Endocytic pathway in mushroom development: role of Le.Rab7 and interacting proteins.

• Other Authors: Lee, Ming Tsung.
• Format: Others
• Language: English; Chinese
• Published: 2006
• Subjects: Shiitake > Growth; Shiitake > Molecular genetics; Endocytosis
• Online Access: http://library.cuhk.edu.hk/record=b5892911; http://repository.lib.cuhk.edu.hk/en/item/cuhk-325669

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