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.iAbstract (Chinese) --- p.iiiAcknowledgements --- p.ivAbbreviations --- p.vTable of Contents --- p.viList of Figures --- p.xiList of Tables --- p.xiiiiChapter Chapter One --- Literature ReviewChapter 1.1 --- Introduction --- p.1Chapter 1.2 --- Life cycle of L. edodes --- p.3Chapter 1.3 --- Environmental Stimuli for primoridum initiation --- p.6Chapter 1.4 --- Two component system (TCS) --- p.7Chapter 1.4.1 --- Modular structure of TCS --- p.7Chapter 1.4.2 --- Overview of the difference of TCS from other signaling cascades --- p.8Chapter 1.4.3 --- Functional roles of TCS in different organisms --- p.9Chapter 1.4.4 --- Molecular studies on histidine kinases --- p.16Chapter 1.5 --- Rationale and Summary --- p.18Chapter Chapter Two --- Screening of Differentially Expressed GenesChapter 2.1 --- Introduction --- p.20Chapter 2.2 --- Materials and Methods --- p.24Chapter 2.2.1 --- Reverse Dot Blot Hybridization --- p.24Chapter 2.2.1.1 --- Construction of primordial cDNA library --- p.24Chapter 2.2.1.2 --- PCR amplification of cDNA clones --- p.24Chapter 2.2.1.3 --- Membrane preparation --- p.24Chapter 2.2.1.4 --- cDNA probe preparation --- p.25Chapter 2.2.1.4.1 --- RNA extraction --- p.25Chapter 2.2.1.4.2 --- RAP-PCR --- p.26Chapter 2.2.1.4.3 --- Purification of RAP-PCR product --- p.27Chapter 2.2.1.4.4 --- Labeling of probe --- p.27Chapter 2.2.1.5 --- Stringent washing and signal detection --- p.27Chapter 2.2.2 --- Confirmation of the expression level of differentially expressed genes by Reverse transcription PCR (RT-PCR) --- p.28Chapter 2.2.2.1 --- Primer Design --- p.28Chapter 2.2.2.2 --- First strand total cDNA synthesis --- p.28Chapter 2.2.2.3 --- Reverse transcription PCR --- p.29Chapter 2.3 --- Results --- p.30Chapter 2.3.1 --- Screening of differentially expressed genes --- p.30Chapter 2.3.2 --- Sequence analysis --- p.30Chapter 2.3.3 --- Confirmations of differential expression of candidate genes by RT-PCR --- p.34Chapter 2.4 --- Discussion --- p.39Chapter 2.4.1 --- Putative roles of differentially expressed genes --- p.39Chapter 2.4.2 --- Confirmation of differential expression --- p.40Chapter Chapter Three --- Characterization and Full Length Sequence Analysis of Le.nikl cloneChapter 3.1 --- Introduction --- p.42Chapter 3.2 --- Materials and Methods --- p.43Chapter 3.2.1 --- Full length sequence of partial Le.nikl cDNA clone by primer walkingChapter 3.2.1.1 --- Primer Design --- p.43Chapter 3.2.2 --- Confirmations of differentially expressed Le.nikl by Northern blot analyses --- p.44Chapter 3.2.2.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.44Chapter 3.2.2.2 --- Northern blotting --- p.45Chapter 3.2.2.3 --- Probe preparation --- p.46Chapter 3.2.2.4 --- "Hybridization, Stringency washes, Signal detection" --- p.46Chapter 3.2.3 --- Cloning of 5' end of Le.nikl --- p.47Chapter 3.2.3.1 --- Amplification of 5' partial end of Le.nikl from primordium cDNA library --- p.47Chapter 3.2.3.2 --- Polishing the PCR products --- p.48Chapter 3.2.3.3 --- Cloning of PCR products --- p.48Chapter 3.2.3.4 --- PCR screening of the transformants --- p.49Chapter 3.2.3.5 --- Sequencing analysis --- p.49Chapter 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.49Chapter 3.3 --- Results --- p.51Chapter 3.3.1 --- Northern analysis of Le.nik1 --- p.51Chapter 3.3.2 --- Sequence analysis of 2.75kb Le.niklChapter 3.4 --- Discussion --- p.63Chapter Chapter Four --- Protein Interaction Study of Response Regulator of Le.NIK1 by Yeast two hybridChapter 4.1 --- Introduction --- p.65Chapter 4.2 --- Materials and Methods --- p.68Chapter 4.2.1 --- "Yeast strains, media, yeast vectors" --- p.68Chapter 4.2.2 --- Bait construction --- p.68Chapter 4.2.2.1 --- "Cloning of bait insert Le.nik1-rec into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.68Chapter 4.2.2.1.1 --- Design of cloning primer --- p.70Chapter 4.2.2.1.2 --- First strand total cDNA synthesis --- p.70Chapter 4.2.2.1.3 --- PCR amplification of bait insert --- p.70Chapter 4.2.2.2 --- Small-scale transformation of GBKT7-Le.nik1-rec plasmid --- p.71Chapter 4.2.2.2.1 --- Preparation of yeast competent cells --- p.71Chapter 4.2.2.2.2 --- Transformation of the GBKT7-Le.nik1-rec plasmid into the yeast strain AH109 --- p.72Chapter 4.2.2.2.3 --- Test for the Self-Activation of DNA-BD fusion --- p.72Chapter 4.2.2.2.4 --- Verification of bait protein expression --- p.72Chapter 4.2.2.2.4.1 --- Yeast protein extraction by TCA method --- p.72Chapter 4.2.2.2.4.2 --- Western blot analyses --- p.73Chapter 4.2.2.2.4.2.1 --- SDS-PAGE --- p.73Chapter 4.2.2.2.4.2.2 --- Western blotting --- p.75Chapter 4.2.2.2.4.2.3 --- Immunodetection --- p.75Chapter 4.2.2.2.4.2.4 --- ECL detection --- p.75Chapter 4.2.2.2.5 --- Test for the toxicity of DNA-BD fusion --- p.76Chapter 4.2.3 --- Prey construction -Total primordium cDNA synthesis --- p.76Chapter 4.2.3.1 --- First-strand cDNA synthesis --- p.76Chapter 4.2.3.2 --- ds cDNA amplification --- p.77Chapter 4.2.3.3 --- ds cDNA purification --- p.77Chapter 4.2.4 --- Yeast two hybrid screening assay --- p.77Chapter 4.2.4.1 --- Yeast competent cells preparation --- p.77Chapter 4.2.4.2 --- Screening by co-transformation --- p.78Chapter 4.2.4.2.1 --- for positive and negative control --- p.78Chapter 4.2.4.2.2 --- for prey ds DNA and pGBKT7-Le.nik1-rec --- p.78Chapter 4.2.4.3 --- Selection for transformants expressing interacting proteins --- p.78Chapter 4.2.5 --- β-galactosidase analysis- colony lift filter assay --- p.79Chapter 4.2.6 --- PCR screening of the prey --- p.80Chapter 4.2.7 --- DNA Sequencing of the prey insert --- p.80Chapter 4.2.8 --- Plasmid Isolation --- p.81Chapter 4.2.8.1 --- Isolation and sequencing of yeast plasmid --- p.81Chapter 4.2.9 --- Confirmations of protein interaction and self-activation test of AD-prey insert by small-scale co-transformation --- p.82Chapter 4.2.10 --- Northern Blot analyses of prey genes --- p.82Chapter 4.3 --- Results --- p.83Chapter 4.3.1 --- DNA-BD fusion construction --- p.83Chapter 4.3.2 --- AD fusion library construction --- p.88Chapter 4.3.3 --- Yeast two hybrid screening assay by co-transformation --- p.88Chapter 4.4 --- Discussion --- p.96Chapter Chapter Five --- Effect of Different Mannitol Osmolarity on Le.nikl Transcriptional ExpressionChapter 5.1 --- Introduction --- p.101Chapter 5.2 --- Materials and Methods --- p.102Chapter 5.2.1 --- "Mycelium inoculum preparation, cultivation mediums and cultivation conditions" --- p.102Chapter 5.2.2 --- RNA extraction --- p.102Chapter 5.2.3 --- Northern analysis --- p.102Chapter 5.3 --- Results --- p.103Chapter 5.4 --- Discussion --- p.112Chapter 5.4.1 --- Effect of high osmolarity on mushroom growth --- p.112Chapter 5.4.2 --- Effect of high osmolarity on gene expression --- p.113Chapter Chapter Six --- General Discussion --- p.114References --- p.11