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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324372 |
| Date | January 2003 |
| Contributors | Wong, Wing Lei., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiv, 129 leaves : ill. ; 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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