Serial analysis of gene expression during mycelium and primordium stages of shiitake mushroom (xianggu) Lentinula edodes.

Chum Wing Yan, Winnie.Thesis submitted in 2002.Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.Includes bibliographical references (leaves 157-178).Abstracts in English and Chinese.English Abstract --- p.iChinese Abstract --- p.iiiAcknowledgements --- p.vAbbreviations --- p.viTable of Contents --- p.viiList of Figures --- p.xList of Tables --- p.xiiiChapter Chapter One --- Literature ReviewChapter 1.1 --- Introduction --- p.1Chapter 1.2 --- Life cycle --- p.2Chapter 1.3 --- Nutritional value --- p.4Chapter 1.4 --- Medicinal value --- p.4Chapter 1.4.1 --- Antitumor ability --- p.5Chapter 1.4.2 --- Antimicrobial ability --- p.5Chapter 1.4.3 --- Hypocholesterolaemic effect --- p.6Chapter 1.4.4 --- Anti-viral effect --- p.6Chapter 1.4.5 --- Anticaries effects --- p.7Chapter 1.6 --- Commercial value --- p.7Chapter 1.6.1 --- Biodecolorization --- p.7Chapter 1.6.2 --- Bioconversion --- p.7Chapter 1.6.3 --- Biodegradation --- p.8Chapter 1.6.4 --- Indicator --- p.9Chapter 1.7 --- Cultivation --- p.9Chapter 1.8 --- Content --- p.10Chapter 1.9 --- Transformation --- p.11Chapter 1.10 --- Gene regulation for growth and fruiting body development --- p.13Chapter 1.11 --- Serial Analysis of Gene Expression --- p.18Chapter 1.11.1 --- Introduction --- p.18Chapter 1.11.2 --- Principles --- p.20Chapter 1.11.3 --- SAGE Application in Cancer and Immunology Studies --- p.22Chapter 1.11.4 --- Improvement of SAGE --- p.23Chapter 1.11.5 --- Bioinformatics --- p.24Chapter 1.12 --- DNA Microarray --- p.27Chapter 1.12.1 --- Introduction --- p.27Chapter 1.12.2 --- Application --- p.28Chapter 1.12.3 --- Method of cDNA Microarray --- p.28Chapter Chapter Two --- Serial Analysis of Gene ExpressionChapter 2.1 --- Introduction --- p.30Chapter 2.2 --- Material and Methods --- p.32Chapter 2.2.1 --- Mushroom cultivation and RNA extraction --- p.32Chapter 2.2.2 --- RNA Quality Estimation --- p.33Chapter 2.2.3 --- mRNA Isolation --- p.34Chapter 2.2.4 --- Serial Analysis of Gene Analysis (SAGE) --- p.34Chapter 2.2.4.1 --- Binding mRNA to magnetic beads for cDNA synthesis --- p.34Chapter 2.2.4.2 --- DNA synthesis verification --- p.35Chapter 2.2.4.3 --- NlaIII digestion --- p.36Chapter 2.2.4.4 --- NlaIII digestion verification --- p.36Chapter 2.2.4.5 --- Adapters ligation --- p.37Chapter 2.2.4.6 --- Cleaving with tagging enzyme --- p.37Chapter 2.2.4.7 --- Ditags creation --- p.38Chapter 2.2.4.8 --- PCR optimization and scale-up --- p.38Chapter 2.2.4.9 --- Polyacrylamide gel electrophoresis --- p.39Chapter 2.2.4.10 --- Eluting DNA from the gel --- p.40Chapter 2.2.4.11 --- NlaIII Cleavage and polyacrylamide gel electrophoresis --- p.40Chapter 2.2.4.12 --- Concatemers Ligation --- p.41Chapter 2.2.4.13 --- Cloning Concatemers into pZErO®-1 --- p.42Chapter 2.2.5 --- One Shot® TOP 10 Electrocomp´ёØ E. Coli transformation --- p.42Chapter 2.2.6 --- PCR Screening and Sequencing --- p.44Chapter 2.2.7 --- Sequence Analysis --- p.44Chapter 2.3 --- Results --- p.45Chapter 2.3.1 --- RNA Extraction --- p.45Chapter 2.3.2 --- cDNA Synthesis --- p.45Chapter 2.3.3 --- NlaIII digestion --- p.45Chapter 2.3.4 --- PCR amplification --- p.46Chapter 2.3.5 --- Gel-purification of the 100bp Ditags --- p.46Chapter 2.3.6 --- Isolation of the 26bp Ditags --- p.46Chapter 2.3.7 --- Concatemers Generation --- p.47Chapter 2.3.8 --- PCR Screening --- p.47Chapter 2.3.9 --- The Abundance and Identity of SAGE Tags --- p.48Chapter 2.4 --- Discussion --- p.86Chapter 2.4.1 --- RNA Extraction --- p.86Chapter 2.4.2 --- cDNA Synthesis and NlaIII digestion --- p.86Chapter 2.4.3 --- PCP amplification --- p.87Chapter 2.4.4 --- SAGE Tags Analysis --- p.88Chapter Chapter Three --- MicroarrayChapter 3.1 --- Introduction --- p.96Chapter 3.2 --- Materials and Methods --- p.97Chapter 3.2.1 --- Microarray chip preparation --- p.97Chapter 3.3.2 --- Sample preparation --- p.97Chapter 3.2.3 --- cDNA Synthesis and Sample labeling --- p.98Chapter 3.2.4 --- cDNA Purification --- p.99Chapter 3.2.5 --- cDNA analysis --- p.99Chapter 3.2.6 --- Array Hybridization --- p.102Chapter 3.2.6.1 --- Sample Preparation --- p.102Chapter 3.2.6.2 --- Hybridization Procedure --- p.102Chapter 3.2.7 --- Stringency Washes --- p.103Chapter 3.2.8 --- Detection with TSA --- p.103Chapter 3.2.9 --- Scanning and Analysis --- p.105Chapter 3.3 --- Results --- p.109Chapter 3.4 --- Discussion --- p.120Chapter Chapter Four --- Full Length SequencingChapter 4.1 --- Introduction --- p.124Chapter 4.2 --- Material and Methods --- p.124Chapter 4.3 --- Results and Discussion --- p.125Chapter Chapter Five --- General Discussion --- p.149Appendix --- p.155References --- p.15