Identification and characterization of differentially expressed genes in dikaryons of lentinula edodes by cDNA microarray.

by Shih Sheung Mei.Thesis submitted in: July 2003.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 206-215).Abstracts in English and Chinese.Abstract --- p.iiAchnoledgements --- p.viAbbreviations --- p.viiiList of contents --- p.vivList of tables --- p.xiiiList of figures --- p.xvChapter Chapter One --- Literature ReviewChapter 1.1 --- Introducation of Lentinula edodes --- p.1Chapter 1.1.1 --- Life cycle of Basidiomycete --- p.1Chapter 1.1.2 --- Differentially Expressed Genes in stages of Lentinula edodes --- p.3Chapter 1.2 --- Relationship of Monokaryons and Dikaryons in Basidiomycetes --- p.4Chapter 1.2.1 --- Mating Type Gene in Filamentous Fungi --- p.4Chapter 1.2.3 --- Dikaryon Formation and Homeodomain Proteins --- p.6Chapter 1.2.4 --- Clamp Connection formation in Dikaryon --- p.9Chapter 1.3 --- Stuctural Protein of Mushroom --- p.11Chapter 1.3.1 --- Hydrophobin --- p.11Chapter 1.3.1.1 --- General Introduction --- p.11Chapter 1.3.1.2 --- Structure of hydrophobin --- p.11Chapter 1.3.1.3 --- Formation of Disulphide bonds and Glycosylation --- p.12Chapter 1.3.1.4 --- Functions of Hydrophobins --- p.13Chapter 1.4 --- Genomics of filamentous fungi --- p.15Chapter 1.5 --- Genetic analysis of filamentous fungi --- p.18Chapter 1.6 --- Objectives of the Project --- p.20Chapter Chapter Two --- Identification of Differentially Expressed Genes in Dikaryons of Lentinula edodes by Microarray of Primordium Expressed Sequence TagsChapter 2.1 --- Introduction --- p.23Chapter 2.2 --- Materials and Methods --- p.27Chapter 2.2.1 --- Construction of EST database --- p.27Chapter 2.2.2 --- Construction of EST Microarray cDNA gene-chip --- p.27Chapter 2.2.2.1 --- Amplification of the primordium EST clones --- p.27Chapter 2.2.2.2 --- Purification of the amplified EST clones --- p.28Chapter 2.2.2.3 --- Spotting of the amplified EST clones onto chips --- p.29Chapter 2.2.3 --- Screening of the Differentially Expressed Genes in Dikaryons by Primordium Microarray --- p.31Chapter 2.2.3.1 --- Mycelium Cultivation and Preparation of Total RNA --- p.31Chapter 2.2.3.2 --- cDNA synthesis and labeling --- p.32Chapter 2.2.3.3 --- cDNA purification --- p.33Chapter 2.2.3.4 --- Probe Storage Conditions --- p.34Chapter 2.2.3.5 --- cDNA analysis --- p.35Chapter 2.2.3.6 --- Microarray hybridization --- p.37Chapter 2.2.3.7 --- Stringency washes --- p.39Chapter 2.2.3.8 --- Detection with TSA --- p.39Chapter 2.2.3.9 --- Microarray scanning and data anlysis --- p.41Chapter 2.3 --- Results --- p.45Chapter 2.3.1 --- Amplification of primordium ESTs --- p.45Chapter 2.3.2 --- Purification of PCR products --- p.45Chapter 2.3.3 --- Data Analysis of Microarray Data --- p.47Chapter 2.3.3.1 --- Generation of Primordium EST Microarray Image for analysis --- p.47Chapter 2.3.3.2 --- Normalization of the Data --- p.49Chapter 2.3.3.3. --- Transciption Profile of Dikaryon compared with Monokaryon --- p.79Chapter 2.3.3.4. --- Differentially Expression of Dikaryon L54 --- p.80Chapter 2.4 --- Discussion --- p.85Chapter Chapter Three --- Enrichment of Genes with Differentially Expression in Dikaryons by Construction of Full-length Subtractive LibraryChapter 3.1 --- Introduction of Subtraction Cloning --- p.93Chapter 3.2 --- Materials and Methods --- p.97Chapter 3.2.1 --- Construction of Full-length Dikaryotic Subtractive library --- p.97Chapter 3.2.1.1 --- Isolation of PolyA+ mRNA of Dikaryon for Subtraction --- p.97Chapter 3.2.1.2 --- Enrichment of Differentially Expressed Genes in Dikaryon L54 by Subtraction with Monokaryons A and B --- p.99Chapter 3.2.1.3 --- First-Strand cDNA Synthesis --- p.102Chapter 3.2.1.4 --- cDNA Amplification by Long-Distance PCR --- p.102Chapter 3.2.1.5 --- Proteinase K Digestion --- p.103Chapter 3.2.1.6 --- Sfi Digestion --- p.104Chapter 3.2.1.7 --- cDNA size fractionation by CHROMA SPIN-400 --- p.104Chapter 3.2.1.8 --- Determination of the Ligation Efficiency --- p.106Chapter 3.2.1.9 --- Ligation of cDNA to lamda TriplEx2 Vector --- p.107Chapter 3.2.1.10 --- Lamda-phage Packaging Reaction --- p.107Chapter 3.2.1.11 --- Titering the Unamplifled Library and Determining the Percentage of Recombinant Clones --- p.108Chapter 3.2.1.12 --- Library Amplification --- p.109Chapter 3.2.1.13 --- Conversion of λTriplEx2 Recombinant Clones to pTriplEx2 Recombinant Plasmids --- p.111Chapter 3.2.2 --- Screening of the Subtractive library --- p.114Chapter 3.2.2.1 --- Verification of the enrichment by Plaque Lifting hybridization --- p.114Chapter 3.2.2.1.1 --- Lifting the Plaques --- p.114Chapter 3.2.2.1.2 --- Synthesis of the Probes for Plaque Lift Hybridization --- p.115Chapter 3.2.2.1.3 --- Hybridization to the Membranes --- p.116Chapter 3.2.2.2 --- Screening the Subtractive library by Macroarray Hybridization --- p.117Chapter 3.2.2.2.1 --- Colony Picking by QPik System --- p.117Chapter 3.2.2.2.2 --- Gridding of Macroarray --- p.118Chapter 3.2.2.2.3 --- Filter Processing of Gridded Membrane --- p.119Chapter 3.2.2.2.4 --- Hybridization to the Macroarray Membrane --- p.120Chapter 3.3 --- Results and Discussion --- p.121Chapter 3.3.1 --- Enrichment of Differentially Expressed Genes in Dikaryon L54 by Subtraction with Monokaryons A and B --- p.121Chapter 3.3.2 --- Construction of the full-length subtractive library --- p.123Chapter 3.3.3 --- Conversion of A TriplEx2 Recombinant Clones to pTriplEx2 Recombinant Plamid --- p.124Chapter 3.3.4 --- Verification the Enrichment of Subtractive library by Plaque lifting Hybridization --- p.125Chapter 3.3.5 --- Screening of the Subtractive library by Macroarray --- p.125Chapter 3.4 --- Discussion --- p.126Chapter Chapter Four --- Identification of Genes with Differentially Expression in Dikaryons by Subtactive cDNA Library MicroarrayChapter 4.1 --- Introduction --- p.135Chapter 4.2 --- Materials and MethodsChapter 4.2.1 --- Selection and Amplification of clonesin SubtractionLlibrary for Microarray screening --- p.140Chapter 4.2.2 --- PCR product Purification --- p.141Chapter 4.2.3 --- Generation of Subtractive Dikaryotic Library Microarray Chip --- p.142Chapter 4.2.4 --- Screening the Differentially Expressed Genesin Dikaryon L54 by the Subtraction Dikaryotic Library cDNA Microarray Analysis --- p.143Chapter 4.2.4.1 --- Preparation of Total RNA --- p.143Chapter 4.2.4.2 --- Synthesis and fluorescent labeling of total cDNA --- p.145Chapter 4.2.4.3 --- Purification of labeled cDNA --- p.146Chapter 4.2.4.4 --- Storage Condition of Probe --- p.147Chapter 4.2.4.5 --- Analysis of labeled total cDNA --- p.148Chapter 4.2.4.6 --- Microarray hybridization --- p.150Chapter 4.2.4.7 --- Stringency washes --- p.152Chapter 4.2.4.8 --- Detection with TSA --- p.153Chapter 4.2.4.9 --- Image generation and data analysis --- p.155Chapter 4.2.5 --- Sequence analysis of clones showing differentially expressed in dikaryons in microarray screening --- p.157Chapter 4.2.5.1 --- Single-pass partial sequencing of 3´ة-end of subtractive cDNA clones --- p.157Chapter 4.2.5.2 --- Compiling dikaryotic EST database --- p.158Chapter 4.2.6 --- Comparison microarray analysis with SAGE analysis of the differentially expressed genes --- p.159Chapter 4.3 --- Results --- p.161Chapter 4.3.1 --- Preparation of clones for microarray hybridization --- p.161Chapter 4.3.2 --- Screening the differentially expressed genesin dikaryon L54 by the subtractive dikaryotic library cDNA microarray analysis --- p.162Chapter 4.3.2.1 --- Image capture and microarray data analysis --- p.162Chapter 4.3.2.2 --- Comparision of dikaryon L54 with monokaryons A and B --- p.163Chapter 4.3.2.3 --- Sequenced and comparison of the differentially expressed genes in dikaryon --- p.166Chapter 4.3.3 --- Comparison microarray analysis with SAGE analysis of the differentially expressed genes --- p.169Chapter Chapter Five --- Conclusion and Future Perpectives --- p.198References --- p.20