Cheng, Chi Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-160). / Abstracts in English and Chinese. / English Abstract --- p.ii / Chinese Abstract --- p.iv / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Literature Review / Chapter 1.1 --- Introduction & Taxonomy --- p.1 / Chapter 1.2 --- Life cycle and morphology --- p.1 / Chapter 1.3 --- Growth requirements --- p.4 / Chapter 1.3.1 --- Nutritional requirements --- p.4 / Chapter 1.3.2 --- Environment factors --- p.5 / Chapter 1.4 --- Fruiting body development in Coprinopsis cinerea --- p.6 / Chapter 1.4.1 --- Physiology of the fruiting process --- p.6 / Chapter 1.4.2 --- Other studies related to the fruiting process --- p.7 / Chapter 1.5 --- Other biological studies in Coprinopsis cinerea --- p.8 / Chapter 1.5.1 --- Meiosis studies --- p.9 / Chapter 1.5.2 --- Mating analyses --- p.10 / Chapter 1.5.3 --- Peroxidase production --- p.11 / Chapter 1.5.4 --- Transformation and gene silencing --- p.12 / Chapter 1.5.5 --- Other studies --- p.13 / Chapter 1.6 --- C. cinerea genome project --- p.13 / Chapter 1.7 --- Transcriptome analyses --- p.14 / Chapter 1.7.1 --- Serial Analysis of Gene Expression (SAGE) --- p.14 / Chapter 1.7.2 --- Analyzing the 5´ةend of transcripts --- p.16 / Chapter 1.7.3 --- Mapping of SAGE tags to the genome --- p.19 / Chapter 1.8 --- High throughput sequencing --- p.20 / Chapter 1.8.1 --- Pyrophosphate sequencing --- p.20 / Chapter 1.8.2 --- Application of pyrosequencing --- p.21 / Chapter 1.9 --- Aims of project --- p.22 / Chapter Chapter 2 --- 5' Serial Analysis of Gene Expression (5' SAGE) from mycelial and primordial stages of C. cinerea / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- 5' SAGE libraries construction --- p.29 / Chapter 2.2.1.1 --- Mushroom mycelium and primordium cultivation --- p.29 / Chapter 2.2.1.2 --- RNA extraction --- p.29 / Chapter 2.2.1.3 --- Isolation of mRNA --- p.30 / Chapter 2.2.1.4 --- cDNA synthesis --- p.31 / Chapter 2.2.1.5 --- Mmel digestion and Polyacrylamide gel electrophoresis --- p.32 / Chapter 2.2.1.6 --- Formation and amplification of ditag --- p.33 / Chapter 2.2.2 --- Identification of lOObp ditag --- p.34 / Chapter 2.2.3 --- High throughput pyrosequencing --- p.35 / Chapter 2.2.4 --- Tags extraction from ditags --- p.35 / Chapter 2.2.5 --- Genome mapping and annotation --- p.36 / Chapter 2.3 --- Results --- p.37 / Chapter 2.3.1 --- 5´ةSAGE libraries construction --- p.37 / Chapter 2.3.1.1 --- cDNA synthesis --- p.37 / Chapter 2.3.1.2 --- Mmel digestion and ditag formation --- p.38 / Chapter 2.3.2 --- Identification of lOObp ditags --- p.39 / Chapter 2.3.3 --- High throughput pyrosequencing --- p.40 / Chapter 2.3.4 --- Tags extraction from ditags --- p.41 / Chapter 2.3.5 --- Genome mapping and annotation --- p.42 / Chapter 2.4 --- Discussion --- p.46 / Chapter 2.4.1 --- 5´ةSAGE libraries construction --- p.46 / Chapter 2.4.2 --- Tags extraction and genome mapping --- p.46 / Chapter 2.4.3 --- Observations based on the genome mapping data --- p.48 / Chapter Chapter 3 --- Validation of expression patterns of 5' SAGE libraries and analysis of differentially expressed genes / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Identification of housekeeping gene by Northern Blot analysis --- p.58 / Chapter 3.2.1.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.58 / Chapter 3.2.1.2 --- Transfer of RNAs --- p.58 / Chapter 3.2.1.3 --- Probe preparation --- p.59 / Chapter 3.2.1.4 --- "Hybridization, Stringency washes and signal detection" --- p.60 / Chapter 3.2.2 --- Quantitative real-time PCR --- p.61 / Chapter 3.2.2.1 --- cDNA synthesis from 2 developmental stages --- p.61 / Chapter 3.2.2.2 --- Primer design and verification --- p.62 / Chapter 3.2.2.3 --- Real time PCR reaction and data analysis --- p.65 / Chapter 3.2.3 --- Gene expression level comparison --- p.65 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Identification of housekeeping gene by Northern Blot analysis --- p.67 / Chapter 3.3.2 --- Quantitative real-time PCR analysis --- p.71 / Chapter 3.3.3 --- Gene expression level comparison --- p.78 / Chapter 3.4 --- Discussion --- p.126 / Chapter 3.4.1 --- Validation of 5´ة SAGE libraries --- p.126 / Chapter 3.4.2 --- Analysis of highly and differentially expressed genes --- p.127 / Chapter Chapter 4 --- General discussion --- p.135 / References --- p.144 / Appendix --- p.161

Coprinopsis cinerea, 亦稀灰蓋鬼傘，是研究擔子菌發育過程的模範生物。它的生命週期短，容易培養，亦有已建立的遺傳和分子生物研究技術。最近，它的完整基因組序列亦被發表。C. cinerea 的子實體萌生和發展是快速而複雜的過程。它受多種因素影響，如交配基因，營養消耗，光照和溫度。然而，我們對於C. cinerea 出菇的基本機制和涉及的分子途徑仍不清楚。在這項研究中，我採用NimbleGen 微陣列，以了解13,320 個C. cinerea 基因模型的表達。此微陣列覆蓋不同發育階段，包括雙核菌絲體，子實體初體，第2階段原基體，年青和成熟子實體。11,815個預測，在至少一個發育階段表達。707個基因模型在出菇的萌生過程有差異表達。我發現一些可能參與子實體的萌生和發展的份子。它們可能在檢測養分、形態、信號轉導和應激反應方面，擔當重要角色。此外，我亦分析了轉錄因子 、蛋白激酶組和細胞色素P450的基因表達模式。 / C. cinerea 的子實體發展是與光暗週期同步。當子實體初體在沒有光的環境培植，使會形成dark stipe。101個基因在dark stipe 表達差異。它們可能參與原基體成熟的過程。此微陣列基因表達數據，對了解菇機制有價格的信息。 / 胞吞作用是真核細胞透過質膜內陷將外來物質攝取的過程。Rab5 和Rab7 分別控制早期和晚期的胞吞作用。C. cinerea 的胞吞作用是組織由110個基因組成。FM4-64 螢光顯示在C. cinerea 菌絲體的胞吞作用是依賴肌動蛋白和能源。從菌絲體到年青子實體，Cc. Rab5的表達維持相同水平，而Cc.Rab7 的表達則不斷增加，兩者在成熟子實體的表達都是最高，原位雜交體技術顯示 Cc.Rab5 和Cc.Rab7 的 mRNA在年青子實體的子實層以及整個子實層上菌摺表達。我在第2階段原基體進行RNA 乾擾，致使Cc.Rab5 和Cc.Rab7的基因表達敲落。這導致原基體的生長遲緩。及後形成的成熟子實體亦有異常形態。因此，我推測Cc. Rab5 和Cc.Rab7參與出菇過程，並影響擔孢子的形成。這些結果表明，胞吞作用在C.Cinerea 子實體發育過程中發揮定一定的作用。 / Coprinopsis cinerea, is a model organism for studying developmental processes in basidiomycetous fungi. It has a short life cycle, easy to be cultivated in laboratory and can be accessed by various genetic and molecular techniques. Recently, its complete genome sequence was released. The fruiting body development in C. cinerea is a rapid yet complicated process. It is under the regulation of various factors such as mating type genes, nutrients depletion, light and temperature. However, the underlying mechanism and molecular events involved during fruiting body initiation and development remains unclear. / In this study, fruiting body developmental stages including mycelium, fruiting initials, stage 2 primordium, young and mature fruiting body, were analyzed with a comprehensive NimbleGen microarray. 11,815 out of 13,320 predicted gene models were expressed in at least one of the stages. 707 genes were differentially expressed during fruiting body initiation. Potential players involved in nutrients sensing, morphogenesis, signaling pathways and stress response were identified. In particular, expression patterns of all transcription factors, kinome and cytochrome P450s were analyzed. / The fruiting body development of C. cinerea is synchronized with the light/dark cycle. Differentially expressed genes were found in dark stipe produced by keeping fruiting initials in complete darkness. 101 genes, which are likely to be involved in maturation of primordium were identified. / Endocytosis is an essential process in eukaryotes through which cells take up extracellular substrates by membrane invaginations. Rab5 and Rab7 control the early and late stage of endocytosis respectively. The C. cinerea endocytic machinery composed of 110 genes models. The endocytic pathway was traced by FM4-64 and was found to be actin- and energy-dependent. Temporal and spatial expressions of Cc.Rab5 and Cc.Rab7 during fruiting body development were studied. Cc.Rab5 expressed constitutively from mycelium to young fruiting body stage, and reached the highest in the mature fruiting body. The expression of Cc.Rab7 increased continually from mycelium to mature fruiting body stage. From the in situ RNA-RNA hybridization results, both transcripts were localized at the hymenium layer in the young fruiting body and throughout the gill tissue of the mature cap. Knock-down of Cc.Rab5 and Cc.Rab7 by siRNA resulted in retarded growth of the stage 2 primordium and abnormal mature fruiting body. Cc.Rab5 and Cc.Rab7 may be involved in the formation of basidiospores. Endocytosis may play some roles during fruiting body development in C. cinerea. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lee, Yung Yung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 156-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Abbreviations --- p.iv / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Importance of fruiting body (mushrooms) production --- p.1 / Chapter 1.2 --- Introduction on Coprinopsis cinerea --- p.1 / Chapter 1.2.1 --- General introduction --- p.1 / Chapter 1.2.2 --- Life cycle and morphology --- p.2 / Chapter 1.2.3 --- Growth conditions for C. cinerea --- p.4 / Chapter 1.3 --- Regulation of fruiting development in C. cinerea --- p.5 / Chapter 1.3.1 --- Regulation by mating types --- p.5 / Chapter 1.3.2 --- Regulation by light-dark cycle --- p.6 / Chapter 1.3.3 --- Regulation by physiological factors --- p.9 / Chapter 1.4 --- Fruiting-specific genes --- p.9 / Chapter 1.5 --- Genome project of C. cinerea --- p.10 / Chapter 1.6 --- Transformation and gene silencing in C. cinerea --- p.11 / Chapter 1.7 --- DNA microarray --- p.12 / Chapter 1.8 --- Endocytosis --- p.13 / Chapter 1.8.1 --- The endocytic pathway --- p.14 / Chapter 1.8.2 --- Rab GTPase --- p.17 / Chapter 1.8.2.1 --- Control of the active and inactive state of Rab proteins --- p.18 / Chapter 1.8.2.2 --- Functions of Rab GTPases in vesicular transport --- p.19 / Chapter 1.8.2.3 --- Rab5 and Rab7 GTPase --- p.20 / Chapter 1.8.3 --- Endocytosis in fungi --- p.21 / Chapter 1.9 --- Aims of project --- p.22 / Chapter Chapter 2 --- Whole genome expression analysis during fruiting body development --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- NimbleGen 12x135K gene expression microarray --- p.26 / Chapter 2.2.1.1 --- Strains and culture conditions --- p.26 / Chapter 2.2.1.2 --- RNA extraction --- p.27 / Chapter 2.2.1.3 --- Overall design of the NimbleGen custom Microarray --- p.28 / Chapter 2.2.1.4 --- Microarray hybridization, data extraction and normalization --- p.28 / Chapter 2.2.2 --- Microarray data analysis, clustering and GO assignment --- p.29 / Chapter 2.2.3 --- Validation of expression patterns of NimbleGen microarray and analysis of differentially expressed genes by quantitative real-time PCR --- p.29 / Chapter 2.2.3.1 --- cDNA synthesis --- p.29 / Chapter 2.2.3.2 --- Primer design and verification --- p.30 / Chapter 2.2.3.3 --- Real time PCR and data analysis --- p.32 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Whole-genome expression during fruiting body development --- p.33 / Chapter 2.3.2 --- Differentially expressed genes during fruiting body initiation --- p.48 / Chapter 2.3.2.1 --- Fruiting body initiation-specific genes --- p.52 / Chapter 2.3.3 --- Gene expression analysis during fruiting body development --- p.53 / Chapter 2.3.4 --- The C. cinerea kinome --- p.55 / Chapter 2.3.5 --- Transcription factors in C. cinerea --- p.60 / Chapter 2.3.6 --- The cytochrome P450 family in C. cinerea --- p.65 / Chapter 2.3.7 --- Validation of NimbleGen microarray data by quantitative real-time PCR --- p.68 / Chapter 2.4 --- Discussion --- p.77 / Chapter Chapter 3 --- Effect of light on gene expression of fruiting body development --- p.92 / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- NimbleGen 12x135K gene expression microarray --- p.93 / Chapter 3.2.2 --- Validation of expression patterns of NimbleGen microarray and analysis of differentially expressed genes by quantitative real-time PCR --- p.93 / Chapter 3.2.2.1 --- cDNA synthesis --- p.94 / Chapter 3.2.2.2 --- Primer design and verification --- p.94 / Chapter 3.3 --- Results --- p.95 / Chapter 3.3.1 --- Differentially expressed genes in dark stipes --- p.95 / Chapter 3.3.2 --- Validation of expression patterns of NimbleGen microarray by real-time PCR --- p.102 / Chapter 3.4 --- Discussion --- p.107 / Chapter Chapter 4 --- Endocytosis in C. cinerea and its role in fruiting body development --- p.111 / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Materials and Methods --- p.112 / Chapter 4.2.1 --- The endosomal machinery of C. cinerea --- p.112 / Chapter 4.2.2 --- Tracing the endocytic pathway sing FM-64 --- p.113 / Chapter 4.2.2.1 --- Strains and culture conditions --- p.113 / Chapter 4.2.2.2 --- FM4-64 internalization in mycelium of C. cinerea --- p.113 / Chapter 4.2.2.3 --- Drug treatment effect on the internalization of FM4-64 dye --- p.114 / Chapter 4.2.3 --- Temporal and spatial expression of Cc.Rab5 and Cc.Rab7 --- p.114 / Chapter 4.2.3.1 --- Cloning of Cc.Rab5 and Cc.Rab7 --- p.114 / Chapter 4.2.3.1.1 --- RNA extraction and cDNA synthesis --- p.114 / Chapter 4.2.3.1.2 --- TA cloning of amplification products and bacterial transformation --- p.115 / Chapter 4.2.3.1.3 --- PCR screening for positive transformants and sequencing --- p.115 / Chapter 4.2.3.2 --- Quantitative real-time PCR --- p.116 / Chapter 4.2.3.2.1 --- RNA extraction and cDNA synthesis --- p.116 / Chapter 4.2.3.2.2 --- Primer design and verification --- p.116 / Chapter 4.2.3.2.3 --- Real time PCR and data analysis --- p.117 / Chapter 4.2.3.3 --- In situ RNA-RNA hybridization --- p.117 / Chapter 4.2.3.3.1 --- Tissue preparation --- p.117 / Chapter 4.2.3.3.2 --- RNA probe synthesis --- p.117 / Chapter 4.2.3.3.3 --- Hybridization, signal development and image viewing --- p.118 / Chapter 4.2.4 --- Knock-down of endogenous Cc.Rab5 and Cc.Rab7 by siRNA --- p.119 / Chapter 4.2.4.1 --- Strains and culture conditions --- p.119 / Chapter 4.2.4.2 --- Production of dsRNA of Cc.Rab5 and Cc.Rab7 --- p.119 / Chapter 4.2.4.3 --- Digestion of dsRNA to give siRNA --- p.120 / Chapter 4.2.4.4 --- Effects of Cc.Rab5 and Cc.Rab7 siRNA on fruiting body development --- p.120 / Chapter 4.2.4.4.1 --- Application of siRNA to C. cinerea culture --- p.120 / Chapter 4.2.4.4.2 --- Validation of the knock-down efficacy by real-time PCR --- p.121 / Chapter 4.3 --- Results --- p.122 / Chapter 4.3.1 --- The endosomal machinery of C. cinerea --- p.122 / Chapter 4.3.2 --- The endocytic pathway of C. cinerea --- p.127 / Chapter 4.3.2.1 --- Time-course of FM4-64 internalization --- p.127 / Chapter 4.3.2.2 --- Validation of active transport of FM4-64 --- p.129 / Chapter 4.3.3 --- Cloning of Cc.Rab5 and Cc.Rab7 --- p.131 / Chapter 4.3.4 --- Temporal expression of Cc.Rab5 and Cc.Rab7 --- p.133 / Chapter 4.3.5 --- Spatial expression of Cc.Rab5 and Cc.Rab7 --- p.136 / Chapter 4.3.6 --- Effects of Cc.Rab5 and Cc.Rab7 knock-down by siRNA --- p.140 / Chapter 4.3.6.1 --- Observation of effect of siRNA on fruiting body development --- p.140 / Chapter 4.3.6.2 --- Validation of the efficacy of external application of siRNA --- p.143 / Chapter 4.4 --- Discussion --- p.146 / Chapter Chapter 5 --- Concluding remarks --- p.152 / References --- p.156 / Appendix --- p.180