Return to search

Two component histidine kinase gene Le.nik1 of Shiitake mushrooms Lentinula edodes.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324372
Date January 2003
ContributorsWong, Wing Lei., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xiv, 129 leaves : ill. ; 30 cm.
RightsUse 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/)

Page generated in 0.0027 seconds