Coprinopsis cinerea is extensively used as a model to study the development of homobasidiomycete fungi. Unraveling the molecular basis of fungal developmental processes would contribute to evolutionary studies, improve the knowledge about multicellularity development, and lead to improvement in the breeding and cultivation of edible or medical homobasidiomycete mushrooms. / I studied the fungal developmental processes in two aspects: 1) the hypothesis of a “developmental hourglass”, and 2) the existence of microRNA-like RNA (milRNA) genes in fungi. / The model of “developmental hourglass” suggests that middevelopment is the most conserved and the most resistant to evolutionary changes. Although extensively explored in animals and plants, such hourglass pattern has not been reported in fungi yet. I tested the hourglass model in C. cinerea using two complementary approaches, the transcriptome age index (TAI) and the transcriptome divergence index (TDI). Both the TAI and the TDI profiles displayed an hourglass pattern over the development of C. cinerea; the young fruiting body stage was the waist that expressed the evolutionarily oldest transcriptome (lowest TAI) and gave the strongest signal of purifying selection (lowest TDI). By cross-kingdom comparisons, it is found that all three kingdoms displayed high expression levels of genes in “information storage and processing” at the waist stage, while genes in “metabolism” became more active later; besides, genes at the waist stage were underrepresented in “signal transduction mechanisms”. / MicroRNA (miRNA) is a group of endogenous non-coding regulatory RNAs of ~22 nt that regulate gene expression in various biological processes such as cell differentiation, development regulation and heterochromatin formation. Past research work reported several simple filamentous fungi to contain milRNAs in their genomes; however, no milRNA has been reported in mushrooms so far. Through computational prediction, I identified 16 putative milRNA genes in C. cinerea. Besides, evolutionary analysis showed that C. cinerea contains Dicer-like proteins (DCLs), which may play roles in milRNA biogenesis. / Both the discovery of a “developmental hourglass” and milRNA genes laid a foundation for analysis of fruiting body formation in fungi, and for evolutionary analysis of multicellular development across kingdoms. / 灰蓋鬼傘（Coprinopsis cinerea）作為一個典型的生物模型，被廣泛地運用在擔子菌生長發育的研究中。從分子層面上解析出真菌生長發育的過程，可以促進進化生物學的研究，提高對多細胞生物演化的認知，以及改善食用菌和藥用菌的育種與培養。 / 我從兩個角度去分析了真菌的生長發育過程：1）真菌的“發育沙漏”假說，2）真菌基因組裡類微RNA（microRNA-like RNA [milRNA]）的基因。 / “發育沙漏”模型指出，發育中期在進化中是最保守的、最能抵抗進化帶來的改變。這個沙漏模型在動物與植物中被廣泛地研究與證實，但是迄今為止，並未有人對其在真菌中的存在進行過探究。我以C. cinerea作為研究模型，採用了兩種互補的方法，transcriptome age index (TAI) 和 transcriptome divergence index (TDI)，探究了發育沙漏在真菌中的存在。兩個指數均在C. cinerea發育過程中呈現出了沙漏的形狀；年輕的子實體（young fruiting body）階段是沙漏的腰身，表達出進化中最古老的基因並給出了最強的淨化選擇（purifying selection）的信號。我將三大真核生物進行了比較，發現在腰身階段時，在“信息儲存和處理”中發揮作用的基因呈現出了高表達量，而“代謝”基因的表達量在後期更高；而且在腰身階段表達的基因中，負責“信號傳遞機制”的基因偏少。 / 微RNA（microRNA [miRNA]）是一類長約22個核苷酸、不轉譯蛋白質的RNA分子。它們可以調節其他基因的表達，在多方面的生理過程中發生作用，比如細胞分化、發育調節、異染色質的形成等等。近幾年的研究表明，一些簡單的絲狀真菌的基因組中含有類微RNA（milRNA）的基因；但是，至今未有報導說蘑菇是否含有此類基因。我運用計算預測的方法在C. cinerea的基因組裡找到了16個可能的類微RNA基因。此外，從進化的角度分析，我發現C. cinerea含有類Dicer蛋白酶（Dicer-like proteins [DCLs]），而這些類Dicer蛋白酶可能在milRNA的產生過程中發揮作用。 / 這篇論文所報導的兩個發現──真菌的“發育漏斗“和類微RNA基因，均為之後子實體發育的研究及多細胞發育的研究奠定了基礎。 / Cheng, Xuanjin. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references. / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / 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.

by Ng Tak Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 151-168). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutraceutical and Medicinal Properties of L. edodes --- p.4 / Chapter 1.2.1 --- Nutritional value --- p.4 / Chapter 1.2.2 --- Hypocholesterolaemic Effect --- p.5 / Chapter 1.2.3 --- Anti-tumor Effect --- p.5 / Chapter 1.2.4 --- Anti-viral Effect --- p.6 / Chapter 1.2.5 --- Immunopotentiating Effect --- p.6 / Chapter 1.3 --- Life cycle of L. edodes --- p.7 / Chapter 1.4 --- Environmental factors affecting mycelial growth and fruit body --- p.11 / Chapter 1.4.1 --- Nutrient requirement --- p.11 / Chapter 1.4.2 --- Physical and chemical factors --- p.12 / Chapter 1.5 --- Molecular studies on mushroom development --- p.15 / Chapter 1.5.1 --- Mating-type genes --- p.15 / Chapter 1.5.2 --- Hydrophobins --- p.19 / Chapter 1.5.3 --- Fruiting regulatory genes --- p.23 / Chapter 1.5.4 --- Molecular studies on fruit body development of I. edodes --- p.24 / Chapter 1.5.4.1 --- Identification of L. edodes genes --- p.24 / Chapter 1.5.4.2 --- Functional characterization of L. edodes genes --- p.27 / Chapter 1.5.4.3 --- Transformation in L. edodes --- p.28 / Chapter Chapter Two --- Expressed Sequence Tags (ESTs) of L. edodes / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Generation of expressed sequence tag --- p.33 / Chapter 2.2.1.1 --- Mushroom cultivation and RNA extraction --- p.33 / Chapter 2.2.1.2 --- Construction of primordium cDNA library --- p.34 / Chapter 2.2.1.3 --- Mass excision of pBK-CMV plasmid --- p.34 / Chapter 2.2.1.4 --- Random screening of mass excised cDNA clone --- p.38 / Chapter 2.2.1.5 --- Isolation of recombinant plasmid --- p.38 / Chapter 2.2.1.6 --- Generation of 3´ة end partially sequence --- p.39 / Chapter 2.2.1.7 --- Sequence analysis --- p.40 / Chapter 2.2.2 --- Reverse dot-blot Hybridization --- p.40 / Chapter 2.2.2.1 --- PCR amplification of cDNA clone --- p.40 / Chapter 2.2.2.2 --- Membrane preparation --- p.40 / Chapter 2.2.2.3 --- cDNA probe preparation --- p.41 / Chapter 2.2.2.4 --- Hybridization --- p.42 / Chapter 2.2.2.5 --- Stringent washing and autoradiography --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Construction of primordium cDNA library --- p.44 / Chapter 2.3.2 --- Screening of recombinant clone --- p.44 / Chapter 2.3.3 --- Isolation and reconfirmation of recombinant plasmid --- p.46 / Chapter 2.3.4 --- Generation of EST --- p.47 / Chapter 2.3.5 --- EST identity --- p.47 / Chapter 2.3.6 --- Reverse dot-blot hybridization --- p.56 / Chapter 2.3.7 --- Analysis of hybridization signal --- p.60 / Chapter 2.4 --- Discussion --- p.71 / Chapter Chapter Three --- Sequence Analysis and Transcriptional Profiling of Genes Encoding GTP-binding Proteins / Chapter 3.1 --- Introduction --- p.78 / Chapter 3.2 --- Materials and Methods --- p.82 / Chapter 3.2.1 --- Sequence manipulation --- p.82 / Chapter 3.2.2 --- Northern blot hybridization --- p.82 / Chapter 3.2.2.1 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.82 / Chapter 3.2.2.2 --- RNA fixation by capillary method --- p.83 / Chapter 3.2.2.3 --- Probe preparation --- p.84 / Chapter 3.2.2.4 --- Hybridization --- p.85 / Chapter 3.2.2.5 --- Stringent washing and autoradiography --- p.85 / Chapter 3.2.3 --- Real-Time SYBR Green RT-PCR --- p.85 / Chapter 3.2.3.1 --- Primer design --- p.85 / Chapter 3.2.3.2 --- RT-PCR reaction --- p.86 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Sequence manipulation --- p.88 / Chapter 3.3.2 --- Transcriptional analysis --- p.103 / Chapter 3.4 --- Discussion --- p.108 / Chapter 3.4.1 --- Heterotrimeric G proteins --- p.108 / Chapter 3.4.2 --- Ras-related protein Rab7 --- p.112 / Chapter 3.4.3 --- Developmentally regulated GTP-binding protein --- p.113 / Chapter Chapter Four --- Yeast Complementation and Over-expression tests of Le.Gβ1 and Le.Gγ1 / Chapter 4.1 --- Introduction --- p.115 / Chapter 4.2 --- Materials and Methods --- p.120 / Chapter 4.2.1 --- "Yeast strains, media and yeast vectors" --- p.120 / Chapter 4.2.2 --- Primer design --- p.121 / Chapter 4.2.3 --- RT-PCR Amplification of Le.Gβ1 and Le.Gγ1 --- p.121 / Chapter 4.2.4 --- Purification of PCR products --- p.122 / Chapter 4.2.5 --- Enzymatic digestion and purification --- p.122 / Chapter 4.2.6 --- Ligation and E. coli transformation --- p.122 / Chapter 4.2.7 --- PCR screening of E. coli transformants --- p.124 / Chapter 4.2.8 --- Plasmids extraction --- p.124 / Chapter 4.2.9 --- Yeast transformation --- p.124 / Chapter 4.2.10 --- Mating test --- p.125 / Chapter 4.3 --- Results --- p.129 / Chapter 4.3.1 --- Cloning of Le.Gβ1 and Le.Gγ1 --- p.129 / Chapter 4.3.2 --- Yeast transformation --- p.129 / Chapter 4.3.3 --- Mating test --- p.130 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter Five --- General Discussion --- p.144 / References --- p.151