Lee Ming Tsung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 160-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutritional values --- p.2 / Chapter 1.3 --- Medicinal values --- p.3 / Chapter 1.3.1 --- Anti-tumor effect --- p.3 / Chapter 1.3.2 --- Anti-viral and anti-caries effect --- p.4 / Chapter 1.3.3 --- Immunopotentiating effect --- p.4 / Chapter 1.3.4 --- Hypocholesterolaemic effect --- p.5 / Chapter 1.4 --- Life cycle and morphology --- p.6 / Chapter 1.5 --- Growth requirements --- p.9 / Chapter 1.5.1 --- Nutritional factors --- p.9 / Chapter 1.5.2 --- Physical and chemical factors --- p.10 / Chapter 1.6 --- Application of L. edodes --- p.12 / Chapter 1.7 --- Endocytosis --- p.13 / Chapter 1.7.1 --- Different types of endocytosis --- p.13 / Chapter 1.7.1.1 --- Phagocytosis --- p.14 / Chapter 1.7.1.2 --- Pinocytosis --- p.15 / Chapter 1.7.1.3 --- Receptor-mediated endocytosis --- p.15 / Chapter 1.7.2 --- The Endocytic Pathway --- p.17 / Chapter 1.7.3 --- Endocytosis in fungi --- p.20 / Chapter 1.7.4 --- Rab GTPases --- p.21 / Chapter 1.7.4.1 --- Control of the active and inactive state of Rab proteins --- p.22 / Chapter 1.7.4.2 --- Regulation of docking and fusion of membrane in endosomal trafficking --- p.23 / Chapter 1.7.4.3 --- Rab7 GTPase --- p.26 / Chapter 1.8 --- Aims of the project --- p.28 / Chapter Chapter 2 --- Protein-protein Interaction Study of Le.Rab7 by in vivo and in vitro Interaction Assay --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Yeast two-hybrid screening --- p.36 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Rab7-pGBK.T7 --- p.36 / Chapter 2.2.1.1.1 --- Bacterial transformation --- p.36 / Chapter 2.2.1.1.2 --- PCR screening for positive transformants --- p.38 / Chapter 2.2.1.1.3 --- Plasmid preparation and confirmation of transformants --- p.38 / Chapter 2.2.1.1.4 --- Sequencing --- p.39 / Chapter 2.2.1.2 --- Confirmation of Le.Rab7 protein expression in yeast --- p.40 / Chapter 2.2.1.2.1 --- Yeast transformation --- p.40 / Chapter 2.2.1.2.2 --- Yeast protein extraction --- p.40 / Chapter 2.2.1.2.3 --- Western Blotting --- p.41 / Chapter 2.2.1.3 --- Yeast Two-hybrid screening by Yeast-mating --- p.42 / Chapter 2.2.1.4 --- Identification of Preys --- p.44 / Chapter 2.2.1.4.1 --- PCR screening for clones grown on plates --- p.44 / Chapter 2.2.1.4.2 --- Colony lift filter assay --- p.45 / Chapter 2.2.1.4.3 --- Sequencing --- p.47 / Chapter 2.2.1.5 --- Confirmation of interaction by Co-transformation assay --- p.47 / Chapter 2.2.1.5.1 --- Plasmid preparation of positive clones --- p.47 / Chapter 2.2.1.5.2 --- Transformation and bacterial plasmid preparation --- p.48 / Chapter 2.2.1.5.3 --- Yeast two-hybrid screening by co-transformation --- p.48 / Chapter 2.2.1.5.4 --- Colony lift filter assay --- p.50 / Chapter 2.2.2 --- Rapid Amplification of cDNA 5'ends --- p.51 / Chapter 2.2.2.1 --- RNA preparation --- p.51 / Chapter 2.2.2.1.1 --- Strains and culture conditions --- p.51 / Chapter 2.2.2.1.2 --- RNA extraction --- p.51 / Chapter 2.2.2.2 --- 5' RACE --- p.52 / Chapter 2.2.2.2.1 --- RNA processing --- p.52 / Chapter 2.2.2.2.2 --- Reverse transcription --- p.53 / Chapter 2.2.2.2.3 --- Nested PCR for 5'RLM-RACE --- p.53 / Chapter 2.2.2.3 --- "Gel analysis of products, TA cloning of RACE product and sequencing" --- p.54 / Chapter 2.2.2.4 --- Cloning of full-length Le.Rab5 --- p.54 / Chapter 2.2.3 --- In vitro protein-protein interaction assay --- p.55 / Chapter 2.2.3.1 --- Plasmid extraction from E.coli --- p.55 / Chapter 2.2.3.2 --- In vitro translation --- p.56 / Chapter 2.2.3.3 --- In vitro co-immunoprecipitation --- p.56 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Yeast two-hybrid analysis by yeast mating assay --- p.57 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Ra67-pGBKT7 --- p.57 / Chapter 2.3.1.1.1 --- PCR screening for positive transformants --- p.57 / Chapter 2.3.1.1.2 --- Plasmid preparation and confirmation of transformants --- p.58 / Chapter 2.3.1.1.3 --- Sequencing --- p.59 / Chapter 2.2.1.2 --- Confirmation of protein expression in yeast --- p.60 / Chapter 2.3.1.2.1 --- Yeast transformation --- p.60 / Chapter 2.3.1.2.2 --- SDS-PAGE and Western blotting of Le.Rab7 in yeast --- p.61 / Chapter 2.2.1.3 --- Yeast two-hybrid screening by yeast mating assay --- p.62 / Chapter 2.2.1.4 --- Identification of Preys --- p.63 / Chapter 2.3.1.4.1 --- PCR screening for clones grown on plates --- p.63 / Chapter 2.3.1.4.2 --- Colony lift assay --- p.65 / Chapter 2.3.1.4.3 --- Sequencing --- p.67 / Chapter 2.3.2 --- Confirmation of interactions by co-transformation assay --- p.70 / Chapter 2.2.2.1 --- Yeast two-hybrid analysis by co-transformation assay --- p.70 / Chapter 2.2.2.2 --- Colony lift filter assay --- p.70 / Chapter 2.2.2.3 --- Selection of prey plasmids for in vitro binding assay --- p.72 / Chapter 2.3.3 --- Rapid amplification of cDNA ends (RACE) --- p.76 / Chapter 2.2.3.1 --- TA cloning of RACE product and sequencing --- p.76 / Chapter 2.2.3.2 --- Cloning of full-length Le.Rab5 --- p.79 / Chapter 2.3.4 --- In vitro protein-protein interaction assay --- p.80 / Chapter 2.4 --- Discussion --- p.82 / Chapter Chapter 3 --- Temporal and Spatial expression of Le.Rab7，Le.Rab5 and Le.RACKl --- p.87 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Northern blot analysis --- p.93 / Chapter 3.2.1.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.93 / Chapter 3.2.1.2 --- Northern blotting --- p.94 / Chapter 3.2.1.2.1 --- Transfer of RNAs --- p.94 / Chapter 3.2.1.2.2 --- Probe preparation --- p.95 / Chapter 3.2.1.2.3 --- "Hybridization, Stringency washes and Signal detection" --- p.96 / Chapter 3.2.2 --- Quantitative RT-PCR --- p.97 / Chapter 3.2.2.1 --- cDNA synthesis from different developmental stages --- p.97 / Chapter 3.2.2.1.1 --- RNA preparation extraction --- p.97 / Chapter 3.2.2.1.2 --- DNase I treatment --- p.97 / Chapter 3.2.2.1.3 --- Reverse transcription --- p.98 / Chapter 3.2.2.2 --- Real time PCR --- p.98 / Chapter 3.2.2.2.1 --- Primer design and verification --- p.98 / Chapter 3.2.2.2.2 --- Real time PCR reaction and data analysis --- p.100 / Chapter 3.2.3 --- In situ RNA-RNA hybridization --- p.101 / Chapter 3.2.3.1 --- Preparation of samples and probes --- p.101 / Chapter 3.2.3.1.1 --- Tissue preparation --- p.101 / Chapter 3.2.3.1.2 --- RNA probe synthesis --- p.101 / Chapter 3.2.3.2 --- Hybridization and Signal development --- p.102 / Chapter 3.2.3.3 --- Image viewing --- p.103 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Northern blot analysis --- p.105 / Chapter 3.3.2 --- Quantitative RT-PCR assays --- p.109 / Chapter 3.3.3 --- In situ RNA-RNA hybridization --- p.113 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- Existence of endocytosis and Protein localization of Le.Rab7 in L. edodes --- p.123 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.2 --- Materials and Methods --- p.127 / Chapter 4.2.1 --- Tracing the endocytie pathway using FM4-64 dye --- p.127 / Chapter 4.2.1.1 --- Strains and culture conditions --- p.127 / Chapter 4.2.1.2 --- FM4-64 internalization in mycelium and gill tissue of L. edodes --- p.127 / Chapter 4.2.2 --- Drug treatment effect on the internalization of FM4-64 dye --- p.128 / Chapter 4.2.3 --- Double labeling with AM4-64 and anti-Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1 --- Synthesis of Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1.1 --- Customization of Le.Rab7 antiserum --- p.129 / Chapter 4.2.3.1.2 --- Validation of anti-Le.Rab7 polyclonal antiserum --- p.129 / Chapter 4.2.3.2 --- Double immunofluorescence labeling --- p.130 / Chapter 4.2.4 --- Immunohistochemistry of young and mature fruiting body --- p.131 / Chapter 4.2.4.1 --- Tissue preparation --- p.131 / Chapter 4.2.4.2 --- Immunohistochemical staining --- p.132 / Chapter 4.2.4.3 --- Image viewing --- p.133 / Chapter 4.3 --- Results --- p.134 / Chapter 4.3.1 --- Presence of endocytosis in L .edodes --- p.134 / Chapter 4.3.2 --- Validation of active transport of FM4-64 --- p.137 / Chapter 4.3.3 --- Dye internalization at specific structures in L. edodes --- p.138 / Chapter 4.3.4 --- Presence of Le.Rab7 protein in the endosomal structures along the endocytic pathway --- p.142 / Chapter 4.3.5 --- Presence of Le.Rab7 protein in the pre- and hymenophore of fruiting body --- p.145 / Chapter 4.4 --- Discussion --- p.148 / Chapter Chapter 5 --- General discussion --- p.152 / References --- p.160
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325669 |
| Date | January 2006 |
| Contributors | Lee, Ming Tsung., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 177 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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