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

香菇是一種重要的藥用蘑菇。數千年來，香菇一直被人們作為食物和藥物來使用。許多研究表明，香菇的提取物具有抗氧化活性，而且他們的抗氧化活性與他們的酚類化合物的含量相關。然而，到目前為止該研究大多集中在對香菇子實體的研究，對香菇菌絲體分泌物的研究就少見報導。 / 在本課題研究中，使用不同的體外抗氧化測定方法和酚類化合物含量測定方法來研究兩種香菇菌絲體分泌物（1358DE 和L5458DE）。實驗結果表明， 在不同的體外抗氧化實驗中1358DE 和L5458DE均具有明顯不同的抗氧化活性。在清除DPPH自由基，清除氫氧根離子，清除超氧陽離子，清除過氧化氫離子，螯合亞鐵離子，還原能力，抑制老鼠紅細胞溶血和抑制脂質過氧化的實驗中，1358DE 和L5458DE的IC50 分別為3.3和132.6; 44.5和 > 1000; 26.9和53.7; 153.6和 >175.0; 176.0和521.0; 26.7和746.4; 47.8和736.9; 3.1和 > 1000 μg/ml。他們的多酚化合物的含量分別為237.33 and 24.08 mg (GAE)/g of DE。實驗資料表明，1358DE的抗氧化活性高於L5458DE，其原因可能是1358DE的酚類化合物含量較高。 / 由於1358DE具有較好的抗氧化活性，採用有機溶劑萃取的方法將其分成水溶性部位和乙酸乙酯部位。體外抗氧化實驗表明，水溶性部位的抗氧化活性與1358DE相近，而乙酸乙酯部位則沒有表現出抗氧化活性。因此，使用聚醯胺柱色譜（可以將多酚類化合物從其他成分中分離出來）對水溶性部位進行進一步的分離，可以得到兩個聚醯胺洗脫部位（P-1和P-2）。與原來的水溶性部位比較，P-1的糖的含量明顯增加，而多酚化合物含量明顯減少，抗氧化活性也明顯降低；相反，P-2的糖的含量明顯減少，而多酚化合物含量明顯增加，抗氧化活性也明顯增加。該實驗結果表明，糖對抗氧化活性的貢獻遠不及多酚化合物。因此，多酚化合物是1358DE的抗氧化活性成分。基質輔助鐳射解吸電離飛行時間質譜和三氯化鐵試劑測定結果表明，P-2是一類水溶性多酚低聚物（WSP），它的分子量在600~1200Da之間。 / 水溶性多酚低聚物（WSP）是1358DE的主要抗氧化活性成分。採用過氧化氫引導細胞毒性的細胞(V79-4)模型來進一步研究WSP的抗氧化活性。在細胞毒性試驗中，在所有測試濃度，WSP在濃度6.25~50 μg/ml均能明顯抑制過氧化氫引致的細胞毒性。此外，WSP還能明顯抑制由過氧化氫引起的丙二醛（MDA）增加和抗氧化酶（SOD，CAT，GSH-Px）的減少。 / 許多抗氧化劑被報導具有抗血管增生活性，該活性與其抗氧化活性相關。由於WSP具有非常好的抗氧化活性，因此，採用斑馬魚模型來研究WSP的抗血管增生活性。在內源性鹼性磷酸酶測定實驗結果表明，WSP在濃度50，100，150，200，250 μg/ml，斑馬魚（野生型）的血管生成明顯分別減低為87.2, 85.6, 74.8, 69.4, and 62.8%（與空白對照組相比）。此外，在螢光顯微鏡下可觀察到WSP在濃度為250μg/ml能明顯抑制螢光斑馬魚（fli1a:EGFP）的節間血管形成。 / 本研究表明，水溶性多酚低聚物（WSP）是香菇菌絲體分泌物的抗氧化成分，WSP不僅具有抗氧化活性，同時還具有抗血管增生活性。此外，本研究結果表明香菇菌絲體分泌物是很好的天然抗氧化劑的來源。 / Shiitake mushroom (Lentinus edodes), known in China as Xiang-gu, is one of the most valuable medicinal mushrooms, and has been used for thousands of years both as food and medicine. Shiitake mushroom extracts have also been found to have antioxidant properties and their antioxidant ability is positively correlated with their phenolic content. However, thus far, investigation of the antioxidant ability of shiitake mushroom has mainly focused on the fruiting body, and the antioxidant properties of its mycelial exudates are rarely reported. / In this study, exudates (DE) secreted from two shiitake mushroom mycelia (strains 1358 and L5458) were evaluated for their antioxidative properties and phenolic content. 1358DE and L5458DE showed distinct antioxidant activity in different in vitro assays, including scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radical, superoxide anions and hydrogen peroxide; the ability to chelate ferrous ions; reducing power; hemolysis inhibition activity in rat erythrocyte; and lipid peroxidation inhibition (IC₅₀ values of 1358DE and L5458DE were 3.3 and 132.6; 44.5 and > 1000; 26.9 and 53.7; 153.6 and >175.0; 176.0 and 521.0; 26.7 and 746.4; 47.8 and 736.9; and 3.1 and > 1000 μg/mL, respectively). Their total phenolic content was 237.33 and 24.08 mg gallic acid equivalent (GAE)/g of dry DE, respectively. Overall, these results show that 1358DE generally possesses better antioxidant properties than L5458DE, possibly due to its larger total phenolic content. / 1358DE were selected to further investigate for its better antioxidant effect. 1358DE was fractionated using water-solvent partition and two fractions [water soluble fraction (WF) and ethyl acetate fraction (EF)] were obtained. The antioxidant effects of WF were similar to those of the original 1358DE, while EF did not possess any antioxidant activities. The WF was further isolated with polyamide column, which can separated the polyphenols from other components, and two sub-fraction (P-1 and P-2) were obtained. After the WF passing through the polyamide column, carbohydrate content in the sub-fraction 1 (P-1) was significantly increased, while its total phenolic content reduced dramatically, and its antioxidant activity decreased. However, the sub-fraction 2 (P-2) was the opposite. Carbohydrate content in P-2 was significantly reduced, while its total phenolic content increased dramatically, and its antioxidant activity increased. Apparently, carbohydrate contributed little to the antioxidant effect than that of the phenolic compounds as shown from this investigation. These results suggest that the antioxidant effect in 1358DE was contributed by the presence of polyphenols. Besides, results from Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ferric trichloride reaction suggested that P-2 was oligomers of water soluble polyphenols (WSP) with the molecular weight of about 600~1200 Da. / The water soluble polyphones (WSP) were the potent antioxidant components in 1358DE and further study its protective effect against the hydrogen peroxide which induced cytotoxicity in V79-4 cells. In the cell viability experiments, pretreatment of WSP at the concentrations of 6.25~50 μg/ml increased the cell viability significantly more than at the presence of H₂O₂ only. Besides, the pretreatment of cells with WSP significantly inhibited the increase of Malondialdehyde (MDA, which is a by-product of lipid peroxidation) production and the decrease of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) activities induced by H₂O₂. / Quite a few antioxidant compounds have been reported that a causative relationship may exist between the anti-angiogenic activity and antioxidant effect. Therefore, a zaebrafish model was using to investigate the anti-angiogenic activity of the WSP because of its excellent antioxidant activity. In quantitative of endogenous alkaline phosphatase (EAP) assay, after the embryos treated with WSP at final concentrations of 50, 100, 150, 200, 250 μg/ml, and the vessel formation were significantly (p < 0.05) reduced to 87.2, 85.6, 74.8, 69.4, and 62.8% of the control value, respectively. Moreover, from the microscope, compare to the control, WSP at the concentration of 250 μg/ml also showed potent inhibition on the intersegmental blood vessels (ISVs) formation in Tg(fli1a:EGFP)y1 zebrafish embryos. Thus, the finding indicated that WSP could inhibit vessel formation in zeabrafish. / Overall, this study revealed that water soluble polyphenols (WSP) was the active components of 1358DE. Besides of the antioxidant effect, the WSP could inhibit vessel formation significantly in zebrafish. The findings indicate that exudates of shiitake mushroom mycelia have good potential as a source of natural antioxidants. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Weihuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 90-103). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.ix / List of Tables --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Oxidation and antioxidant --- p.1 / Chapter 1.1.1 --- Reactive oxygen species (ROS) --- p.1 / Chapter 1.1.2 --- Sources of ROS --- p.1 / Chapter 1.1.3 --- The role of ROS in normal physiology --- p.2 / Chapter 1.1.4 --- Oxidative damage to DNA, lipids and proteins --- p.2 / Chapter 1.1.5 --- Antioxidant defense systems in vivo --- p.4 / Chapter 1.1.6 --- Sources of antioxidants --- p.6 / Chapter 1.2 --- Assessment of antioxidant capacity in vitro and in vivo (Antioxidant methodology) --- p.9 / Chapter 1.2.1 --- Assessment of Free Radical Scavenging Capacity in vitro --- p.9 / Chapter 1.2.2 --- Antioxidant capacity in cultured Cells --- p.10 / Chapter 1.2.3 --- Antioxidant capacity in vivo --- p.11 / Chapter 1.3 --- Mushrooms --- p.12 / Chapter 1.3.1 --- Mushroom life cycle --- p.12 / Chapter 1.3.2 --- Nutritional and medicinal values of mushroom --- p.14 / Chapter 1.4 --- Shiitake mushroom (Lentinus edodes) --- p.15 / Chapter 1.5 --- Objectives --- p.17 / Chapter Chapter 2 --- Antioxidant activity and total phenolic content in Shiitake mycelial exudates --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.2 --- Materials and methods --- p.19 / Chapter 2.2.1 --- Materials --- p.19 / Chapter 2.2.2 --- Sample preparation --- p.20 / Chapter 2.2.3 --- In vitro antioxidant activity assays --- p.20 / Chapter 2.2.4 --- Determination of total phenolic content --- p.25 / Chapter 2.2.5 --- Statistical analysis --- p.25 / Chapter 2.3 --- Results and discussion --- p.25 / Chapter 2.3.1 --- Antioxidant activity --- p.25 / Chapter 2.3.2 --- Total phenolic content --- p.37 / Chapter 2.3.3 --- Antioxidant activity (IC₅₀ values) and phenolic content --- p.37 / Chapter 2.4 --- Conclusion --- p.39 / Chapter Chapter 3 --- The antioxidant components of Shiitake mycelial exudates (1358DE) --- p.42 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.42 / Chapter 3.2.1 --- Materials --- p.42 / Chapter 3.2.2 --- Sample preparation --- p.43 / Chapter 3.2.3 --- HPLC analytical condition --- p.43 / Chapter 3.2.4 --- Sample fractionated using solvent-water partition and polyamide column chromatographic method guided by in vitro antioxidant assays --- p.43 / Chapter 3.2.5 --- Determination of total phenolic content --- p.44 / Chapter 3.2.6 --- Determination of the contents of carbohydrate --- p.45 / Chapter 3.2.7 --- MALDI-TOF MS analysis --- p.45 / Chapter 3.2.8 --- Statistical analysis --- p.46 / Chapter 3.3 --- Results and discussions --- p.46 / Chapter 3.3.1 --- HPLC analytical results --- p.46 / Chapter 3.3.2 --- Samples fractionation using solvent-water partition --- p.47 / Chapter 3.3.3 --- Water soluble fraction (WF) was further isolated using polyamide column chromatographic --- p.50 / Chapter 3.3.4 --- Molecular weight determination of P-2 --- p.54 / Chapter 3.4 --- Conclusion --- p.56 / Chapter Chapter 4 --- Antioxidative effect of water soluble polyphenols (WSP) in Shiitake mycelial exudates (1358DE) against H2O2-induced cytotoxicity in V79-4 cells --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Sample preparation, materials and methods --- p.58 / Chapter 4.2.1 --- Preparation of the water soluble polyphenols (WSP) --- p.58 / Chapter 4.2.2 --- Materials --- p.58 / Chapter 4.2.3 --- Cell culture and treatment --- p.59 / Chapter 4.2.4 --- MTT assay --- p.60 / Chapter 4.2.5 --- Lactate dehydrogenase (LDH) release assay --- p.60 / Chapter 4.2.6 --- Assay for lipid peroxidation measuring the malondialdehyde (MDA) --- p.61 / Chapter 4.2.7 --- Assay for antioxidant enzymes --- p.62 / Chapter 4.2.8 --- Protein determination --- p.62 / Chapter 4.2.9 --- Statistical analysis --- p.63 / Chapter 4.3 --- Results and discussion --- p.63 / Chapter 4.3.1 --- Cytotoxicty of WSP in V79-4 cells --- p.63 / Chapter 4.3.2 --- Determined the time of WSP pretreatment in V79-4 cell against H₂O₂-induced cytotoxicity --- p.63 / Chapter 4.3.3 --- Protective effect of WSP treated cell against H₂O₂-induced cytotoxicity --- p.64 / Chapter 4.3.3 --- Inhibition of WSP on lipid peroxidation --- p.66 / Chapter 4.3.4 --- Effects of WSP on antioxidant enzyme activities --- p.66 / Chapter 4.4 --- Conclusion --- p.72 / Chapter Chapter 5 --- Anti-angiogenic property of water soluble polyphenols (WSP) in Shiitake mycelial exudates (1358DE) --- p.73 / Chapter 5.1 --- Introduction --- p.73 / Chapter 5.1.1 --- Angiogenesis --- p.73 / Chapter 5.1.2 --- Angiogenesis as a therapeutic target --- p.73 / Chapter 5.1.3 --- Tumors angiogenesis --- p.75 / Chapter 5.1.4 --- Reactive oxygen species (ROS) and tumor angiogenesis --- p.75 / Chapter 5.1.5 --- Anti-angiogenic effects of polyphenols --- p.76 / Chapter 5.1.6 --- Experimental model for studying anti-angiogenic agents --- p.76 / Chapter 5.2 --- Sample preparation, materials and methods --- p.78 / Chapter 5.2.1 --- Preparation of the water soluble polyphenols (WSP) --- p.78 / Chapter 5.2.2 --- Materials --- p.78 / Chapter 5.2.3 --- Methods --- p.79 / Chapter 5.2.4 --- Statistical analysis --- p.80 / Chapter 5.3 --- Results --- p.81 / Chapter 5.3.1 --- Anti-angiogenic effect of WSP on zebrafish model --- p.81 / Chapter 5.3.2 --- Microscopic imaging --- p.82 / Chapter 5.4 --- Discussion and conclusion --- p.82 / Chapter Chapter6 --- Conclusions --- p.84 / Chapter 6.1 --- Conclusion --- p.86 / Chapter 6.2 --- Future works --- p.88 / References --- p.89 / Chapter Appendix 1 --- Publication --- p.104

Lee Ming Tsung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 160-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutritional values --- p.2 / Chapter 1.3 --- Medicinal values --- p.3 / Chapter 1.3.1 --- Anti-tumor effect --- p.3 / Chapter 1.3.2 --- Anti-viral and anti-caries effect --- p.4 / Chapter 1.3.3 --- Immunopotentiating effect --- p.4 / Chapter 1.3.4 --- Hypocholesterolaemic effect --- p.5 / Chapter 1.4 --- Life cycle and morphology --- p.6 / Chapter 1.5 --- Growth requirements --- p.9 / Chapter 1.5.1 --- Nutritional factors --- p.9 / Chapter 1.5.2 --- Physical and chemical factors --- p.10 / Chapter 1.6 --- Application of L. edodes --- p.12 / Chapter 1.7 --- Endocytosis --- p.13 / Chapter 1.7.1 --- Different types of endocytosis --- p.13 / Chapter 1.7.1.1 --- Phagocytosis --- p.14 / Chapter 1.7.1.2 --- Pinocytosis --- p.15 / Chapter 1.7.1.3 --- Receptor-mediated endocytosis --- p.15 / Chapter 1.7.2 --- The Endocytic Pathway --- p.17 / Chapter 1.7.3 --- Endocytosis in fungi --- p.20 / Chapter 1.7.4 --- Rab GTPases --- p.21 / Chapter 1.7.4.1 --- Control of the active and inactive state of Rab proteins --- p.22 / Chapter 1.7.4.2 --- Regulation of docking and fusion of membrane in endosomal trafficking --- p.23 / Chapter 1.7.4.3 --- Rab7 GTPase --- p.26 / Chapter 1.8 --- Aims of the project --- p.28 / Chapter Chapter 2 --- Protein-protein Interaction Study of Le.Rab7 by in vivo and in vitro Interaction Assay --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Yeast two-hybrid screening --- p.36 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Rab7-pGBK.T7 --- p.36 / Chapter 2.2.1.1.1 --- Bacterial transformation --- p.36 / Chapter 2.2.1.1.2 --- PCR screening for positive transformants --- p.38 / Chapter 2.2.1.1.3 --- Plasmid preparation and confirmation of transformants --- p.38 / Chapter 2.2.1.1.4 --- Sequencing --- p.39 / Chapter 2.2.1.2 --- Confirmation of Le.Rab7 protein expression in yeast --- p.40 / Chapter 2.2.1.2.1 --- Yeast transformation --- p.40 / Chapter 2.2.1.2.2 --- Yeast protein extraction --- p.40 / Chapter 2.2.1.2.3 --- Western Blotting --- p.41 / Chapter 2.2.1.3 --- Yeast Two-hybrid screening by Yeast-mating --- p.42 / Chapter 2.2.1.4 --- Identification of Preys --- p.44 / Chapter 2.2.1.4.1 --- PCR screening for clones grown on plates --- p.44 / Chapter 2.2.1.4.2 --- Colony lift filter assay --- p.45 / Chapter 2.2.1.4.3 --- Sequencing --- p.47 / Chapter 2.2.1.5 --- Confirmation of interaction by Co-transformation assay --- p.47 / Chapter 2.2.1.5.1 --- Plasmid preparation of positive clones --- p.47 / Chapter 2.2.1.5.2 --- Transformation and bacterial plasmid preparation --- p.48 / Chapter 2.2.1.5.3 --- Yeast two-hybrid screening by co-transformation --- p.48 / Chapter 2.2.1.5.4 --- Colony lift filter assay --- p.50 / Chapter 2.2.2 --- Rapid Amplification of cDNA 5'ends --- p.51 / Chapter 2.2.2.1 --- RNA preparation --- p.51 / Chapter 2.2.2.1.1 --- Strains and culture conditions --- p.51 / Chapter 2.2.2.1.2 --- RNA extraction --- p.51 / Chapter 2.2.2.2 --- 5' RACE --- p.52 / Chapter 2.2.2.2.1 --- RNA processing --- p.52 / Chapter 2.2.2.2.2 --- Reverse transcription --- p.53 / Chapter 2.2.2.2.3 --- Nested PCR for 5'RLM-RACE --- p.53 / Chapter 2.2.2.3 --- "Gel analysis of products, TA cloning of RACE product and sequencing" --- p.54 / Chapter 2.2.2.4 --- Cloning of full-length Le.Rab5 --- p.54 / Chapter 2.2.3 --- In vitro protein-protein interaction assay --- p.55 / Chapter 2.2.3.1 --- Plasmid extraction from E.coli --- p.55 / Chapter 2.2.3.2 --- In vitro translation --- p.56 / Chapter 2.2.3.3 --- In vitro co-immunoprecipitation --- p.56 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Yeast two-hybrid analysis by yeast mating assay --- p.57 / Chapter 2.2.1.1 --- Confirmation of the clones Le.Ra67-pGBKT7 --- p.57 / Chapter 2.3.1.1.1 --- PCR screening for positive transformants --- p.57 / Chapter 2.3.1.1.2 --- Plasmid preparation and confirmation of transformants --- p.58 / Chapter 2.3.1.1.3 --- Sequencing --- p.59 / Chapter 2.2.1.2 --- Confirmation of protein expression in yeast --- p.60 / Chapter 2.3.1.2.1 --- Yeast transformation --- p.60 / Chapter 2.3.1.2.2 --- SDS-PAGE and Western blotting of Le.Rab7 in yeast --- p.61 / Chapter 2.2.1.3 --- Yeast two-hybrid screening by yeast mating assay --- p.62 / Chapter 2.2.1.4 --- Identification of Preys --- p.63 / Chapter 2.3.1.4.1 --- PCR screening for clones grown on plates --- p.63 / Chapter 2.3.1.4.2 --- Colony lift assay --- p.65 / Chapter 2.3.1.4.3 --- Sequencing --- p.67 / Chapter 2.3.2 --- Confirmation of interactions by co-transformation assay --- p.70 / Chapter 2.2.2.1 --- Yeast two-hybrid analysis by co-transformation assay --- p.70 / Chapter 2.2.2.2 --- Colony lift filter assay --- p.70 / Chapter 2.2.2.3 --- Selection of prey plasmids for in vitro binding assay --- p.72 / Chapter 2.3.3 --- Rapid amplification of cDNA ends (RACE) --- p.76 / Chapter 2.2.3.1 --- TA cloning of RACE product and sequencing --- p.76 / Chapter 2.2.3.2 --- Cloning of full-length Le.Rab5 --- p.79 / Chapter 2.3.4 --- In vitro protein-protein interaction assay --- p.80 / Chapter 2.4 --- Discussion --- p.82 / Chapter Chapter 3 --- Temporal and Spatial expression of Le.Rab7，Le.Rab5 and Le.RACKl --- p.87 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Northern blot analysis --- p.93 / Chapter 3.2.1.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.93 / Chapter 3.2.1.2 --- Northern blotting --- p.94 / Chapter 3.2.1.2.1 --- Transfer of RNAs --- p.94 / Chapter 3.2.1.2.2 --- Probe preparation --- p.95 / Chapter 3.2.1.2.3 --- "Hybridization, Stringency washes and Signal detection" --- p.96 / Chapter 3.2.2 --- Quantitative RT-PCR --- p.97 / Chapter 3.2.2.1 --- cDNA synthesis from different developmental stages --- p.97 / Chapter 3.2.2.1.1 --- RNA preparation extraction --- p.97 / Chapter 3.2.2.1.2 --- DNase I treatment --- p.97 / Chapter 3.2.2.1.3 --- Reverse transcription --- p.98 / Chapter 3.2.2.2 --- Real time PCR --- p.98 / Chapter 3.2.2.2.1 --- Primer design and verification --- p.98 / Chapter 3.2.2.2.2 --- Real time PCR reaction and data analysis --- p.100 / Chapter 3.2.3 --- In situ RNA-RNA hybridization --- p.101 / Chapter 3.2.3.1 --- Preparation of samples and probes --- p.101 / Chapter 3.2.3.1.1 --- Tissue preparation --- p.101 / Chapter 3.2.3.1.2 --- RNA probe synthesis --- p.101 / Chapter 3.2.3.2 --- Hybridization and Signal development --- p.102 / Chapter 3.2.3.3 --- Image viewing --- p.103 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Northern blot analysis --- p.105 / Chapter 3.3.2 --- Quantitative RT-PCR assays --- p.109 / Chapter 3.3.3 --- In situ RNA-RNA hybridization --- p.113 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- Existence of endocytosis and Protein localization of Le.Rab7 in L. edodes --- p.123 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.2 --- Materials and Methods --- p.127 / Chapter 4.2.1 --- Tracing the endocytie pathway using FM4-64 dye --- p.127 / Chapter 4.2.1.1 --- Strains and culture conditions --- p.127 / Chapter 4.2.1.2 --- FM4-64 internalization in mycelium and gill tissue of L. edodes --- p.127 / Chapter 4.2.2 --- Drug treatment effect on the internalization of FM4-64 dye --- p.128 / Chapter 4.2.3 --- Double labeling with AM4-64 and anti-Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1 --- Synthesis of Le.Rab7 antibody --- p.129 / Chapter 4.2.3.1.1 --- Customization of Le.Rab7 antiserum --- p.129 / Chapter 4.2.3.1.2 --- Validation of anti-Le.Rab7 polyclonal antiserum --- p.129 / Chapter 4.2.3.2 --- Double immunofluorescence labeling --- p.130 / Chapter 4.2.4 --- Immunohistochemistry of young and mature fruiting body --- p.131 / Chapter 4.2.4.1 --- Tissue preparation --- p.131 / Chapter 4.2.4.2 --- Immunohistochemical staining --- p.132 / Chapter 4.2.4.3 --- Image viewing --- p.133 / Chapter 4.3 --- Results --- p.134 / Chapter 4.3.1 --- Presence of endocytosis in L .edodes --- p.134 / Chapter 4.3.2 --- Validation of active transport of FM4-64 --- p.137 / Chapter 4.3.3 --- Dye internalization at specific structures in L. edodes --- p.138 / Chapter 4.3.4 --- Presence of Le.Rab7 protein in the endosomal structures along the endocytic pathway --- p.142 / Chapter 4.3.5 --- Presence of Le.Rab7 protein in the pre- and hymenophore of fruiting body --- p.145 / Chapter 4.4 --- Discussion --- p.148 / Chapter Chapter 5 --- General discussion --- p.152 / References --- p.160

Cai Yi Jin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 118-128). / Chapter 1. --- Introduction / Chapter 1.1 --- Importance and Cultivation history of edible mushroom --- p.1 / Chapter 1.2 --- Variety and structure of growth substrates for mushroom --- p.4 / Chapter 1.3 --- Mushroom growth and substrate-degrading enzymes --- p.8 / Chapter 1.4 --- Purpose of study --- p.15 / Chapter 2. --- Methods and Materials / Chapter 2.1 --- Organisms --- p.17 / Chapter 2.2 --- Media --- p.17 / Chapter 2.3 --- Culture conditions --- p.21 / Chapter 2.3.1 --- Growth temperature --- p.21 / Chapter 2.3.2 --- Growth Studies --- p.21 / Chapter 2.3.2.1 --- Effect of pH on mycelial growth --- p.21 / Chapter 2.3.2.2 --- Effect of different carbon sources on mycelial growth --- p.21 / Chapter 2.3.2.3 --- Effect of lignin-related phenolic monomers and tannin derivatives on fungal growth --- p.22 / Chapter 2.3.3 --- Culture conditions for production of extracellular enzymes --- p.23 / Chapter 2.3.3.1 --- Tyrosinase --- p.23 / Chapter 2.3.3.2 --- Laccase --- p.23 / Chapter 2.3.3.3 --- Manganese-dependent Peroxidase and Lignin Peroxidase --- p.23 / Chapter 2.3.3.4 --- Cellulytic and Xylanolytic enzymes --- p.24 / Chapter 2.3.3.5 --- Lipase --- p.25 / Chapter 2.3.4 --- Culture conditions for studying properties of cellulases of V. volvacea --- p.26 / Chapter 2.3.4.1 --- CMCase --- p.26 / Chapter 2.3.4.2 --- "CMCase, FPase and β-Glucosidase" --- p.26 / Chapter 2.3.4.3 --- β-Glucosidase --- p.26 / Chapter 2.4 --- Enzyme assay --- p.27 / Chapter 2.4.1 --- Tyrosinase --- p.27 / Chapter 2.4.2 --- Laccase --- p.27 / Chapter a. --- o-Tolidine Method --- p.27 / Chapter b. --- ABTS Method --- p.28 / Chapter c. --- Syringaldazine Method --- p.28 / Chapter 2.4.3 --- Lignin peroxidase --- p.29 / Chapter 2.4.4 --- Manganese-dependent peroxidase --- p.29 / Chapter 2.4.5 --- Exoglucanase (avicelase) --- p.30 / Chapter 2.4.6 --- Endoglucanase (carboxymethylcellulase or CMCase) --- p.31 / Chapter 2.4.7 --- Filter paper digesting enzyme (FPase) --- p.32 / Chapter 2.4.8 --- P-Glucosidase --- p.32 / Chapter 2.4.9 --- Xylanase --- p.34 / Chapter 2.4.10 --- β-Xylosidase --- p.34 / Chapter 2.4.11 --- Lipase --- p.36 / Chapter 2.5 --- Other analytical methods --- p.36 / Chapter 2.5.1 --- Determination of phenol oxidase activity by the Bavendamm reaction --- p.36 / Chapter 2.5.2 --- Qualitative evaluation of CMCase by Congo red staining --- p.37 / Chapter 2.5.3 --- Effect of phenolic monomers and tannic acid on CMCase activity of V. volvacea --- p.38 / Chapter 2.5.4 --- Protein determination --- p.39 / Chapter 2.5.5 --- Non-denaturing gel electrophoresis pattern of fungal laccases --- p.39 / Chapter 2.6 --- Chemicals --- p.39 / Chapter 3. --- Results / Chapter 3.1 --- Growth and Nutritional characteristics --- p.44 / Chapter 3.1.1 --- Fungal growth on defined and non-defined culture media --- p.44 / Chapter 3.1.2 --- Effect of carbon source on fungal --- p.45 / Chapter 3.1.3 --- Effect of pH on fungal growth --- p.45 / Chapter 3.2 --- Effect of lignin-related phenolic monomers and tannin derivatives on fungal growth --- p.45 / Chapter 3.2.1 --- Effect of lignin-related phenolic monomers on fungal growth --- p.45 / Chapter 3.2.2 --- Effect of tannin derivatives on fungal growth --- p.61 / Chapter 3.3 --- Phenol Oxidase --- p.67 / Chapter 3.3.1 --- Phenol oxidase --- p.67 / Chapter 3.3.1.1 --- Guaiacol-reacting enzyme --- p.67 / Chapter 3.3.1.2 --- o-Anisidine oxidizing enzyme --- p.68 / Chapter 3.3.2 --- Tyrosinase --- p.69 / Chapter 3.3.3 --- Laccase --- p.69 / Chapter 3.3.3.1 --- "Laccase detected by o-Tolidine, ABTS Syringaldazine" --- p.69 / Chapter 3.3.3.2 --- Effect of pH on laccase activity --- p.69 / Chapter 3.4 --- Lignin-Transforming Enzymes --- p.73 / Chapter 3.4.1 --- Lignin peroxidase (LP) --- p.73 / Chapter 3.4.2 --- Manganese-dependent peroxidase (MnP) --- p.74 / Chapter 3.5 --- Cellulases --- p.78 / Chapter 3.5.1. --- Cellulases of V. volvacea --- p.78 / Chapter 3.5.1.1 --- Qualitative estimation of cellulose-degrading enzymes of V. volvacea grown on different substrates --- p.78 / Chapter 3.5.1.2 --- Influence of pH and temperature --- p.79 / Chapter 3.5.1.3 --- Cellulolytic activities in cultures grown on cellulose --- p.83 / Chapter 3.5.1.4 --- Cellulolytic activities in cultures grown on paddy straw --- p.91 / Chapter 3.5.1.5 --- β-Glucosidase activity in cultures grown on cellobiose --- p.91 / Chapter 3.5.1.6 --- Effect of lignin-related phenolic monomers and tannic acid on CMCase of V. volvacea --- p.95 / Chapter 3.5.2 --- Cellulases of P.sajor-caju --- p.96 / Chapter 3.5.3 --- Cellulases of L. edodes --- p.96 / Chapter 3.6 --- Xylanase --- p.96 / Chapter 3.6.1 --- "Xylanase of V. volvacea, strain V34" --- p.96 / Chapter 3.6.2 --- Xylanase of P.sajor-caju --- p.100 / Chapter 3.6.3 --- Xylanase of L. edodes --- p.100 / Chapter 3.7 --- Lipase of V. volvacea --- p.103 / Chapter 4. --- Discussion / Chapter 4.1. --- Carbon nutrition and pH for fungal growth --- p.104 / Chapter 4.1.1 --- Carbon nutrition --- p.104 / Chapter 4.1.2 --- pH --- p.104 / Chapter 4.2 --- "Effect of lignin-related phenolic monomers and tannin derivatives on fungal growth of L. edodes, P. sajor-caju and V, volvacea" --- p.105 / Chapter 4.2.1 --- Lignin-related phenolic monomers --- p.105 / Chapter 4.2.2 --- Tannin derivatives --- p.107 / Chapter 4.3 --- "Production of phenoloxidases by V. volvacea, L. edodes and P. sajor-caju" --- p.108 / Chapter 4.3.1 --- Guaiacol- and Anisidine reacting enzymes and Tyrosinase --- p.108 / Chapter 4.3.2 --- Laccase --- p.109 / Chapter 4.4. --- "Lignin-degrading Enzymes of V. volvacea, P. sajor-caju and L. edodes" --- p.110 / Chapter 4.5. --- "Cellulolytic and Hemicellulolytic Activity of V. volvacea, P.sajor-caju and L. edodes" --- p.113 / References --- p.118 / Appendix1 --- p.129