Effect of stress on fruit body initiation of shiitake mushroom Lentinula edodes.

Tjia Wai Mui.Thesis submitted in: July 2002.Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.Includes bibliographical references (leaves 123-140).Abstracts in English and Chinese.Abstract (English) --- p.iAbstract (Chinese) --- p.iiiAcknowledgement --- p.ivAbbreviations --- p.vTable of Contents --- p.viList of Figures --- p.xList of Tables --- p.xiiChapter Chapter One --- Literature ReviewChapter 1.1 --- Introduction --- p.1Chapter 1.2 --- Growth of L. edodes --- p.3Chapter 1.2.1 --- Life cycle of L. edodes --- p.3Chapter 1.2.2 --- Growth parameters of L. edodes --- p.6Chapter 1.2.2.1 --- Temperature --- p.6Chapter 1.2.2.2 --- Relative humidity --- p.7Chapter 1.2.2.3 --- Moisture content in substrate --- p.7Chapter 1.2.2.4 --- Light --- p.8Chapter 1.2.2.5 --- pH --- p.8Chapter 1.3 --- Cultivation of L. edodes --- p.9Chapter 1.3.1 --- History and development of artificial cultivation --- p.9Chapter 1.3.2 --- Use of forced fruiting --- p.11Chapter 1.4 --- Molecular studies of stress on fungi --- p.12Chapter 1.4.1 --- Studies of temperature stress in mushroom --- p.12Chapter 1.4.2 --- Studies of molecular chaperones in fungi --- p.13Chapter 1.4.2.1 --- Role of molecular chaperones --- p.13Chapter 1.4.2.2 --- Heat shock protein 70 (Hsp70) and their cochaperones --- p.13Chapter 1.4.2.3 --- Other chaperones --- p.15Chapter 1.4.2.4 --- Molecular chaperones and development --- p.16Chapter 1.5 --- Prospectus --- p.19Chapter Chapter Two --- The Effect of Stress on the Growth of L. edodesChapter 2.1 --- Introduction --- p.23Chapter 2.2 --- Materials and Methods --- p.24Chapter 2.2.1 --- Strain and culture conditions --- p.24Chapter 2.2.2 --- Stress treatments --- p.24Chapter 2.2.3 --- Data collection --- p.25Chapter 2.2.4 --- Data analysis --- p.25Chapter 2.3 --- Results --- p.27Chapter 2.3.1 --- Reliability analysis --- p.27Chapter 2.3.2 --- Descriptive statistics --- p.28Chapter 2.3.3 --- Independent t-test (ANOVA) --- p.33Chapter 2.4 --- Discussion --- p.37Chapter Chapter Three --- Sequence Analysis of selected Stress GenesChapter 3.1 --- Introduction --- p.39Chapter 3.2 --- Materials and Methods --- p.40Chapter 3.2.1 --- Isolation of stress genes --- p.40Chapter 3.2.1.1 --- Construction of primordial cDNA library --- p.40Chapter 3.2.1.2 --- Screening of cDNA clones --- p.40Chapter 3.2.2 --- Sequence analyses of stress genes --- p.41Chapter 3.2.2.1 --- Amplification and purification of cDNA insert --- p.41Chapter 3.2.2.2 --- Full length DNA cycle sequencing --- p.42Chapter 3.2.2.3 --- Sequence analyses --- p.43Chapter 3.2.3 --- Screening of LeSSA (Inducible HSP70) --- p.45Chapter 3.2.3.1 --- PCR screening of LeSSA by degenerate primers and LeSSB specific primers --- p.45Chapter 3.2.3.2 --- Screening of LeSSA from cDNA library by hybridization --- p.49Chapter 3.3 --- Results --- p.51Chapter 3.3.1 --- Sequence analyses --- p.51Chapter 3.3.1.1 --- LeSSB --- p.51Chapter 3.3.1.2 --- LeMge1 --- p.57Chapter 3.3.1.3 --- LeSTI1 --- p.62Chapter 3.3.1.4 --- LeTCP1β --- p.69Chapter 3.3.1.5 --- LeTCP1γ --- p.74Chapter 3.3.2 --- Failure of isolating LeSSA (Inducible HSP70) --- p.80Chapter 3.4 --- Discussion --- p.82Chapter 3.4.1 --- Sequence analyses --- p.82Chapter 3.4.2 --- Screening of LeSSA --- p.84Chapter Chapter Four --- Characterization of stress genes upon different stressesChapter 4.1 --- Introduction --- p.86Chapter 4.2 --- Materials and Methods --- p.87Chapter 4.2.1 --- Strain and culture conditions --- p.87Chapter 4.2.2 --- Stress treatments --- p.87Chapter 4.2.3 --- Isolation of total RNAs --- p.87Chapter 4.2.4 --- Reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.88Chapter 4.2.4.1 --- Reverse transcription --- p.88Chapter 4.2.4.2 --- PCR amplification by specific primers of stress genes --- p.89Chapter 4.2.5 --- Northern blot analyses --- p.91Chapter 4.2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.91Chapter 4.2.5.2 --- Northern blotting --- p.91Chapter 4.2.5.3 --- Preparation of probes --- p.92Chapter 4.2.5.4 --- Hybridization and stringency washes --- p.93Chapter 4.2.6 --- Isolation of total protein --- p.94Chapter 4.2.7 --- Quantification of protein by Bradford method --- p.95Chapter 4.2.8 --- Western blot analyses --- p.95Chapter 4.2.8.1 --- Sodium dodecyl sulfate ´ؤ polyacrylamide gel electrophoresis (SDS-PAGE) --- p.95Chapter 4.2.8.2 --- Western blotting --- p.96Chapter 4.2.8.3 --- Immunodetection --- p.98Chapter 4.2.8.4 --- ECL detection --- p.98Chapter 4.3 --- Results --- p.99Chapter 4.3.1 --- Reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.99Chapter 4.3.2 --- Northern blot hybridization --- p.106Chapter 4.3.2.1 --- Establishing an internal control --- p.106Chapter 4.3.2.2 --- Dig-labelling of stress genes --- p.106Chapter 4.3.2.3 --- Northern blot hybridizaton of stress genes --- p.106Chapter 4.3.3 --- Western blot hybridization --- p.111Chapter 4.4 --- Discussions --- p.113Chapter Chapter Five --- General Discussions --- p.118References --- p.12