by Wong Wing Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 118-129). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Life cycle of L. edodes --- p.3 / Chapter 1.3 --- Environmental Stimuli for primoridum initiation --- p.6 / Chapter 1.4 --- Two component system (TCS) --- p.7 / Chapter 1.4.1 --- Modular structure of TCS --- p.7 / Chapter 1.4.2 --- Overview of the difference of TCS from other signaling cascades --- p.8 / Chapter 1.4.3 --- Functional roles of TCS in different organisms --- p.9 / Chapter 1.4.4 --- Molecular studies on histidine kinases --- p.16 / Chapter 1.5 --- Rationale and Summary --- p.18 / Chapter Chapter Two --- Screening of Differentially Expressed Genes / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and Methods --- p.24 / Chapter 2.2.1 --- Reverse Dot Blot Hybridization --- p.24 / Chapter 2.2.1.1 --- Construction of primordial cDNA library --- p.24 / Chapter 2.2.1.2 --- PCR amplification of cDNA clones --- p.24 / Chapter 2.2.1.3 --- Membrane preparation --- p.24 / Chapter 2.2.1.4 --- cDNA probe preparation --- p.25 / Chapter 2.2.1.4.1 --- RNA extraction --- p.25 / Chapter 2.2.1.4.2 --- RAP-PCR --- p.26 / Chapter 2.2.1.4.3 --- Purification of RAP-PCR product --- p.27 / Chapter 2.2.1.4.4 --- Labeling of probe --- p.27 / Chapter 2.2.1.5 --- Stringent washing and signal detection --- p.27 / Chapter 2.2.2 --- Confirmation of the expression level of differentially expressed genes by Reverse transcription PCR (RT-PCR) --- p.28 / Chapter 2.2.2.1 --- Primer Design --- p.28 / Chapter 2.2.2.2 --- First strand total cDNA synthesis --- p.28 / Chapter 2.2.2.3 --- Reverse transcription PCR --- p.29 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Screening of differentially expressed genes --- p.30 / Chapter 2.3.2 --- Sequence analysis --- p.30 / Chapter 2.3.3 --- Confirmations of differential expression of candidate genes by RT-PCR --- p.34 / Chapter 2.4 --- Discussion --- p.39 / Chapter 2.4.1 --- Putative roles of differentially expressed genes --- p.39 / Chapter 2.4.2 --- Confirmation of differential expression --- p.40 / Chapter Chapter Three --- Characterization and Full Length Sequence Analysis of Le.nikl clone / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and Methods --- p.43 / Chapter 3.2.1 --- Full length sequence of partial Le.nikl cDNA clone by primer walking / Chapter 3.2.1.1 --- Primer Design --- p.43 / Chapter 3.2.2 --- Confirmations of differentially expressed Le.nikl by Northern blot analyses --- p.44 / Chapter 3.2.2.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.44 / Chapter 3.2.2.2 --- Northern blotting --- p.45 / Chapter 3.2.2.3 --- Probe preparation --- p.46 / Chapter 3.2.2.4 --- "Hybridization, Stringency washes, Signal detection" --- p.46 / Chapter 3.2.3 --- Cloning of 5' end of Le.nikl --- p.47 / Chapter 3.2.3.1 --- Amplification of 5' partial end of Le.nikl from primordium cDNA library --- p.47 / Chapter 3.2.3.2 --- Polishing the PCR products --- p.48 / Chapter 3.2.3.3 --- Cloning of PCR products --- p.48 / Chapter 3.2.3.4 --- PCR screening of the transformants --- p.49 / Chapter 3.2.3.5 --- Sequencing analysis --- p.49 / Chapter 3.2.3.6 --- PCR for confirmation of the 5' sequence originated from Le.nikl gene instead of genes from the same gene family --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Northern analysis of Le.nik1 --- p.51 / Chapter 3.3.2 --- Sequence analysis of 2.75kb Le.nikl / Chapter 3.4 --- Discussion --- p.63 / Chapter Chapter Four --- Protein Interaction Study of Response Regulator of Le.NIK1 by Yeast two hybrid / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Materials and Methods --- p.68 / Chapter 4.2.1 --- "Yeast strains, media, yeast vectors" --- p.68 / Chapter 4.2.2 --- Bait construction --- p.68 / Chapter 4.2.2.1 --- "Cloning of bait insert Le.nik1-rec into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.68 / Chapter 4.2.2.1.1 --- Design of cloning primer --- p.70 / Chapter 4.2.2.1.2 --- First strand total cDNA synthesis --- p.70 / Chapter 4.2.2.1.3 --- PCR amplification of bait insert --- p.70 / Chapter 4.2.2.2 --- Small-scale transformation of GBKT7-Le.nik1-rec plasmid --- p.71 / Chapter 4.2.2.2.1 --- Preparation of yeast competent cells --- p.71 / Chapter 4.2.2.2.2 --- Transformation of the GBKT7-Le.nik1-rec plasmid into the yeast strain AH109 --- p.72 / Chapter 4.2.2.2.3 --- Test for the Self-Activation of DNA-BD fusion --- p.72 / Chapter 4.2.2.2.4 --- Verification of bait protein expression --- p.72 / Chapter 4.2.2.2.4.1 --- Yeast protein extraction by TCA method --- p.72 / Chapter 4.2.2.2.4.2 --- Western blot analyses --- p.73 / Chapter 4.2.2.2.4.2.1 --- SDS-PAGE --- p.73 / Chapter 4.2.2.2.4.2.2 --- Western blotting --- p.75 / Chapter 4.2.2.2.4.2.3 --- Immunodetection --- p.75 / Chapter 4.2.2.2.4.2.4 --- ECL detection --- p.75 / Chapter 4.2.2.2.5 --- Test for the toxicity of DNA-BD fusion --- p.76 / Chapter 4.2.3 --- Prey construction -Total primordium cDNA synthesis --- p.76 / Chapter 4.2.3.1 --- First-strand cDNA synthesis --- p.76 / Chapter 4.2.3.2 --- ds cDNA amplification --- p.77 / Chapter 4.2.3.3 --- ds cDNA purification --- p.77 / Chapter 4.2.4 --- Yeast two hybrid screening assay --- p.77 / Chapter 4.2.4.1 --- Yeast competent cells preparation --- p.77 / Chapter 4.2.4.2 --- Screening by co-transformation --- p.78 / Chapter 4.2.4.2.1 --- for positive and negative control --- p.78 / Chapter 4.2.4.2.2 --- for prey ds DNA and pGBKT7-Le.nik1-rec --- p.78 / Chapter 4.2.4.3 --- Selection for transformants expressing interacting proteins --- p.78 / Chapter 4.2.5 --- β-galactosidase analysis- colony lift filter assay --- p.79 / Chapter 4.2.6 --- PCR screening of the prey --- p.80 / Chapter 4.2.7 --- DNA Sequencing of the prey insert --- p.80 / Chapter 4.2.8 --- Plasmid Isolation --- p.81 / Chapter 4.2.8.1 --- Isolation and sequencing of yeast plasmid --- p.81 / Chapter 4.2.9 --- Confirmations of protein interaction and self-activation test of AD-prey insert by small-scale co-transformation --- p.82 / Chapter 4.2.10 --- Northern Blot analyses of prey genes --- p.82 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- DNA-BD fusion construction --- p.83 / Chapter 4.3.2 --- AD fusion library construction --- p.88 / Chapter 4.3.3 --- Yeast two hybrid screening assay by co-transformation --- p.88 / Chapter 4.4 --- Discussion --- p.96 / Chapter Chapter Five --- Effect of Different Mannitol Osmolarity on Le.nikl Transcriptional Expression / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials and Methods --- p.102 / Chapter 5.2.1 --- "Mycelium inoculum preparation, cultivation mediums and cultivation conditions" --- p.102 / Chapter 5.2.2 --- RNA extraction --- p.102 / Chapter 5.2.3 --- Northern analysis --- p.102 / Chapter 5.3 --- Results --- p.103 / Chapter 5.4 --- Discussion --- p.112 / Chapter 5.4.1 --- Effect of high osmolarity on mushroom growth --- p.112 / Chapter 5.4.2 --- Effect of high osmolarity on gene expression --- p.113 / Chapter Chapter Six --- General Discussion --- p.114 / References --- p.118

by Chan Ngar Lei Annie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 90-102. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF FIGURES --- p.vii / LIST OF TABLES --- p.ix / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 2. --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Economic and Biotechnological Significance of Lentinus edodes --- p.5 / Chapter 2.1.1 --- General Review --- p.5 / Chapter 2.1.2 --- Nutritional and Medicinal Values --- p.7 / Chapter 2.1.3 --- Lignocellulose Degradation and Utilization --- p.9 / Chapter 2.2 --- Biological Background --- p.11 / Chapter 2.2.1 --- Life Cycle --- p.11 / Chapter 2.2.2 --- Patterns of Sexuality --- p.12 / Chapter 2.3 --- Genetic Improvement of Lentinus edodes --- p.16 / Chapter 2.3.1 --- Introduction --- p.16 / Chapter 2.3.2 --- Mutagenic Agents --- p.17 / Chapter 2.3.3 --- Genetic Markers and Strain Improvement --- p.20 / Chapter 3. --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- Biological Materials --- p.24 / Chapter 3.2 --- Media --- p.25 / Chapter 3.2.1 --- Complete Medium (CM) --- p.25 / Chapter 3.2.2 --- Complete Medium with Yeast Extract (CM with Y.E.) --- p.25 / Chapter 3.2.3 --- Complete Fruiting Medium (CF) --- p.25 / Chapter 3.2.4 --- Complete Migration Medium (CMM) --- p.25 / Chapter 3.2.5 --- Minimal Medium (MM) --- p.25 / Chapter 3.2.6 --- Carboxymethyl Cellulose - Leatham Medium (CMC - Leatham) --- p.26 / Chapter 3.2.7 --- Potato Dextrose Agar (PDA) --- p.26 / Chapter 3.3 --- Anti-metabolites for Screening of Resistant Mutants --- p.27 / Chapter 3.4 --- Characterization of Monokaryons --- p.28 / Chapter 3.4.1 --- Isolation of Monosporous Mycelia --- p.28 / Chapter 3.4.2 --- Assessment of Mycelial Growth --- p.28 / Chapter 3.4.3 --- Determination of Mating Type of Monosporous Mycelia --- p.28 / Chapter 3.5 --- Mutagenesis and Isolation of Mutants --- p.29 / Chapter 3.5.1 --- Effects of Homogenization on Growth --- p.29 / Chapter 3.5.2 --- Determination of the Ultraviolet Irradiation Killing Curve --- p.29 / Chapter 3.5.3 --- Isolation of High Temperature Tolerant Strains --- p.30 / Chapter 3.5.4 --- Isolation of Auxotrophic Mutants --- p.30 / Chapter 3.5.5 --- Isolation of Anti-metabolite Resistant Mutants --- p.33 / Chapter 3.6 --- Characterization of Anti-metabolite Resistant Mutants --- p.33 / Chapter 3.6.1 --- "Measurements of Growth Rate, Anti-metabolites Resistance and Osmotic Sensitivity" --- p.33 / Chapter 3.6.2 --- Determination of Dominance Relationships --- p.34 / Chapter 3.6.3 --- Detection of Extracellular Enzymes --- p.34 / Chapter 4. --- RESULTS --- p.36 / Chapter 4.1 --- Radial Growth Rate of Monosporous Cultures --- p.36 / Chapter 4.2 --- Mating Reactions in Lentinus edodes --- p.36 / Chapter 4.3 --- Effects of Homogenization on Growth of L. edodes --- p.46 / Chapter 4.4 --- Determination of Ultraviolet (UV) Killing Curve --- p.46 / Chapter 4.5 --- Selection of High Temperature Tolerant Strains --- p.50 / Chapter 4.6 --- Auxotrophic Mutants Isolation and Identification --- p.50 / Chapter 4.7 --- Selection of Anti-metabolite Resistant Mutants --- p.52 / Chapter 4.8 --- Characterization of Cycloheximide Resistant Mutants V --- p.63 / Chapter 4.8.1 --- "Growth Rate, Anti-metabolite Resistance and Osmotic Sensitivity of Cycloheximide Resistant Mutants" --- p.63 / Chapter 4.8.2 --- Dominance Tests for Cycloheximide Resistant Strains --- p.66 / Chapter 4.8.3 --- Detection of Extracellular Enzymes --- p.73 / Chapter 5. --- DISCUSSION --- p.78 / Chapter 5.1 --- Mating Reactions in Lentinus edodes --- p.78 / Chapter 5.2 --- Isolation of Auxotrophic and Anti-metabolites Resistant Mutants --- p.81 / Chapter 5.3 --- Characterization of Cycloheximide Resistant Mutants of Lentinus edodes --- p.84 / CONCLUSION --- p.88 / REFERENCES --- p.90

by Leung Sze Wan, Grace. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 183-200). / Abstract also in Chinese. / Abstract --- p.iii / Acknowledgment --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xv / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- The life of Lentinula edodes --- p.3 / Chapter 1.3 --- Biochemical and molecular studies on mushroom development --- p.6 / Chapter 1.3.1 --- From monokaryotic to dikaryotic mycelium --- p.7 / Chapter 1.3.2 --- Initiation and differentiation of the primordium --- p.11 / Chapter 1.3.3 --- Growth and maturation of the fruit body --- p.21 / Chapter 1.4 --- Prospectus --- p.25 / Chapter Chapter Two --- Isolation of Genes Differentially Expressed During the Development of Lentinula edodes by RAP-PCR / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.32 / Chapter 2.2.1 --- Strains and culture conditions --- p.32 / Chapter 2.2.2 --- Isolation of total RNAs --- p.32 / Chapter 2.2.3 --- RNA fingerprinting by RAP-PCR --- p.34 / Chapter 2.2.4 --- PCR reamplification of RAP products --- p.34 / Chapter 2.2.5 --- Reverse dot-blot analysis --- p.35 / Chapter 2.2.5.1 --- Membrane preparation --- p.35 / Chapter 2.2.5.2 --- Probe preparation --- p.36 / Chapter 2.2.5.3 --- Hybridization --- p.37 / Chapter 2.2.5.4 --- Stringency washes and autoradiography --- p.38 / Chapter 2.2.6 --- Cloning and sequencing of differentially expressed genes --- p.38 / Chapter 2.2.6.1 --- Ligation of inserts into pCR-Script vector --- p.38 / Chapter 2.2.6.2 --- Transformation --- p.39 / Chapter 2.2.6.3 --- PCR screening of white colonies --- p.41 / Chapter 2.2.6.4 --- Extraction of plasmid DNA --- p.41 / Chapter 2.2.6.5 --- DNA cycle sequencing --- p.42 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Total RNA isolation --- p.44 / Chapter 2.3.2 --- RNA fingerprints --- p.48 / Chapter 2.3.3 --- Reverse dot-blot analysis --- p.53 / Chapter 2.3.4 --- Cloning and sequencing of selected RAP-products --- p.61 / Chapter 2.3.5 --- Sequence analyses --- p.61 / Chapter 2.4 --- Discussion --- p.77 / Chapter Chapter Three --- Expression Pattern Analysis by Northern Blot Hybridization / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.2 --- Materials and Methods --- p.85 / Chapter 3.2.1 --- Primer design & PCR amplification of Le.ras fragment --- p.85 / Chapter 3.2.2 --- Primer design & PCR amplification of L. edodes GAPDH gene --- p.85 / Chapter 3.2.3 --- Cloning & sequencing of L edodes GAPDH gene --- p.86 / Chapter 3.2.4 --- Northern blot analysis --- p.87 / Chapter 3.2.4.1 --- RNA extraction by Tri-reagent --- p.87 / Chapter 3.2.4.2 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.88 / Chapter 3.2.4.3 --- Northern blotting --- p.88 / Chapter 3.2.4.4 --- Preparation of probes --- p.89 / Chapter 3.2.4.5 --- Hybridization and stringency washes --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Establishing an internal control for expression level studies I: Le.ras --- p.91 / Chapter 3.3.2 --- Establishing an internal control for expression level studies II: GAPDH gene --- p.91 / Chapter 3.3.3 --- Northern blot hybridizations of RAP-fragments --- p.92 / Chapter 3.4 --- Discussion --- p.101 / Chapter Chapter Four --- Obtaining Full-length cDNA of L. edodes MAP kinase and Cyclin B by Rapid Ampification of cDNA Ends (RACE) / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.1.1 --- Principles of 3´ة RACE --- p.107 / Chapter 4.1.2 --- Principles of 5' RACE --- p.109 / Chapter 4.2 --- Materials and Methods --- p.112 / Chapter 4.2.1 --- Isolation of Total RNA --- p.112 / Chapter 4.2.2 --- 3'RACE --- p.112 / Chapter 4.2.2.1 --- First Strand cDNA Synthesis --- p.112 / Chapter 4.2.2.2 --- Amplification of the Target cDNA --- p.113 / Chapter 4.2.2.3 --- Reamplification and nested amplification of 3'RACE products --- p.114 / Chapter 4.2.3 --- 5'RACE --- p.115 / Chapter 4.2.3.1 --- First Strand cDNA Synthesis --- p.115 / Chapter 4.2.3.2 --- GlassMax DNA Isolation Spin Cartridge Purification of cDNA --- p.115 / Chapter 4.2.3.3 --- TdT Tailing of cDNA --- p.116 / Chapter 4.2.3.4 --- PCR of dC-tailed cDNA --- p.116 / Chapter 4.2.3.5 --- Nested Amplification --- p.117 / Chapter 4.2.4 --- Cloning and sequencing of RACE products --- p.117 / Chapter 4.2.5 --- Obtaining full-length cDNA of L. edodes MAPK --- p.118 / Chapter 4.2.5.1 --- Design of primers --- p.118 / Chapter 4.2.5.2 --- PCR amplification of LeMAPK --- p.118 / Chapter 4.2.5.3 --- Sequencing of full-length LeMAPK --- p.119 / Chapter 4.3 --- Results --- p.120 / Chapter 4.3.1 --- RACE of L. edodes Cyclin B and MAPK --- p.120 / Chapter 4.3.2 --- Sequences of Cyclin B and MAPK RACE products --- p.127 / Chapter 4.3.3 --- PCR product and sequence of L. edodes MAPK full-length cDNA --- p.131 / Chapter 4.4 --- Discussion --- p.139 / Chapter Chapter Five --- Functional Analysis of LeMAPK by Yeast Complementation Tests / Chapter 5.1 --- Introduction --- p.144 / Chapter 5.2 --- Materials and Methods --- p.152 / Chapter 5.2.1 --- The construct --- p.152 / Chapter 5.2.2 --- Yeast strains and media --- p.153 / Chapter 5.2.3 --- Yeast transformation --- p.154 / Chapter 5.2.4 --- Monitoring the Expression of HA-MAPK by Western Analysis --- p.155 / Chapter 5.2.4.1 --- Western blot analysis with anti-HA antibody --- p.155 / Chapter 5.2.4.2 --- Preparation of Cell Lysate from Yeast --- p.155 / Chapter 5.2.4.3 --- Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) --- p.156 / Chapter 5.2.4.4 --- Western Blotting --- p.157 / Chapter 5.2.4.5 --- Immunodetection --- p.158 / Chapter 5.2.4.6 --- ECL detection --- p.158 / Chapter 5.2.5 --- Complementation test of LeMAPK on yeast fus3Δkss1Δ double mutant --- p.159 / Chapter 5.2.5.1 --- Mating test --- p.159 / Chapter 5.2.5.2 --- Haploid invasive growth / Chapter 5.3 --- Results --- p.161 / Chapter 5.3.1 --- The construct - from E. coli to yeast --- p.161 / Chapter 5.3.2 --- The expression of LeMAPK in yeast and the complementation tests --- p.166 / Chapter 5.4 --- Discussion --- p.170 / Chapter Chapter Six --- General Discussion --- p.175 / References --- p.183

Wong, Man Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-146). / Abstracts in English and Chinese. / Abstract --- p.iii / 摘要 --- p.v / Acknowledgments --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / List of appendix --- p.xv / Abbreviations --- p.xvi / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Background of Lentinula edodes --- p.1 / Chapter 1.2 --- Life cycle and mating system of Lentinula edodes --- p.1 / Chapter 1.3 --- Breeding and strain improvement --- p.5 / Chapter 1.4 --- Application of molecular markers --- p.6 / Chapter 1.5 --- Objectives and long term significance --- p.9 / Chapter Chapter 2 --- Genome survey sequencing and preliminary analysis --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.1.1 --- Genome sequencing of basidiomycetes --- p.11 / Chapter 2.1.2 --- Polymerase chain reaction-single strand conformational polymorphism --- p.12 / Chapter 2.1.3 --- Sequencing chemistry --- p.13 / Chapter 2.2 --- Materials and methods --- p.15 / Chapter 2.2.1 --- Strain and DNA extraction --- p.15 / Chapter 2.2.2 --- PCR-SSCP analysis --- p.15 / Chapter 2.2.3 --- Shotgun sequencing and sequence assembly --- p.17 / Chapter 2.2.4 --- Comparison with 5 basidiomycetes --- p.17 / Chapter 2.3 --- Results --- p.19 / Chapter 2.3.1 --- PCR-SSCP --- p.19 / Chapter 2.3.2 --- Shotgun sequencing and assembly --- p.21 / Chapter 2.3.3 --- Comparison with 5 basidiomycetes --- p.22 / Chapter 2.4 --- Discussion --- p.30 / Chapter Chapter 3 --- Cloning of A mating-type locus of Lentinula edodes --- p.33 / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Materials and methods --- p.35 / Chapter 3.2.1 --- Genome sequencing and assembly --- p.35 / Chapter 3.2.2 --- Genomic screening of A-mating type genes --- p.35 / Chapter 3.2.3 --- Gap filling and sequence confirmation --- p.36 / Chapter 3.2.4 --- Alignment of overlapping sequences to give contiguous sequence --- p.37 / Chapter 3.2.5 --- Open reading frame prediction and protein homolog search --- p.37 / Chapter 3.2.6 --- Conserved domain search --- p.37 / Chapter 3.2.7 --- Testing for polymorphism --- p.38 / Chapter 3.3 --- Results --- p.39 / Chapter 3.3.1 --- Genomic screening of A-mating type genes --- p.39 / Chapter 3.3.2 --- Gap filling and sequence confirmation --- p.45 / Chapter 3.3.3 --- Protein homologs and putative protein domains --- p.48 / Chapter 3.3.4 --- Polymorphism of A mating-type genes --- p.53 / Chapter 3.4 --- Discussion --- p.55 / Chapter 3.4.1 --- Genome mining of the A mating-type locus of L. edodes --- p.55 / Chapter 3.4.2 --- Genomic structure of the A mating-type region in L. edodes --- p.55 / Chapter 3.4.3 --- Functional protein domains in A mating-type genes --- p.56 / Chapter 3.4.4 --- Polymorphism of A mating- type locus --- p.58 / Chapter 3.4.5 --- Conclusion and future perspectives --- p.59 / Chapter Chapter 4 --- Simple sequence repeat (SSR) markers development --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Materials and methods --- p.62 / Chapter 4.2.1 --- Strains --- p.62 / Chapter 4.2.2 --- Datasets for SSRs mining --- p.63 / Chapter 4.2.3 --- in silico detection of SSR motifs and primer design --- p.63 / Chapter 4.2.4 --- SSR amplification --- p.64 / Chapter 4.2.5 --- Cloning and sequencing of PCR products --- p.64 / Chapter 4.2.6 --- Testing for polymorphism --- p.65 / Chapter 4.3 --- Results --- p.66 / Chapter 4.3.1 --- in silico detection of SSR motifs and primer design --- p.66 / Chapter 4.3.2 --- SSR amplification --- p.69 / Chapter 4.3.3 --- SSR polymorphism --- p.83 / Chapter 4.4 --- Discussion --- p.86 / Chapter 4.4.1 --- Efficiency of in silico detection of SSR motifs and primer design --- p.86 / Chapter 4.4.2 --- Effectiveness and polymorphism of SSR primer pairs --- p.89 / Chapter 4.4.3 --- Conclusion and future perspectives --- p.90 / Chapter Chapter 5 --- High-throughput sequencing of AP-PCR amplicons for SCAR markers development and phylogenetic analysis --- p.91 / Chapter 5.1 --- Introduction --- p.91 / Chapter 5.2 --- Materials and methods --- p.94 / Chapter 5.2.1 --- Strains --- p.94 / Chapter 5.2.2 --- AP-PCR analysis --- p.94 / Chapter 5.2.3 --- Re-amplification of AP-PCR amplicons --- p.96 / Chapter 5.2.4 --- GS-FLX sequencing --- p.96 / Chapter 5.2.5 --- Strain-specific sequences identification --- p.97 / Chapter 5.2.6 --- SCAR marker analysis --- p.97 / Chapter 5.2.7 --- Phylogenetic analysis --- p.99 / Chapter 5.3 --- Results --- p.100 / Chapter 5.3.1 --- AP-PCR analysis --- p.100 / Chapter 5.3.2 --- Re-amplification of AP-PCR amplicons --- p.100 / Chapter 5.3.3 --- GS-FLX sequencing and strain-specific sequence identification --- p.103 / Chapter 5.3.4 --- SCAR marker analysis --- p.106 / Chapter 5.3.5 --- Phylogenetic analysis --- p.108 / Chapter 5.4 --- Discussion --- p.111 / Chapter 5.4.1 --- Sensitivity of band detection --- p.111 / Chapter 5.4.2 --- SCAR marker development --- p.111 / Chapter 5.4.3 --- Phylogenetic analysis --- p.113 / Chapter 5.4.4 --- Conclusion --- p.114 / Chapter Chapter 6 --- Concluding remarks --- p.115 / Chapter 6.1 --- Project summary --- p.115 / Chapter 6.2 --- Future perspectives --- p.119 / Appendix --- p.121 / References --- p.141

Au, Chun Hang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Hui Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 166-172). / Abstracts in English and Chinese. / Abstract --- p.iii / Acknowledgments --- p.vi / Abbreviations --- p.vii / Table of Contents --- p.viii / Table of Figures --- p.xiii / Table of Tables --- p.xviii / Chapter Chapter 1. --- Literature Reviews / Chapter 1.1 --- Functional Genomics and Its Developments --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- "Transcriptomics, Proteomics and Metabolomics" --- p.1 / Chapter 1.1.3 --- Gene-perturbing Strategies --- p.3 / Chapter 1.1.4 --- Applications of Functional Genomics --- p.4 / Chapter 1.2 --- Serial Analysis of Gene Expression (SAGE) and Generation of Longer cDNA Fragments from SAGE tags for Gene Identification (GLGI) --- p.6 / Chapter 1.2.1 --- Introduction --- p.6 / Chapter 1.2.2 --- Principles and Methods of SAGE --- p.6 / Chapter 1.2.3 --- Data Analysis --- Bioinformatics --- p.9 / Chapter 1.2.4 --- Applications of SAGE --- p.9 / Chapter 1.2.5 --- Modifications of SAGE --- p.10 / Chapter 1.2.6 --- Principles and Methods of GLGI --- p.11 / Chapter 1.2.7 --- Applications and Improvements of GLGI --- p.14 / Chapter 1.3 --- Transformation --- p.15 / Chapter 1.3.1 --- Introduction --- p.15 / Chapter 1.3.2 --- Different Methods of Transformation --- p.15 / Chapter 1.3.2.1 --- General Transformation Strategy --- p.15 / Chapter 1.3.2.2 --- Polyethylene Glycol (PEG)-mediated Transformation --- p.16 / Chapter 1.3.2.3 --- Restriction Enzyme Mediated Integration (REMI) --- p.16 / Chapter 1.3.2.4 --- Electroporation --- p.17 / Chapter 1.3.2.5 --- Particle Bombardment --- p.17 / Chapter 1.3.3 --- The Future Needs of Transformation --- p.18 / Chapter 1.4 --- RNA Silencing --- p.20 / Chapter 1.4.1 --- Introduction --- p.20 / Chapter 1.4.2 --- Major Components and Principles of RNAi --- p.21 / Chapter 1.4.3 --- Applications of RNA Silencing --- p.23 / Chapter 1.5 --- The Target Organism Lentinula edodes --- p.25 / Chapter 1.5.1 --- Introduction --- p.25 / Chapter 1.5.2 --- The Life Cycle of L. edodes --- p.26 / Chapter 1.5.3 --- Biochemical and Molecular Studies on L. edodes --- p.27 / Chapter 1.5.4 --- Prospectus --- p.29 / Chapter Chapter 2. --- Development of Methods for Studying Gene Function in Lentinula edodes / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.32 / Chapter 2.2.1 --- Cultivation of Lentinula edodes --- p.32 / Chapter 2.2.2 --- Proplast Release and Regeneration --- p.32 / Chapter 2.2.3 --- Preparation of Plasmid DNA --- p.33 / Chapter 2.2.4 --- Selectable Marker …Bialaphos --- p.35 / Chapter 2.2.5 --- Transformation --- p.35 / Chapter 2.2.5.1 --- Electroporation --- p.35 / Chapter 2.2.5.2 --- PEG-mediated Transformation --- p.36 / Chapter 2.3 --- Results --- p.37 / Chapter 2.3.1 --- Cultivation of Lentinula edodes --- p.37 / Chapter 2.3.2 --- Proplast Release and Regeneration --- p.37 / Chapter 2.3.3 --- Preparation of Plasmid DNA --- p.43 / Chapter 2.3.4 --- Selectable Marker--- Bialaphos --- p.43 / Chapter 2.3.5 --- Transformation --- p.46 / Chapter 2.3.5.1 --- Electroporation --- p.46 / Chapter 2.3.5.2 --- PEG-mediated Transformation --- p.46 / Chapter 2.4 --- Discussions and Conclusions --- p.57 / Chapter Chapter 3. --- Identification of Interested Genes in Expression Profile of SAGE using GLGI Method. / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.1.1 --- Results of SAGE Analysis --- p.61 / Chapter 3.1.2 --- Use of GLGI Method for Extension of SAGE Tags --- p.63 / Chapter 3.1.3 --- 5´ة Extension of GLGI (5'GLGI) --- p.65 / Chapter 3.1.3.1 --- Introduction --- p.65 / Chapter 3.1.3.2 --- "Overall strategy of 5, GLGI Method" --- p.67 / Chapter 3.1.3.3 --- Two-Steps PCR Method --- p.69 / Chapter 3.2 --- Generation of Longer cDNA Fragments from SAGE tags for Gene Identification (GLGI) --- p.71 / Chapter 3.2.1 --- Materials and Methods (GLGI Analysis) --- p.71 / Chapter 3.2.1.1 --- Total RNA Extraction --- p.71 / Chapter 3.2.1.2 --- Messenger RNA (mRNA) Extraction --- p.72 / Chapter 3.2.1.3 --- Preparation of 3´ة cDNA for GLGI --- p.73 / Chapter 3.2.1.4 --- NIaIII digestion of double strand cDNA --- p.74 / Chapter 3.2.1.5 --- PCR amplification of the 3'-cDNAs (Optional) --- p.77 / Chapter 3.2.1.6 --- GLGI Amplification of The Target Template --- p.80 / Chapter 3.2.1.7 --- DNA Cloning (Optional) --- p.82 / Chapter 3.2.1.8 --- Sequencing of GLGI PCR products --- p.85 / Chapter 3.2.2 --- 5' Materials and Methods (5' GLGI Analysis) --- p.86 / Chapter 3.2.2.1 --- Preparation of unique antisense primers --- p.86 / Chapter 3.2.2.2 --- 5' extension of GLGI products --- p.87 / Chapter 3.2.2.3 --- DNA Cloning (Optional) --- p.89 / Chapter 3.2.2.4 --- Sequencing of 5' GLGI PCR products --- p.89 / Chapter 3.2.3 --- Results (GLGI Analysis) --- p.90 / Chapter 3.2.3.1 --- Total RNA Extraction --- p.90 / Chapter 3.2.3.2 --- Messenger RNA Extraction --- p.90 / Chapter 3.2.3.3 --- Preparation of 3' cDNA for GLGI --- p.90 / Chapter 3.2.3.4 --- NIaIII digestion of double strand cDNA --- p.94 / Chapter 3.2.3.5 --- GLGI Amplification of The Target Template --- p.94 / Chapter 3.2.3.6 --- Sequencing of GLGI PCR products --- p.103 / Chapter 3.2.4 --- Results (5' GLGI Analysis) --- p.111 / Chapter 3.2.4.1 --- 5' extension of GLGI products --- p.111 / Chapter 3.2.4.2 --- Sequencing of 5´ة GLGI PCR products --- p.116 / Chapter 3.3 --- Discussions and Conclusions --- p.126 / Chapter 3.3.1 --- GLGI amplification of the target template --- p.126 / Chapter 3.3.2 --- 5' extension of GLGI products --- p.129 / Chapter 3.3.3 --- Two-Steps PCR Method --- p.130 / Chapter 3.3.4 --- Sequencing results of GLGI method and 5' GLGI method --- p.131 / Chapter Chapter 4. --- Identification of Unknown EST Using PCR Method With cDNA Library / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.2 --- Materials and Methods --- p.134 / Chapter 4.2.1 --- Extension of 5' end of EST sequence by PCR method --- p.134 / Chapter 4.2.2 --- Purification of PCR products --- p.136 / Chapter 4.2.3 --- Sequencing of Extended EST products --- p.136 / Chapter 4.3 --- Results --- p.137 / Chapter 4.3.1 --- Extension of 5' end of EST sequence by PCR method --- p.137 / Chapter 4.3.2 --- Sequencing of Extended EST products --- p.137 / Chapter 4.4 --- Discussions and Conclusions --- p.147 / Chapter Chapter 5. --- General Discussions --- p.151 / Appendix I --- p.156 / Reference --- p.166

Xing, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-120). / Abstracts in English and Chinese. / ABSTRACT OF THESIS ENTITLED: --- p.I / 論文摘要 --- p.Ill / ACKNOWLEDGEMENTS --- p.IV / ABBREVIATIONS --- p.V / CONTENTS --- p.VI / LIST OF FIGURES --- p.X / LIST OF TABLES --- p.XII / Chapter CHAPTER 1: --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- Pectic substances --- p.1 / Chapter 1.1.2 --- Structure and classification of pectins --- p.2 / Chapter 1.1.3 --- Classification of pectinases --- p.4 / Chapter 1.1.4 --- Application of pectinases --- p.5 / Chapter 1.1.5 --- Production of pectinases --- p.5 / Chapter 1.2 --- Lentinula edodes as a source of pectinolytic enzymes --- p.12 / Chapter 1.2.1 --- Taxonomy and Life cycle of L. edodes --- p.12 / Chapter 1.2.2 --- Pectin-degrading enzymes in L edodes --- p.13 / Chapter 1.3 --- Expression systems for fungal pectinolytic enzymes --- p.16 / Chapter 1.4 --- Gene expression analysis --- p.19 / Chapter 1.5 --- Objectives and Long-term significance --- p.20 / Chapter CHAPTER 2: P --- EGTINASES IN L. EDODES --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and Methods --- p.25 / Chapter 2.2.1 --- Fungal strains and growth conditions --- p.25 / Chapter 2.3.2 --- Gene models --- p.25 / Chapter 2.2.4 --- Enzyme activity assays --- p.26 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Alignment of 24 candidate pectin-degradation gene models --- p.30 / Chapter 2.3.2 --- Conserved domains in protein sequences of 24 gene models --- p.30 / Chapter 2.3.3 --- Signal Peptide prediction of pectin-degradation gene models --- p.30 / Chapter 2.3.4 --- Pectinases activities in L. edodes --- p.31 / Chapter 2.3.5 --- Growth of mycelia of L. edodes on pectin and non-pectin media --- p.31 / Chapter 2.4 --- Discussion --- p.48 / Chapter 2.4.1 --- Elimination of non-pectinolytic genes --- p.48 / Chapter 2.4.2 --- Conserved domains and active sites of 6 polygalacturonases --- p.49 / Chapter 2.4.3 --- Pectinases activities in L. edodes --- p.49 / Chapter 2.4.4 --- Effect of pectin on the growth of mycelia in L. edodes --- p.50 / Chapter 2.5 --- Conclusion --- p.51 / Chapter CHAPTER 3: --- TRANSCRIPTIONAL PROFILING OF PECTINASES GENES IN L. EDODES --- p.52 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.2 --- Strain cultivation --- p.57 / Chapter 3.2.3 --- RNA extraction and first strand cDNA synthesis --- p.58 / Chapter 3.2.4 --- Quantitative RT-PCR --- p.58 / Chapter 3.2.5 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.2 --- RNA quality of various samples in L. edodes --- p.62 / Chapter 3.3.3 --- Transcription of 14 putative pectinases genes --- p.62 / Chapter 3.3.4 --- Transcription profiling of pectinases genes during the development of L. edodes --- p.62 / Chapter 3.3.5 --- Transcriptional levels of pectinases genes in mycelia of L. edodes grown in different media --- p.63 / Chapter 3.4 --- Discussions --- p.73 / Chapter 3.4.2 --- Transcription profiling of pectinases genes in L. edodes during four developmental stages --- p.73 / Chapter 3.4.3 --- Differential transcriptional levels of pectinases genes in L. edodes mycelia grown in two media --- p.73 / Chapter 3.4.4 --- Effect of pectic substrates on the pectinases genes transcription in mycelia of L. edodes --- p.74 / Chapter 3.5 --- Conclusion --- p.76 / Chapter CHAPTER 4: --- CLONING OF PECTINASES GENES AND THEIR HETEROLOGOUS EXPRESSION IN PICHIA PASTORIS --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.2 --- Strain cultivation --- p.79 / Chapter 4.2.3 --- RNA extraction and first strand cDNA synthesis --- p.79 / Chapter 4.2.4 --- Cloning and sequencing of pectinases genes --- p.80 / Chapter 4.2.5 --- Subcloning and expression vector construction --- p.80 / Chapter 4.2.6 --- Growth of Pichia pastoris strains --- p.81 / Chapter 4.2.7 --- Transformation into P. pastoris and vivo screening of multiple inserts --- p.81 / Chapter 4.2.8 --- Expression of recombinant P. pastoris strains --- p.82 / Chapter 4.2.9 --- RNA extraction and transcription analysis of pectinases genes in recombinant Pichia strains --- p.83 / Chapter 4.2.10 --- Enzyme activity assays --- p.83 / Chapter 4.2.11 --- SDS-PAGE --- p.84 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.2 --- RT-PCR for full-length cDNA of 13 pectinases genes --- p.88 / Chapter 4.3.3 --- Cloning and sequences analysis of 4 putative pectinases genes --- p.88 / Chapter 4.3.4 --- Construction of expression vectors of pectinases genes and transformation to P. pastoris --- p.88 / Chapter 4.3.5 --- Screening of multiple inserts clones --- p.88 / Chapter 4.3.6 --- Recombination and integration of pectinases genes in P. pastoris --- p.89 / Chapter 4.3.7 --- Transcription and expression of pectinases genes in recombinant Pichia strains. --- p.89 / Chapter 4.4 --- Discussion --- p.104 / Chapter 4.4.2 --- cDNA sequences of 4 pectinases genes --- p.104 / Chapter 4.4.3 --- Heterologous expression of 2 pectinases genes in P. pastoris --- p.104 / Chapter 4.4.4 --- Characterization of the pectinases expressed by recombinant Pichia strains --- p.106 / Chapter 4.5 --- Conclusion --- p.108 / Chapter CHAPTER 5: --- CONCLUDING REMARKS --- p.109 / REFERENCES --- p.121

by Chu Kin Kan Astley. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves xi-xx (3rd gp.)). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.iv / Table of Contents --- p.v / List of Tables --- p.ix / List of Figures --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Popularity of Shiitake Mushroom --- p.1 / Chapter 1.2 --- Inheritance of Genetic Materials in L.edodes --- p.1 / Chapter 1.3 --- Genetic Markers and Linkage Maps of L.edodes --- p.2 / Chapter 1.4 --- Aims of Study --- p.5 / Chapter Chapter 2 --- Mapping of Sequence Tagged Sites (STSs) and Expressed Sequence Tags (ESTs) on the Linkage Map of L.edodes by PCR-Single Strand Conformational Polymorphism (SSCP) Test / Chapter 2.1 --- Literature Review --- p.7 / Chapter 2.1.1 --- Construction of Genetic Linkage Map --- p.7 / Chapter 2.1.2 --- Logarithm of the Odds (LOD) Score --- p.8 / Chapter 2.1.3 --- MAPMAKER Program --- p.10 / Chapter 2.1.4 --- Sequence Tagged Site (STS) and Expressed Sequence Tag (EST) --- p.11 / Chapter 2.1.5 --- Polymerase Chain Reaction - Single Strand Conformational Polymorphism (PCR-SSCP) --- p.13 / Chapter 2.2 --- Material and Methods --- p.18 / Chapter 2.2.1. --- Detection of the STS and EST markers with PCR-SSCP test --- p.18 / Chapter 2.2.1.1 --- Biological Material and Growth Conditions --- p.18 / Chapter 2.2.1.2 --- DNA Samples Preparation --- p.18 / Chapter 2.2.1.3 --- PCR Primers Designation --- p.20 / Chapter 2.2.1.4 --- PCR Amplification --- p.21 / Chapter 2.2.1.5 --- SSCP Test --- p.21 / Chapter 2.2.1.6 --- Silver Staining of the Polyacrylamide Gel --- p.22 / Chapter 2.2.2. --- Mapping of the STS and EST Markers --- p.22 / Chapter 2.2.2.1 --- Biological Material and Growth Conditions --- p.22 / Chapter 2.2.2.2 --- SSI DNA Preparation --- p.22 / Chapter 2.2.2.3 --- PCR-SSCP Test among SSIs --- p.24 / Chapter 2.2.2.4 --- Chi Square (X2) Test --- p.24 / Chapter 2.2.2.5 --- LOD Score Test and Mapping of the Markers --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Detection of the STS and EST Markers from PCR-SSCP Test --- p.27 / Chapter 2.3.1.1 --- DNA Sample Preparation --- p.27 / Chapter 2.3.1.2 --- Primers Designed for STS and EST Amplification --- p.27 / Chapter 2.3.1.3 --- Size Differences between Experimental and Expected PCR Products --- p.38 / Chapter 2.3.1.4 --- Markers of PCR Polymorphism (PCRP) --- p.38 / Chapter 2.3.1.5 --- Markers of PCR-SSCP and PCR Length Polymorphism (PCR-LP) --- p.42 / Chapter 2.3.2 --- Mapping of the STS/EST Markers --- p.49 / Chapter 2.3.2.1 --- DNA Templates of SSIs --- p.49 / Chapter 2.3.2.2 --- Polymorphism Profiles of STSs and ESTs among the L54-SSIs --- p.49 / Chapter 2.3.2.3 --- Chi-square Test --- p.55 / Chapter 2.3.2.4 --- Repeated EST Markers --- p.58 / Chapter 2.3.2.5 --- Anonymous Expressed Sequence Tag - EST31 --- p.58 / Chapter 2.3.2.6 --- Linkage Analysis and Mapping of Markers --- p.59 / Chapter 2.3.2.6.1 --- Linkage Relationships between the16 STS/EST Markers --- p.59 / Chapter 2.3.2.6.2 --- Mapping of the STS/EST Markers onto the RAPD Linkage Map --- p.62 / Chapter 2.4 --- Discussion --- p.69 / Chapter 2.4.1 --- DNA Template Preparation --- p.69 / Chapter 2.4.2 --- Size Difference Between Expected and Experimental PCR Product --- p.69 / Chapter 2.4.3 --- PCR Polymorphism (PCRP) --- p.70 / Chapter 2.4.4 --- PCR-LP --- p.70 / Chapter 2.4.5 --- PCR-SSCP --- p.71 / Chapter 2.4.5.1 --- Primer Designed for PCR-SSCP --- p.71 / Chapter 2.4.5.2 --- Markers Producing Efficiency of PCR-SSCP Test --- p.72 / Chapter 2.4.6 --- Linkage Map of L.edodes --- p.73 / Chapter 2.4.6.1 --- Map Distance --- p.73 / Chapter 2.4.6.2 --- Linkage Groups --- p.74 / Chapter 2.4.6.3 --- Map Markers --- p.75 / Chapter Chapter 3 --- Mapping of Agronomic Features of L.edodes / Chapter 3.1 --- Literature Review --- p.77 / Chapter 3.1.1 --- Aroma Feature of L.edodes --- p.77 / Chapter 3.1.1.1 --- Volatile Compounds in Shiitake (L.edodes) Mushroom --- p.77 / Chapter 3.1.1.2 --- Fragrance Signature of Shiitake Mycelium --- p.79 / Chapter 3.1.2 --- Mapping of Quantitative Trait Loci (QTL) --- p.84 / Chapter 3.1.2.1 --- Complex Traits --- p.84 / Chapter 3.1.2.2 --- Quantitative Traits Locus (QTL) --- p.85 / Chapter 3.1.2.3 --- Maximum-likelihood Estimate in QTL mapping --- p.86 / Chapter 3.1.2.4 --- MAPMAKER/QTL --- p.87 / Chapter 3.2 --- Material and Methods --- p.88 / Chapter 3.2.1 --- Aroma feature of Mycelium --- p.88 / Chapter 3.2.1.1 --- Preliminary Screening of Volatiles in the SSI Mycelia of L.edodes --- p.88 / Chapter 3.2.1.1.1 --- Biological Material and Growth Conditions --- p.88 / Chapter 3.2.1.1.2 --- Volatile Extraction from SSI Mycelia --- p.88 / Chapter 3.2.1.1.3 --- Screening of Volatile Compounds with GC-MS --- p.89 / Chapter 3.2.1.2 --- Quantification of the Target Aromatic Volatile in the Mycelia of SSI and Parents --- p.90 / Chapter 3.2.1.2.1 --- Sample Preparations --- p.90 / Chapter 3.2.1.2.2 --- Quantification of the Target Volatile --- p.90 / Chapter 3.2.2 --- Measurement of Mycelial Growth --- p.91 / Chapter 3.2.3 --- Observation of Pigment Secretion during Mycelial Growth --- p.91 / Chapter 3.2.4 --- Locating Putative QTL on the Genetic Map of L.edodes --- p.92 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Aroma Feature --- p.93 / Chapter 3.3.1.1 --- Preliminary Screening of Volatiles in Mycelia of L.edodes --- p.93 / Chapter 3.3.1.2 --- Quantification of l-octen-3-ol in SSI Mycelia --- p.103 / Chapter 3.3.1.2.1 --- Sample Preparation --- p.103 / Chapter 3.3.1.2.2 --- l-Octen-3-ol contents in SSI Mycelia --- p.103 / Chapter 3.3.1.3 --- Mapping of QTL for l-octen-3-ol level on the genetic map --- p.106 / Chapter 3.3.2 --- Mycelial Growth Rate (MGR) --- p.111 / Chapter 3.3.2.1 --- Measurement of Mycelial Growth Rate --- p.111 / Chapter 3.3.2.2 --- Mapping of QTL for MGR on the genetic map --- p.111 / Chapter 3.3.3 --- Pigment Secretion form SSI mycelia --- p.116 / Chapter 3.4 --- Discussion --- p.118 / Chapter 3.4.1 --- Significance of the QTLs --- p.118 / Chapter 3.4.2 --- QTL for Aroma Feature --- p.119 / Chapter 3.4.2.1 --- Trait of Aroma: l-octen-3-ol level --- p.119 / Chapter 3.4.2.2 --- QTL of l-octen-3-ol level --- p.120 / Chapter 3.4.3 --- Mycelial Growth Rate (MGR) --- p.123 / Chapter 3.4.4 --- Pigment Secretion --- p.125 / Chapter Chapter 4 --- General Discussion and Conclusions --- p.127 / Chapter 4.1 --- Future Works --- p.127 / Chapter 4.1.1 --- Mapping of L.edodes Genes --- p.127 / Chapter 4.1.2 --- Characterizing and Mapping of Agronomic Traits --- p.128 / Chapter 4.2 --- Conclusions --- p.128 / Referencesxi

by Ng Tak Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 151-168). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutraceutical and Medicinal Properties of L. edodes --- p.4 / Chapter 1.2.1 --- Nutritional value --- p.4 / Chapter 1.2.2 --- Hypocholesterolaemic Effect --- p.5 / Chapter 1.2.3 --- Anti-tumor Effect --- p.5 / Chapter 1.2.4 --- Anti-viral Effect --- p.6 / Chapter 1.2.5 --- Immunopotentiating Effect --- p.6 / Chapter 1.3 --- Life cycle of L. edodes --- p.7 / Chapter 1.4 --- Environmental factors affecting mycelial growth and fruit body --- p.11 / Chapter 1.4.1 --- Nutrient requirement --- p.11 / Chapter 1.4.2 --- Physical and chemical factors --- p.12 / Chapter 1.5 --- Molecular studies on mushroom development --- p.15 / Chapter 1.5.1 --- Mating-type genes --- p.15 / Chapter 1.5.2 --- Hydrophobins --- p.19 / Chapter 1.5.3 --- Fruiting regulatory genes --- p.23 / Chapter 1.5.4 --- Molecular studies on fruit body development of I. edodes --- p.24 / Chapter 1.5.4.1 --- Identification of L. edodes genes --- p.24 / Chapter 1.5.4.2 --- Functional characterization of L. edodes genes --- p.27 / Chapter 1.5.4.3 --- Transformation in L. edodes --- p.28 / Chapter Chapter Two --- Expressed Sequence Tags (ESTs) of L. edodes / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Generation of expressed sequence tag --- p.33 / Chapter 2.2.1.1 --- Mushroom cultivation and RNA extraction --- p.33 / Chapter 2.2.1.2 --- Construction of primordium cDNA library --- p.34 / Chapter 2.2.1.3 --- Mass excision of pBK-CMV plasmid --- p.34 / Chapter 2.2.1.4 --- Random screening of mass excised cDNA clone --- p.38 / Chapter 2.2.1.5 --- Isolation of recombinant plasmid --- p.38 / Chapter 2.2.1.6 --- Generation of 3´ة end partially sequence --- p.39 / Chapter 2.2.1.7 --- Sequence analysis --- p.40 / Chapter 2.2.2 --- Reverse dot-blot Hybridization --- p.40 / Chapter 2.2.2.1 --- PCR amplification of cDNA clone --- p.40 / Chapter 2.2.2.2 --- Membrane preparation --- p.40 / Chapter 2.2.2.3 --- cDNA probe preparation --- p.41 / Chapter 2.2.2.4 --- Hybridization --- p.42 / Chapter 2.2.2.5 --- Stringent washing and autoradiography --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Construction of primordium cDNA library --- p.44 / Chapter 2.3.2 --- Screening of recombinant clone --- p.44 / Chapter 2.3.3 --- Isolation and reconfirmation of recombinant plasmid --- p.46 / Chapter 2.3.4 --- Generation of EST --- p.47 / Chapter 2.3.5 --- EST identity --- p.47 / Chapter 2.3.6 --- Reverse dot-blot hybridization --- p.56 / Chapter 2.3.7 --- Analysis of hybridization signal --- p.60 / Chapter 2.4 --- Discussion --- p.71 / Chapter Chapter Three --- Sequence Analysis and Transcriptional Profiling of Genes Encoding GTP-binding Proteins / Chapter 3.1 --- Introduction --- p.78 / Chapter 3.2 --- Materials and Methods --- p.82 / Chapter 3.2.1 --- Sequence manipulation --- p.82 / Chapter 3.2.2 --- Northern blot hybridization --- p.82 / Chapter 3.2.2.1 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.82 / Chapter 3.2.2.2 --- RNA fixation by capillary method --- p.83 / Chapter 3.2.2.3 --- Probe preparation --- p.84 / Chapter 3.2.2.4 --- Hybridization --- p.85 / Chapter 3.2.2.5 --- Stringent washing and autoradiography --- p.85 / Chapter 3.2.3 --- Real-Time SYBR Green RT-PCR --- p.85 / Chapter 3.2.3.1 --- Primer design --- p.85 / Chapter 3.2.3.2 --- RT-PCR reaction --- p.86 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Sequence manipulation --- p.88 / Chapter 3.3.2 --- Transcriptional analysis --- p.103 / Chapter 3.4 --- Discussion --- p.108 / Chapter 3.4.1 --- Heterotrimeric G proteins --- p.108 / Chapter 3.4.2 --- Ras-related protein Rab7 --- p.112 / Chapter 3.4.3 --- Developmentally regulated GTP-binding protein --- p.113 / Chapter Chapter Four --- Yeast Complementation and Over-expression tests of Le.Gβ1 and Le.Gγ1 / Chapter 4.1 --- Introduction --- p.115 / Chapter 4.2 --- Materials and Methods --- p.120 / Chapter 4.2.1 --- "Yeast strains, media and yeast vectors" --- p.120 / Chapter 4.2.2 --- Primer design --- p.121 / Chapter 4.2.3 --- RT-PCR Amplification of Le.Gβ1 and Le.Gγ1 --- p.121 / Chapter 4.2.4 --- Purification of PCR products --- p.122 / Chapter 4.2.5 --- Enzymatic digestion and purification --- p.122 / Chapter 4.2.6 --- Ligation and E. coli transformation --- p.122 / Chapter 4.2.7 --- PCR screening of E. coli transformants --- p.124 / Chapter 4.2.8 --- Plasmids extraction --- p.124 / Chapter 4.2.9 --- Yeast transformation --- p.124 / Chapter 4.2.10 --- Mating test --- p.125 / Chapter 4.3 --- Results --- p.129 / Chapter 4.3.1 --- Cloning of Le.Gβ1 and Le.Gγ1 --- p.129 / Chapter 4.3.2 --- Yeast transformation --- p.129 / Chapter 4.3.3 --- Mating test --- p.130 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter Five --- General Discussion --- p.144 / References --- p.151