Identification and characterization of genes differentially expressed during fruit body development of shiitake mushroom (Xianggu) Lentinula edodes.

• Other Authors: Leung, Sze Wan Grace.
• Format: Others
• Language: English; Chinese
• Published: 1998
• Subjects: Shiitake > Genetics; Shiitake > Growth
• Online Access: http://library.cuhk.edu.hk/record=b5896301; http://repository.lib.cuhk.edu.hk/en/item/cuhk-322271

by Leung Sze Wan, Grace. === Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. === Includes bibliographical references (leaves 183-200). === Abstract also in Chinese. === Abstract --- p.iii === Acknowledgment --- p.v === Abbreviations --- p.vi === Table of contents --- p.vii === List of Figures --- p.xii === List of Tables --- p.xv === Chapter Chapter One --- Literature Review === Chapter 1.1 --- Introduction --- p.1 === Chapter 1.2 --- The life of Lentinula edodes --- p.3 === Chapter 1.3 --- Biochemical and molecular studies on mushroom development --- p.6 === Chapter 1.3.1 --- From monokaryotic to dikaryotic mycelium --- p.7 === Chapter 1.3.2 --- Initiation and differentiation of the primordium --- p.11 === Chapter 1.3.3 --- Growth and maturation of the fruit body --- p.21 === Chapter 1.4 --- Prospectus --- p.25 === Chapter Chapter Two --- Isolation of Genes Differentially Expressed During the Development of Lentinula edodes by RAP-PCR === Chapter 2.1 --- Introduction --- p.27 === Chapter 2.2 --- Materials and Methods --- p.32 === Chapter 2.2.1 --- Strains and culture conditions --- p.32 === Chapter 2.2.2 --- Isolation of total RNAs --- p.32 === Chapter 2.2.3 --- RNA fingerprinting by RAP-PCR --- p.34 === Chapter 2.2.4 --- PCR reamplification of RAP products --- p.34 === Chapter 2.2.5 --- Reverse dot-blot analysis --- p.35 === Chapter 2.2.5.1 --- Membrane preparation --- p.35 === Chapter 2.2.5.2 --- Probe preparation --- p.36 === Chapter 2.2.5.3 --- Hybridization --- p.37 === Chapter 2.2.5.4 --- Stringency washes and autoradiography --- p.38 === Chapter 2.2.6 --- Cloning and sequencing of differentially expressed genes --- p.38 === Chapter 2.2.6.1 --- Ligation of inserts into pCR-Script vector --- p.38 === Chapter 2.2.6.2 --- Transformation --- p.39 === Chapter 2.2.6.3 --- PCR screening of white colonies --- p.41 === Chapter 2.2.6.4 --- Extraction of plasmid DNA --- p.41 === Chapter 2.2.6.5 --- DNA cycle sequencing --- p.42 === Chapter 2.3 --- Results --- p.44 === Chapter 2.3.1 --- Total RNA isolation --- p.44 === Chapter 2.3.2 --- RNA fingerprints --- p.48 === Chapter 2.3.3 --- Reverse dot-blot analysis --- p.53 === Chapter 2.3.4 --- Cloning and sequencing of selected RAP-products --- p.61 === Chapter 2.3.5 --- Sequence analyses --- p.61 === Chapter 2.4 --- Discussion --- p.77 === Chapter Chapter Three --- Expression Pattern Analysis by Northern Blot Hybridization === Chapter 3.1 --- Introduction --- p.82 === Chapter 3.2 --- Materials and Methods --- p.85 === Chapter 3.2.1 --- Primer design & PCR amplification of Le.ras fragment --- p.85 === Chapter 3.2.2 --- Primer design & PCR amplification of L. edodes GAPDH gene --- p.85 === Chapter 3.2.3 --- Cloning & sequencing of L edodes GAPDH gene --- p.86 === Chapter 3.2.4 --- Northern blot analysis --- p.87 === Chapter 3.2.4.1 --- RNA extraction by Tri-reagent --- p.87 === Chapter 3.2.4.2 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.88 === Chapter 3.2.4.3 --- Northern blotting --- p.88 === Chapter 3.2.4.4 --- Preparation of probes --- p.89 === Chapter 3.2.4.5 --- Hybridization and stringency washes --- p.90 === Chapter 3.3 --- Results --- p.91 === Chapter 3.3.1 --- Establishing an internal control for expression level studies I: Le.ras --- p.91 === Chapter 3.3.2 --- Establishing an internal control for expression level studies II: GAPDH gene --- p.91 === Chapter 3.3.3 --- Northern blot hybridizations of RAP-fragments --- p.92 === Chapter 3.4 --- Discussion --- p.101 === Chapter Chapter Four --- Obtaining Full-length cDNA of L. edodes MAP kinase and Cyclin B by Rapid Ampification of cDNA Ends (RACE) === Chapter 4.1 --- Introduction --- p.105 === Chapter 4.1.1 --- Principles of 3´ة RACE --- p.107 === Chapter 4.1.2 --- Principles of 5' RACE --- p.109 === Chapter 4.2 --- Materials and Methods --- p.112 === Chapter 4.2.1 --- Isolation of Total RNA --- p.112 === Chapter 4.2.2 --- 3'RACE --- p.112 === Chapter 4.2.2.1 --- First Strand cDNA Synthesis --- p.112 === Chapter 4.2.2.2 --- Amplification of the Target cDNA --- p.113 === Chapter 4.2.2.3 --- Reamplification and nested amplification of 3'RACE products --- p.114 === Chapter 4.2.3 --- 5'RACE --- p.115 === Chapter 4.2.3.1 --- First Strand cDNA Synthesis --- p.115 === Chapter 4.2.3.2 --- GlassMax DNA Isolation Spin Cartridge Purification of cDNA --- p.115 === Chapter 4.2.3.3 --- TdT Tailing of cDNA --- p.116 === Chapter 4.2.3.4 --- PCR of dC-tailed cDNA --- p.116 === Chapter 4.2.3.5 --- Nested Amplification --- p.117 === Chapter 4.2.4 --- Cloning and sequencing of RACE products --- p.117 === Chapter 4.2.5 --- Obtaining full-length cDNA of L. edodes MAPK --- p.118 === Chapter 4.2.5.1 --- Design of primers --- p.118 === Chapter 4.2.5.2 --- PCR amplification of LeMAPK --- p.118 === Chapter 4.2.5.3 --- Sequencing of full-length LeMAPK --- p.119 === Chapter 4.3 --- Results --- p.120 === Chapter 4.3.1 --- RACE of L. edodes Cyclin B and MAPK --- p.120 === Chapter 4.3.2 --- Sequences of Cyclin B and MAPK RACE products --- p.127 === Chapter 4.3.3 --- PCR product and sequence of L. edodes MAPK full-length cDNA --- p.131 === Chapter 4.4 --- Discussion --- p.139 === Chapter Chapter Five --- Functional Analysis of LeMAPK by Yeast Complementation Tests === Chapter 5.1 --- Introduction --- p.144 === Chapter 5.2 --- Materials and Methods --- p.152 === Chapter 5.2.1 --- The construct --- p.152 === Chapter 5.2.2 --- Yeast strains and media --- p.153 === Chapter 5.2.3 --- Yeast transformation --- p.154 === Chapter 5.2.4 --- Monitoring the Expression of HA-MAPK by Western Analysis --- p.155 === Chapter 5.2.4.1 --- Western blot analysis with anti-HA antibody --- p.155 === Chapter 5.2.4.2 --- Preparation of Cell Lysate from Yeast --- p.155 === Chapter 5.2.4.3 --- Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) --- p.156 === Chapter 5.2.4.4 --- Western Blotting --- p.157 === Chapter 5.2.4.5 --- Immunodetection --- p.158 === Chapter 5.2.4.6 --- ECL detection --- p.158 === Chapter 5.2.5 --- Complementation test of LeMAPK on yeast fus3Δkss1Δ double mutant --- p.159 === Chapter 5.2.5.1 --- Mating test --- p.159 === Chapter 5.2.5.2 --- Haploid invasive growth === Chapter 5.3 --- Results --- p.161 === Chapter 5.3.1 --- The construct - from E. coli to yeast --- p.161 === Chapter 5.3.2 --- The expression of LeMAPK in yeast and the complementation tests --- p.166 === Chapter 5.4 --- Discussion --- p.170 === Chapter Chapter Six --- General Discussion --- p.175 === References --- p.183