.30 / Chapter 2.12 --- Protein precipitation by methanol-chloroform --- p.32 / Chapter 2.13 --- Western blot analysis of proteins from Protein-DNA binding assay --- p.33 / Chapter 2.14 --- Tubulin immunolocalization of root tips --- p.33 / Chapter 2.15 --- Bioinformatics analysis --- p.34 / Chapter Chapter 3 --- Results --- p.36 / Chapter 3.1 --- Plant transformation vectors construction --- p.36 / Chapter 3.2 --- Rice transformation --- p.38 / Chapter 3.3 --- Transgenic plants screening --- p.39 / Chapter 3.4 --- Analysis of the codon usages of CeHCP-3 gene in C. elegans and O. sativa --- p.42 / Chapter 3.5 --- In vitro Protein-DNA binding assays --- p.44 / Chapter 3.6 --- Subcellular localization study of LnCENH3 in rice --- p.46 / Chapter 3.7 --- Chromosome morphology of the transgenic rice expression LnCENH3 --- p.47 / Chapter 3.8 --- Tubulin immunolocalization of LnCENH3-GFP transgenic rice --- p.49 / Chapter 3.9 --- Cloning of CENH3s in Luzula genus --- p.51 / Chapter 3.10 --- Bioinformatics analysis of Luzula CENH3s --- p.53 / Chapter Chapter 4 --- Discussion --- p.57 / Chapter 4.1 --- Expression of fusion proteins in rice --- p.57 / Chapter 4.2 --- Incorporation of LnCENH3-GFP in nucleosomes --- p.57 / Chapter 4.3 --- Expression pattern of LnCENH3-GFP in rice --- p.58 / Chapter 4.4 --- Formation of additional kinetochores on transgenic rice chromosome --- p.60 / Chapter 4.5 --- Deletion of 8 amino acids in LeCENH3 --- p.62 / Chapter Chapter 5 --- Conclusion --- p.65 / References --- p.66 / Chapter 5.1 --- Appendix --- p.77

Sze, Wing Ho Angel. / "December 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 90-112). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review / Chapter 2.1. --- Carotenoids in plants --- p.5 / Chapter 2.2 --- Carotenoid biosynthesis in plants --- p.7 / Chapter 2.3. --- Carotenoids in animals --- p.11 / Chapter 2.4. --- Vitamin A deficiency (VAD) --- p.13 / Chapter 2.5. --- Recommended requirement of vitamin A --- p.15 / Chapter 2.6. --- Bioavailability and bioconversion of dietary carotenoids --- p.17 / Chapter 2.7. --- Efforts to improve carotenoid contents in food crops --- p.19 / Chapter 2.8. --- Carotenoid catabolism --- p.20 / Chapter 2.9. --- Carotenoid cleavage dioxygenase (CCD) --- p.21 / Chapter 2.10. --- Carotenoid-derived phytohormones --- p.24 / Chapter 2.11. --- "CCD and carotenoid-derived colors, aromas and flavors" --- p.27 / Chapter Chapter 3. --- Hypothesis and Objectives --- p.35 / Chapter Chapter 4. --- Materials and methods / Chapter 4.1. --- General cloning and sequencing --- p.36 / Chapter 4.2. --- Extraction of RNA and DNase treatment --- p.36 / Chapter 4.3. --- Reverse transcription --- p.37 / Chapter 4.4. --- Real-time quantitative RT-PCR --- p.39 / Chapter 4.5. --- Cloning of OsCCD1 cDNA --- p.40 / Chapter 4.6. --- Bacterial in vivo assay of OsCCD 1 activity --- p.41 / Chapter 4.7. --- Construction of OsCCD1 RNAi constructs --- p.42 / Chapter 4.8. --- "Construction of ""Super-Golden"" rice constructs" --- p.46 / Chapter 4.8.1. --- "Construction of ""GluC-Y1-Nos"" cassette" --- p.46 / Chapter 4.8.2. --- "Construction of ""Gt1-TCN"" cassette" --- p.46 / Chapter 4.8.3. --- "Construction of""pGT-PCC""" --- p.47 / Chapter 4.8.4. --- "Construction of ""pGYGC""" --- p.47 / Chapter 4.9. --- Rice transformation --- p.54 / Chapter 4.10. --- Detection of transgene --- p.57 / Chapter 4.10.1. --- Southern blot --- p.57 / Chapter 4.10.2. --- HPLC analysis of carotenoids in seeds --- p.59 / Chapter Chapter 5. --- Results / Chapter 5.1. --- Expression profiles of carotenogenic genes in rice endosperms --- p.62 / Chapter 5.2. --- Expression of CCDs in developing rice seeds --- p.64 / Chapter 5.3. --- Features of OsCCD1 --- p.68 / Chapter 5.4. --- Characterization of OsCCD1-knock down transgenic rice --- p.72 / Chapter 5.5. --- "Construction of ""Super-Golden"" rice" --- p.78 / Chapter 5.6. --- Phenotypic characterization of PCC transgenic rice --- p.79 / Chapter 5.7. --- HPLC analysis on seed carotenoid content --- p.80 / Chapter Chapter 6. --- Discussion --- p.82 / Chapter Chapter 7. --- Conclusion --- p.89 / References --- p.90

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy