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

• Other Authors: Shih, Sheung Mei.
• Format: Others
• Language: English; Chinese
• Published: 2004
• Subjects: Shiitake > Molecular genetics; Plant gene expression; DNA microarrays
• Online Access: http://library.cuhk.edu.hk/record=b5896199; http://repository.lib.cuhk.edu.hk/en/item/cuhk-324710

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