Rozprawy doktorskie na temat „Mushroom shiitake”

CAPES
O cultivo de cogumelos comestíveis vem ganhando espaço no meio agrícola em todo o país, contribuindo assim com uma nova opção de produto no mercado de alimentos. A atividade está ligada a manejos culturais e artesanais com princípios ambientalmente sustentáveis e gera renda para a pequena propriedade rural. Neste contexto, o presente estudo avaliou as potencialidades do cultivo artesanal do Shiitake como uma atividade para agroecossistemas sustentáveis. Para tanto, foi realizado um resgate do histórico da implantação e evolução do cultivo artesanal do cogumelo Shiitake em propriedades consideradas modelos, localizadas nos municípios de Pato Branco, Guarapuava e São José dos Pinhais no estado do Paraná e Frei Rogério em Santa Catarina. Na sequência, uma análise comparativa entre os sistemas de cultivo das propriedades e o aproveitamento de áreas de fragmentos florestais ou reflorestamento foi conduzido. Foram avaliados também aspectos de produção, renda da atividade e pluralismo do estabelecimento rural, bem como, efetuada uma caracterização dos parâmetros de qualidade nutricional dos cogumelos produzidos nas diferentes propriedades. Os resultados obtidos revelam potencialidades econômicas na produção do Shiitake. A atividade também possui potenciais para o aproveitamento sustentável de áreas de mata nativa e de reflorestamento, contribuindo para a otimização do uso do espaço físico na pequena propriedade. A cultura artesanal do Shiitake interage de forma sistêmica, aproveitando à madeira oriunda do próprio estabelecimento rural, evitando a contaminação através de agroquímicos no produto e no ambiente e auxiliando na renovação da biomassa do agroecossistema. Por fim, ficou constada a elevada qualidade nutricional dos cogumelos produzidos nos diferentes substratos utilizados nas propriedades estudadas. As amostras de Shiitake apresentaram elevados conteúdos de proteínas, fibras, minerais e baixos conteúdos de gordura.
Edible mushroom cultivation has been gaining ground in the agricultural environment throughout the country, creating a new product option in the food market. This activity is linked to cultural and artisanal management with environmentally sustainable principles and generates income for small farms. In this context, the present study evaluated the potential for artisanal cultivation of Shiitake as an activity for sustainable agroecosystems. Initially, the historic of implementation and evolution of artisanal cultivation of Shiitake mushroom on properties considered models in Pato Branco, Guarapuava and São José dos Pinhais (Paraná) and Frei Rogerio, (Santa Catarina) were carried out. Consequently, a comparative analysis between the cultivation systems of these properties and the use of small wooded areas and reforested areas was conducted. Aspects of production, income and pluralism of the farms, were also performed. At the end, the nutritional quality parameters of mushrooms produced in different properties were evaluated. The results show economic potential in Shiitake production. The activity also has potential for the sustainable exploitation of native forest and reforested areas, contributing to the optimal use of physical space small properties. In artisanal cultivation of Shiitake there is a systemic interaction verified by the use of wood from the same farm, assisting in the renewal of biomass of the agroecosystem. Finally, it was found that mushrooms produced on different substrates used in the studied properties have high nutritional quality. Shiitake samples exhibited high contents of protein, fiber, minerals and low fat content.

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O presente trabalho teve como principais objetivos: conhecer a atividade antioxidante do shiitake (Lentinus edodes) e do cogumelo do sol (Agaricus blazei) conforme os métodos radical livre DPPH e sistema -caroteno/ácido linoléico; avaliar a estabilidade oxidativa do óleo de soja adicionado dos extratos que apresentaram maior atividade antioxidante e a influência dos extratos de cogumelos na retenção de tocoferóis em óleo de soja, quando submetido ao teste de estocagem acelerada. Os extratos de cogumelos foram obtidos sob diferentes condições: solventes de diferentes polaridades e dois tipos de extração, lenta (shaker, 3 horas, 120 rpm) e rápida (liquidificador, 30 minutos). Os solventes utilizados foram: água, metanol:água (1:1), etanol:água (1:1), metanol e etanol. Independente de qual variedade apresentou maior atividade antioxidante, ambas foram selecionadas para serem aplicadas ao óleo de soja. Os tratamentos: Controle (óleo de soja sem antioxidantes), TBHQ (óleo de soja + 100 mg/kg de TBHQ), BHT (óleo de soja + 100 mg/kg de BHT), Shiitake (óleo de soja + 3.500 mg/kg de extrato de shiitake) e Cogumelo do sol (óleo de soja + 3.500 mg/kg de extrato de cogumelo do sol) foram preparados e submetidos ao teste de estabilidade oxidativa por meio do Rancimat (100ºC) com fluxo de ar a 20L/h e ao teste de estocagem acelerada em estufa, a 60ºC durante 15 dias. As amostras foram recolhidas a cada 3 dias e analisadas quanto ao índice de peróxidos, dienos conjugados, ácidos graxos livres e tocoferóis naturalmente presentes no óleo de soja. Além disso, as amostras também foram analisadas diariamente quanto ao ganho de massa. Os resultados obtidos das determinações analíticas foram submetidos às análises de variância, em esquema fatorial, no delineamento inteiramente casualizado. Os extratos metanólicos, de ambas as variedades, independente do tipo...
The present work had as its main goals: cognize the antioxidant activity of shiitake (Lentinus edodes) and cogumelo do sol (Agaricus blazei) according to the method of free radicals DPPH and the system -carotene/acid linoleic; evaluate the oxidative stability of soybean oil added of the extracts that presented higher antioxidant activity and asses the mushroom’s extract influence over the retention of tocopherols in soybean oil, when subjected to the accelerated storage test. The mushroom’s extracts were obtained under different conditions: solvents of different polarities and two types of extraction, slow (shaker, 3 hours, 120 rpm) and fast (blender, 30 minutes). The solvents used, in decreasing order of polarity, were: water, methanol: water (1:1), ethanol: water (1:1), methanol and ethanol. Irrespective of which variety had shown higher antioxidant activity, both were selected to be applied to the soybean oil. The treatments: Control (soybean oil without antioxidants), TBHQ (soybean oil + 100 mg/kg de TBHQ), BHT (soybean oil + 100 mg/kg of BHT), Shiitake (soybean oil + 3,500 mg/kg of extract of the shiitake) and cogumelo do sol (soybean oil + 3,500 mg/kg of extract of the cogumelo do sol) were prepared and subjected to the oxidative stability’s test through the Rancimat (100ºC) with airflow at 20 L/h and to the accelerated storage test in an oven, at 60ºC for 15 days. The samples were collected every 3 days and analyzed regarding the index of peroxides, conjugated dienes, free fatty acids and tocopherols naturally found in the soybean oil added to the mushroom’s extracts. Beyond that, the samples were also analyzed daily regarding the weight gain. The results obtained from the analytics determinations were submitted to the variance analysis, in a factorial scheme, over a full randomized lineation. The methanolic extracts of both varieties, irrespective from... (Complete abstract click electronic access below)

Orientador: Neuza Jorge
Banca: Odair Zenebon
Banca: Elizeu Trabuco
Resumo: O presente trabalho teve como principais objetivos: conhecer a atividade antioxidante do shiitake (Lentinus edodes) e do cogumelo do sol (Agaricus blazei) conforme os métodos radical livre DPPH e sistema -caroteno/ácido linoléico; avaliar a estabilidade oxidativa do óleo de soja adicionado dos extratos que apresentaram maior atividade antioxidante e a influência dos extratos de cogumelos na retenção de tocoferóis em óleo de soja, quando submetido ao teste de estocagem acelerada. Os extratos de cogumelos foram obtidos sob diferentes condições: solventes de diferentes polaridades e dois tipos de extração, lenta (shaker, 3 horas, 120 rpm) e rápida (liquidificador, 30 minutos). Os solventes utilizados foram: água, metanol:água (1:1), etanol:água (1:1), metanol e etanol. Independente de qual variedade apresentou maior atividade antioxidante, ambas foram selecionadas para serem aplicadas ao óleo de soja. Os tratamentos: Controle (óleo de soja sem antioxidantes), TBHQ (óleo de soja + 100 mg/kg de TBHQ), BHT (óleo de soja + 100 mg/kg de BHT), Shiitake (óleo de soja + 3.500 mg/kg de extrato de shiitake) e Cogumelo do sol (óleo de soja + 3.500 mg/kg de extrato de cogumelo do sol) foram preparados e submetidos ao teste de estabilidade oxidativa por meio do Rancimat (100ºC) com fluxo de ar a 20L/h e ao teste de estocagem acelerada em estufa, a 60ºC durante 15 dias. As amostras foram recolhidas a cada 3 dias e analisadas quanto ao índice de peróxidos, dienos conjugados, ácidos graxos livres e tocoferóis naturalmente presentes no óleo de soja. Além disso, as amostras também foram analisadas diariamente quanto ao ganho de massa. Os resultados obtidos das determinações analíticas foram submetidos às análises de variância, em esquema fatorial, no delineamento inteiramente casualizado. Os extratos metanólicos, de ambas as variedades, independente do tipo... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present work had as its main goals: cognize the antioxidant activity of shiitake (Lentinus edodes) and cogumelo do sol (Agaricus blazei) according to the method of free radicals DPPH and the system -carotene/acid linoleic; evaluate the oxidative stability of soybean oil added of the extracts that presented higher antioxidant activity and asses the mushroom's extract influence over the retention of tocopherols in soybean oil, when subjected to the accelerated storage test. The mushroom's extracts were obtained under different conditions: solvents of different polarities and two types of extraction, slow (shaker, 3 hours, 120 rpm) and fast (blender, 30 minutes). The solvents used, in decreasing order of polarity, were: water, methanol: water (1:1), ethanol: water (1:1), methanol and ethanol. Irrespective of which variety had shown higher antioxidant activity, both were selected to be applied to the soybean oil. The treatments: Control (soybean oil without antioxidants), TBHQ (soybean oil + 100 mg/kg de TBHQ), BHT (soybean oil + 100 mg/kg of BHT), Shiitake (soybean oil + 3,500 mg/kg of extract of the shiitake) and cogumelo do sol (soybean oil + 3,500 mg/kg of extract of the cogumelo do sol) were prepared and subjected to the oxidative stability's test through the Rancimat (100ºC) with airflow at 20 L/h and to the accelerated storage test in an oven, at 60ºC for 15 days. The samples were collected every 3 days and analyzed regarding the index of peroxides, conjugated dienes, free fatty acids and tocopherols naturally found in the soybean oil added to the mushroom's extracts. Beyond that, the samples were also analyzed daily regarding the weight gain. The results obtained from the analytics determinations were submitted to the variance analysis, in a factorial scheme, over a full randomized lineation. The methanolic extracts of both varieties, irrespective from... (Complete abstract click electronic access below)
Mestre

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As pesquisas sobre o cultivo axênico de shiitake e produção de inóculo, para as condições brasileiras, são escassas. O estudo do crescimento miceliano visa compreender os aspectos físicos, químicos e ambientais que causam alterações no processo desse crescimento. O objetivo deste trabalho foi o de estudar a influência de aspectos nutricionais e a interferência do substrato na cinética de crescimento de linhagens de Lentinula edodes. Os materiais utilizados foram duas linhagens de Lentinula edodes: L17 e L55 da micoteca do Módulo de Cogumelos da FCA e substratos à base de serragem (S) e bagaço de cana-de-açúcar (B) com a adição de três quantidades de farelos de arroz e de trigo (metade de cada): 0, 10 e 20%, perfazendo 6 tratamentos, os quais foram utilizados na cinética da área de crescimento miceliano em meio de cultura e do volume de crescimento em substrato. Dos resultados obtidos foram extraídas as seguintes conclusões: A cinética de crescimento miceliano em superfície (área), independente das linhagens e substratos, seguiu um modelo matemático representado por uma equação exponencial. Os parâmetros estimados gama tiveram uma relação com a velocidade final instantânea de crescimento em área. A cinética de crescimento miceliano em volume, independente das linhagens e substratos, seguiu um modelo matemático representado por equação logarítmica. Os parâmetros estimados betas, gama e delta, não apresentaram relação com a velocidade de crescimento em volume. Ocorreram interações significativas entre linhagens, substratos base e quantidades de farelos, tanto na cinética de crescimento em superfície quanto em volume. A linhagem L55 se apresentou mais adaptada à metodologia adotada por ser utilizada em cultivo axênico.
In Brazil there was little research related to Shiitake axenic culture. It was researched in this experiment the physical, chemical and environmental aspects in relation to different strains of Lentinula edodes. The aim of this research was to understand the substratum effects in the kinetics of the Shiitake mycelium growth. It was used two Shiitake strains and two different base substrate (eucalyptus sawdust and sugar cane bagasse) varying in three proportions of the supplements. The supplements, a blend of rice and wheat brans, were added in the proportion of 0, 10 and 20% of the base substrate. The experiment was composed of six treatments. It was concluded that the mycelium kinetics growth in culture medium followed a mathematical model that were represented by exponential equation. Gamma parameters were directly proportional to the instantaneous growth velocity in area. The mycelium growth kinetics in volume had no effect relation to the strains and substrate and it followed a mathematical model represented by logarithmic equation. Beta, gamma and delta parameters didn't show any correlation with the growth velocity in volume. There were significant differences between the strains and the mycelium growth in the supplemented substrate. The strain L55 was better adapted than L17.

Orientador: Marlene Rita de Queiroz
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agrícola
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Resumo: A finalidade principal deste trabalho foi reunir subsídios para orientar os processos desecagem, armazenagem e reidratarão do cogumelo Shiitake. A pesquisa foi realizada durante o ano de 2002 no Laboratório de Tecnologia Pós-Colheita/Secagem da Faculdade de Engenharia Agrícola da Universidade Estadual de Campinas e consistiu em duas etapas: secagem e armazenagem por 3 meses. Os fatores estudados foram: na secagem - geometria de corte (Shiitake inteiro e fatiado), temperatura de secagem (50ºC e 70°C), teor de umidade final (5% e 15%); na armazenagem - geometria de corte (Shiitake inteiro e fatiado), teor de umidade inicial (5% e 15%), embalagem (com e sem saco de polipropileno) e tempo de armazenagem (3 meses); na reidratação - tempo de imersão em água (8 tempos), geometria de corte (Shiitake inteiro e fatiado), teor de umidade inicial (5% e 15%) e tempo de armazenagem (3 meses). Os parâmetros de qualidade estudados foram: cor, textura, massa, teor de umidade, capacidade de reidratação. Os parâmetros de secagem estudados foram: cinéticas experimentais de secagem. As cinéticas de secagem mostraram que a secagem ocorreu no período de taxa decrescente e que a secagem dos cogumelos fatiados secos à 70ºC ocorreu em menor tempo que os demais tratamentos. A temperatura de secagem de 70ºC proporcionou menor escurecimento e os resultados do efeito da secagem sobre a textura do cogumelo foram pouco conclusivos. O tempo de armazenagem afetou a qualidade dos cogumelos ocorrendo maior dureza, gomosidade e escurecimento. Entretanto, o uso da embalagem retardou o escurecimento. Cogumelos fatiados armazenados com 15% de umidade inicial e embalados obtiveram pouca variação de mastigabilidade, teor de umidade e massa durante a armazenagem. O tempo de armazenagem provocou redução da capacidade de reidratação para os cogumelos com 5% de umidade inicial. Os cogumelos fatiados, com 5% de umidade inicial e embalados obtiveram valores de reidratação superiores aos demais tratamentos
Abstract: Mushroom (Shiitake), drying, storage and re-hydration: effect on quality parameter process determinations. To orient drying, storage and re-hydration processes, accumulated subsidies by studying some parameters on mushroom Shiitake. During three months, drying, storage and re-hydration processes, were studied on mushroom in 2002 at Drying/Post-harvesting Technology Lab from Agricultural Engineering School of the Campinas State University. In drying process were studied: drying temperature (50º and 70 ºC), final moisture content (5% and 15%), cutting geometry (mushroom whole and sliced); in re-hydration process besides drying temperature and cutting geometry, were assessed also, water immersion (8 times), initial moisture content (5%% and 15%) and storage time (3 months); in packing up process; (with/without polypropylene bag), storage time (3 months) and initial moisture content were evaluated. Color, texture, mass, moisture content, and re-hydration capacity, were also evaluated. Drying kinetics showed that drying occurred at increased rate in the period and sliced mushroom dried at 70º C, occurred in lesser time than other treatments. Drying temperature at 70 ºC had less darkening and the results on mushroom texture drying effects were not conclusive. Drying time affected mushroom quality, occurring great hardness, gummosis and darkening. However, packing up use, delayed the darkening. Stored sliced and wrapped mushrooms with 15% initial moisture content had less, chew property variation, moisture content and mass during storage. Storage time provoked rehydration capacity decrease on mushrooms with 5% of initial moisture. Sliced and wrapped mushrooms, with 5% initial moisture, had re-hydration values greater than other treatments
Mestrado
Tecnologia Pós-Colheita
Mestre em Engenharia Agrícola

Orientador: Augusto Ferreira da Eira
Banca: José Raimundo de Souza Passos
Banca: Nelson Barros Colauto
Resumo: As pesquisas sobre o cultivo axênico de shiitake e produção de inóculo, para as condições brasileiras, são escassas. O estudo do crescimento miceliano visa compreender os aspectos físicos, químicos e ambientais que causam alterações no processo desse crescimento. O objetivo deste trabalho foi o de estudar a influência de aspectos nutricionais e a interferência do substrato na cinética de crescimento de linhagens de Lentinula edodes. Os materiais utilizados foram duas linhagens de Lentinula edodes: L17 e L55 da micoteca do Módulo de Cogumelos da FCA e substratos à base de serragem (S) e bagaço de cana-de-açúcar (B) com a adição de três quantidades de farelos de arroz e de trigo (metade de cada): 0, 10 e 20%, perfazendo 6 tratamentos, os quais foram utilizados na cinética da área de crescimento miceliano em meio de cultura e do volume de crescimento em substrato. Dos resultados obtidos foram extraídas as seguintes conclusões: A cinética de crescimento miceliano em superfície (área), independente das linhagens e substratos, seguiu um modelo matemático representado por uma equação exponencial. Os parâmetros estimados gama tiveram uma relação com a velocidade final instantânea de crescimento em área. A cinética de crescimento miceliano em volume, independente das linhagens e substratos, seguiu um modelo matemático representado por equação logarítmica. Os parâmetros estimados betas, gama e delta, não apresentaram relação com a velocidade de crescimento em volume. Ocorreram interações significativas entre linhagens, substratos base e quantidades de farelos, tanto na cinética de crescimento em superfície quanto em volume. A linhagem L55 se apresentou mais adaptada à metodologia adotada por ser utilizada em cultivo axênico.
Abstract: In Brazil there was little research related to Shiitake axenic culture. It was researched in this experiment the physical, chemical and environmental aspects in relation to different strains of Lentinula edodes. The aim of this research was to understand the substratum effects in the kinetics of the Shiitake mycelium growth. It was used two Shiitake strains and two different base substrate (eucalyptus sawdust and sugar cane bagasse) varying in three proportions of the supplements. The supplements, a blend of rice and wheat brans, were added in the proportion of 0, 10 and 20% of the base substrate. The experiment was composed of six treatments. It was concluded that the mycelium kinetics growth in culture medium followed a mathematical model that were represented by exponential equation. Gamma parameters were directly proportional to the instantaneous growth velocity in area. The mycelium growth kinetics in volume had no effect relation to the strains and substrate and it followed a mathematical model represented by logarithmic equation. Beta, gamma and delta parameters didn't show any correlation with the growth velocity in volume. There were significant differences between the strains and the mycelium growth in the supplemented substrate. The strain L55 was better adapted than L17.
Mestre

Produtos naturais são cada vez mais vendidos como suplementos dietéticos devido a várias das suas propriedades terapêuticas, enfatizando a pesquisa para novos medicamentos. Este trabalho mostra que extratos aquosos de Lentinula edodes, Pleurotus sajor-caju e Agaricus blazei exercem atividade citotóxica, através do ensaio da redução do sal de brometo tetrazólio, nas linhagens celulares humanas de carcinoma de laringe (Hep-2) e carcinoma cervical (HeLa). Os extratos foram obtidos inicialmente em três diferentes temperaturas (4C, 22ºC e 50C). Entretanto, a temperatura ambiente foi escolhida para a realização dos outros experimentos já que apresentou os melhores resultados na redução da viabilidade celular. Ensaios bioquímicos realizados em paralelo indicaram uma maior quantidade de polifenóis nos extratos do L. edodes nas três temperaturas de extração e também uma melhor capacidade de eliminação do radical 2,2-difenil-1-picrilhidrazilo. Os resultados para a atividade citotóxica revelaram que o extrato de P. sajor-caju foi mais eficiente do que os extratos de L. edodes e A. blazei, nas linhagens testadas. Modificações morfológicas observadas nas células foram confirmadas pela coloração de Giemsa, após o tratamento com os extratos, sugerindo a inibição da proliferação e indução da apoptose dose/dependente. O tipo de morte celular foi testada com o método da anexina V acoplada a um fluorocromo mais iodeto de propídio, confirmando que tanto os extratos de L. edodes e P. sajor-caju induzem a apoptose tardia. Ensaios da composição química obtidos por cromatografia gasosa acoplada ao espectrômetro de massas mostraram a presença de ácidos graxos nos extratos, com uma prevalência do ácido palmítico no extrato de L. edodes e P. sajorcaju e ácido esteárico no extrato de A. blazei. A quantificação dos carboidratos e proteínas indicaram uma maior quantidade de carboidratos no L. edodes, e o A. blazei apresentou um maior teor de proteínas. Estes resultados indicam que os extratos aquosos dos cogumelos L. edodes, P. sajor-caju e A. Blazei são potenciais fontes antioxidantes, ricos em proteínas e ácidos graxos e possuem atividade anticancerígena por indução da apoptose. No entanto, novos estudos são necessários para explorar seus usos terapêuticos e para elucidar o seu modo de ação no organismo.
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Natural products are increasingly sold as dietary supplements due to several of its therapeutic properties, emphasizing the search for new drugs. This work shows that aqueous extracts of Lentinula edodes, Pleurotus sajor-caju and Agaricus blazei, exert cytotoxic activity by testing the reduction of tetrazolium bromide salt in human cell lines of laryngeal carcinoma (Hep-2) and cervix carcinoma (HeLa). The extracts were obtained initially at three different temperatures (4C, 22ºC and 50C), however ambient temperature was chosen to carry out other experiments that have shown the best results in the reduction of cell viability. Biochemical assays performed in parallel showed an increased amount of polyphenols in the extracts of L. edodes the three extraction temperatures and also a better ability to eliminate the radical 2,2-diphenyl-1- picrylhydrazyl. The results for the cytotoxic activity revealed that the extract of P. sajor-caju was more efficient than that of L. edodes and A. blazei, the strains tested. Observed morphological changes in the cells was confirmed by Giemsa staining after treatment with the extracts, suggesting that the inhibition of proliferation and induction of apoptosis dose/dependent. The type of cell death was assayed by the method of annexin V bound to a fluorochrome and propidium iodide further confirming that both of L. edodes and P. sajor-caju induce apoptosis late. Testing the chemical composition obtained by gas chromatography mass spectrometer showed the presence of fatty acids in the extracts, with a prevalence of palmitic acid in the extract of L. edodes and P. sajor-caju and stearic acid extract of A. blazei. The quantification of carbohydrates and proteins indicated a greater amount of the carbohydrates L. edodes and A. blazei showed a higher protein content. These results indicate that the aqueous extracts of the mushroom L. edodes , P. sajor-caju and A. blazei, are potential sources antioxidants, high in protein and fatty acids and have anticancer activity by induction of apopt sis. However, further studies are needed to explore their therapeutic use and to elucidate its systemic effects.

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Mycelium growth, production and bromatologicals characteristics of shiitake in function of lineages and chemical and physical properties of eucalyptus clones and species were evaluated. In Experiment 1, mycelium growth of two Lentinula edodes (Berk.) Pegler (LE-95/01 and LE-96/18) species in culture mediums prepared with sawdust extract from seven species (E. saligna, E. grandis, E. urophylla, E. camaldulensis, E. citriodora, E. paniculata e E. pellita) and three eucalyptus clones (hybrid E. grandis x E. urophylla) was analyzed. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 10 repetitions, being that each repetition corresponded to one Petri dish. In Experiment 2, mycelium growth of eight L. edodes lineages (LE-96/17, LE-95/02, LE-95/07, LE-98/55, LE-96/18, LE-95/01, LE-96/13 and LE-98/47) in culture mediums prepared with sawdust extract from Eucalyptus spp was evaluated. The experimental design was totally randomized, with 8 treatments and 8 repetitions, being that each repetition corresponded to one Petri dish. In Experiment 3, production and bromatological characterization of two Lentinula edodes lineages cultivated in seven species and three clones of eucalyptus was evaluated. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 40 repetitions, being that each repetition corresponded to one log. In Experiment 4, physical and chemical properties of seven species and three clones of eucalyptus before and after the cultivation of two L. edodes lineages was evaluated. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 9 repetitions, being that each repetition corresponded to one log. The culture medium that provided highest averages of mycelium growth of L. edodes lineages LE-95/01 and LE-96/18 was the one with... (Complete abstract click electronic access below)

Abstract: Mycelium growth, production and bromatologicals characteristics of shiitake in function of lineages and chemical and physical properties of eucalyptus clones and species were evaluated. In Experiment 1, mycelium growth of two Lentinula edodes (Berk.) Pegler (LE-95/01 and LE-96/18) species in culture mediums prepared with sawdust extract from seven species (E. saligna, E. grandis, E. urophylla, E. camaldulensis, E. citriodora, E. paniculata e E. pellita) and three eucalyptus clones (hybrid E. grandis x E. urophylla) was analyzed. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 10 repetitions, being that each repetition corresponded to one Petri dish. In Experiment 2, mycelium growth of eight L. edodes lineages (LE-96/17, LE-95/02, LE-95/07, LE-98/55, LE-96/18, LE-95/01, LE-96/13 and LE-98/47) in culture mediums prepared with sawdust extract from Eucalyptus spp was evaluated. The experimental design was totally randomized, with 8 treatments and 8 repetitions, being that each repetition corresponded to one Petri dish. In Experiment 3, production and bromatological characterization of two Lentinula edodes lineages cultivated in seven species and three clones of eucalyptus was evaluated. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 40 repetitions, being that each repetition corresponded to one log. In Experiment 4, physical and chemical properties of seven species and three clones of eucalyptus before and after the cultivation of two L. edodes lineages was evaluated. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 9 repetitions, being that each repetition corresponded to one log. The culture medium that provided highest averages of mycelium growth of L. edodes lineages LE-95/01 and LE-96/18 was the one with... (Complete abstract click electronic access below)
Orientador: Marli Teixeira de Almeida Minhoni
Coorientador: José Luiz Stape
Banca: Edson Luiz Furtado
Banca: Claudio Angeli Sansígolo
Banca: Luiz Antônio Graciolli
Banca: Arailde Fontes Urben
Doutor

Lentinula edodes é um cogumelo comestível que possui qualidades nutricionais, terapêuticas e medicinais. Além disso, muitos estudos na área médica têm comprovado que o cogumelo possui efeito antibiótico sobre microrganismos patogênicos ao homem. Na área agrícola, alguns trabalhos realizados com o cogumelo demonstraram possíveis efeitos no controle de fitopatógenos. O presente trabalho teve como objetivo demonstrar a produção de substâncias antimicrobianas por L. edodes ativas sobre Colletotrichum sublineolum, agente causal da antracnose em sorgo, Alternaria solani, responsável pela pinta preta do tomateiro, Xanthomonas axonopodis pv. passiflorae, agente causal da mancha bacteriana em maracujazeiro e Tobacco mosaic virus (TMV), causador de mosaico foliar em fumo. Para os testes com C. sublineolum e A. solani foram utilizados extratos aquosos de L. edodes, obtidos a partir de basidiocarpos desidratados em pó, dos isolados LE JAB-K, LE 96/22, LE 96/17 e LE 95/01. Os resultados evidenciaram que o extrato aquoso de basidiocarpos do isolado LE 96/22 inibiu o crescimento micelial in vitro e a formação de apressórios por C. sublineolum. Já os extratos dos isolados LE JAB-K e LE 95/01 apresentaram efeito inibitório na germinação de conídios e na formação de apressórios do patógeno. Em contrapartida, os extratos aquosos de basidiocarpos dos diferentes isolados de L. edodes não apresentaram efeito inibitório na germinação dos conídios e no crescimento micelial de A. solani. Por sua vez, os extratos aquosos de basidiocarpos a 20% (v/v) e o filtrado do crescimento micelial de L. edodes, misturados à suspensão de X. axonopodis pv. passiflorae, exibiram redução na multiplicação bacteriana. Todos os extratos aquosos de basidiocarpos dos diferentes isolados testados na multiplicação da bactéria mostraram-se termolábeis, quando autoclavados a 121 °C por 20 min. Em experimentos com plantas de fumo, os extratos aquosos de basidiocarpos dos isolados LE 96/17 e LE 96/22 adicionados à suspensão contendo partículas do TMV reduziram significativamente a ocorrência de lesões locais nas folhas. O extrato aquoso do isolado LE 96/22 apresentou compostos antivirais de natureza termoestável. Finalmente, o extrato aquoso de basidiocarpos do isolado LE 96/22, o qual apresentou a maior atividade antimicrobiana, foi purificado parcialmente por cromatografia de troca aniônica (CTA). O pico V apresentou efeito inibitório no crescimento micelial de C. sublineolum. Por sua vez, a multiplicação de X. axonopodis pv. passiflorae foi inibida pelos picos IV, V e VII. Já os picos I, II e III, obtidos em CTA por gradiente linear de NaCl e o pico I obtido em CTA pelo método “step wise”, reduziram significativamente a infectividade do TMV em plantas de fumo. Com base nesses resultados, evidencia-se a ação de preparações de L. edodes sobre fitopatógenos, o que demonstra o uso potencial do mesmo no controle de agentes causais de doenças infecciosas em plantas.
Lentinula edodes is an edible mushroom that has nutritious, therapeutical and medicinal qualities. Moreover, many studies in the medical area have shown that the mushroom exhibits antibiotic effects on pathogenic microorganism to the man. In the agricultural area, work carried out with the mushroom has demonstrated its possible effects to control phytopathogens. The objective of the present work was to demonstrate the productionof antimicrobial substances of L. edodes active on Colletotrichum sublineolum, causal agent of anthracnose in sorghum, Alternaria solani, responsible for the black spot of the tomato plants, X. axonopodis pv. passiflorae, causal agent of the bacterial spot in passion fruit plants and on Tobacco mosaic virus, causal agent of the mosaic in tobacco plants. For the test with C. sublineolum and A. solani aqueous extracts were obtained from dehydrated fruiting bodies from the shiitake isolates LE JAB-K, LE 96/22, LE 96/17 and LE 95/01. The results showed that the fruiting body aqueous extract from isolate LE 96/22 inhibited micelial growth and appressorium formation by C. sublineolum. The aqueous extracts of isolates LE JAB-K and LE 95/01 exhibited inhibitory effect on conidium germination and on formation of appressorium by the patogen. On the other hand, the extracts of the different isolates of L. edodes did not exhibit inhibitory effect on conidium germination and micelial growth of A. solani. The aqueous extracts of fruiting bodies at 20% (v/v) concentration and filtrate of the micelial growth of L. edodes, when mixed to the suspension of X. axonopodis pv. passiflorae, exhibited decreased on bacterial multiplication. All the aqueous extracts of fruiting bodies tested from the different isolates in the bacterial multiplication were thermobile, when heated at 121 °C for 20 min. In experiments with tobacco plants, the aqueous extracts of fruiting bodies of isolates LE 96/17 and LE 96/22 when added to the suspension of TMV reduced the amount of local lesions on the leaves. When the aqueous extracts of LE 96/22 were heated the antiviral nature was not lost. Finally, the aqueous extract of fruiting bodies from isolate LE 96/22 that presented major antimicrobial activity was partially purified by anion exchange chromatography (AEC). The peak V exhibited inhibitory effect on micelial growth of C. sublineolum. Multiplication of X. axonopodis pv. passiflorae was inhibited by peaks IV, V and VII. Regarding TMV infectivity, peaks I, II and III, obtained in CTA through linear gradient of NaCl, and peak I also obtained through CTA by the method "step wise", significantly reduced virus infectivity in tobacco plants. Based upon these results, it is shown that preparations of L. edodes can interfere whith phytopathogen multiplication, demonstrating its potential to control plant diseases.

Os cogumelos Lentinula edodes (shiitake) e Agaricus blazei (cogumelo-do-sol) apresentam substâncias no corpo de frutificação (basidiocarpo) e no micélio com atividades antibióticas e imuno-regulatórias, havendo uma série de relatos sobre a atuação das mesmas no controle de doenças em animais. Em vegetais, não há informações sobre o efeito protetor do cogumelo-do-sol contra fitopatógenos. No caso de shiitake, embora pouco numerosos, os estudos mostraram o potencial do cogumelo para o controle de doenças de plantas, tais como a murcha bacteriana do tomateiro, a murcha de feijão-lima, além de doenças fúngicas em sorgo e da bacteriose do maracujazeiro. Os objetivos do presente trabalho foram o de avaliar o efeito de diferentes preparações obtidas a partir de L. edodes e de A. blazei em patossistemas agrícolas, visando o controle de moléstias de interesse econômico como a antracnose do pepineiro, a mancha bacteriana do tomateiro e o endurecimento dos frutos do maracujazeiro. Obtida a proteção, os estudos buscaram elucidar o modo de ação das preparações de interesse, bem como purificá-las parcialmente, na tentativa de se concentrar o princípio ativo. Em plantas de pepino, extratos aquosos de basidiocarpos, obtidos a partir de diferentes isolados dos cogumelos, reduziram a severidade da antracnose, na dependência da concentração do extrato. Os extratos não afetaram adversamente o agente causal da doença, Colletotrichum lagenarium, mas provocaram o acúmulo de peroxidases e quitinases nas folhas tratadas e sistemicamente. Utilizando-se precipitação fracionada do extrato aquoso bruto de basidiocarpos de shiitake com sulfato de amônio e cromatografia de troca aniônica, obteve-se uma fração de proteínas, apresentando massa molecular de 29 a 35 kDa, com atividade elicitora de peroxidases em cotilédones de pepino. Em plantas de tomate, o isolado ABL 99/28 de A. blazei foi quem, em média, conferiu maior proteção contra Xanthomonas vesicatoria, a qual foi dependente das concentrações de extrato do cogumelo e de células bacterianas empregadas nos testes. Novamente, o extrato aquoso de basidiocarpos do isolado efetivo não atuou diretamente sobre o patógeno, mas desencadeou o aumento na atividade de b-1,3- glucanases nas folhas tratadas, sugerindo que o mecanismo de ação em pepineiro e tomateiro envolveu a indução de resistência. Já no caso do maracujazeiro, os extratos de basidiocarpos, obtidos a partir de diferentes isolados de ambos os cogumelos, protegeram localmente plantas inoculadas mecanicamente com o Passion fruit woodiness vírus (PWV), por reduzirem a infectividade viral, o que foi comprovado em testes conduzidos com Chenopodium quinoa, hospedeiro de lesão local do vírus. Entretanto, não houve proteção sistêmica em plantas de maracujá, nos experimentos de inoculação mecânica, reduzindo as possibilidades do uso dos cogumelos para o controle dessa virose no campo. De forma geral, os resultados mostraram que os cogumelos L. edodes e A. blazei apresentam compostos que ativam as respostas de defesa em plantas e podem auxiliar no controle de doenças vegetais, dependendo da natureza do agente causal.
The mushrooms Lentinula edodes and Agaricus blazei have substances in the fruiting body and in the mycelia exhibiting antibiotic activity and others able to stimulate the immune system in animals. There are many reports about the performance of these substances in the control of animal diseases. In vegetables, there are no information about the protecting effect of the royal mushroom against plant pathogens. In the case of shiitake, although few in number, the studies showed the potential of the mushroom for the control of plant diseases, such as tomato bacterial wilt, sorghum leave spots and bacterial disease of the passion fruit plant. The objectives of the present work were to evaluate the effect of different preparations from L. edodes and A. blazei to control the diseases cucumber anthracnose, tomato bacterial spot and passion fruit woodiness. As the protection of the plants was obtained, the studies tried to elucidate the way of action of the preparations, as well as partially purify them, in an attempt to concentrate the active compound. In cucumber plants, fruiting body aqueous extracts, from different mushroom isolates, reduced anthracnose severity, depending upon the extract concentration. The extracts did not affect adversely the disease causal agent, Colletotrichum lagenarium, but induced the peroxidase and quitinase accumulation in the treated leaves and systemically. By using fractional precipitation of the shiitake fruiting body aqueous extracts with ammonium sulfate, and anion exchange chromatography, a protein fraction exhibiting molecular mass around 29 to 35 kDa and peroxidase elicitor activity in cucumber cotyledons was obtained. In tomato plants, the isolate ABL 99/28 of A. blazei was the one that, on average, gave higher protection against Xanthomonas vesicatoria, which was dependent upon the extract and bacterial cell concentrations. Again, the fruiting body aqueous extract of ABL 99/28 did not act directly onto the pathogen, but it caused an increase in b-1,3-glucanase activity in the treated leaves, suggesting that the mushroom action in cucumber and tomato plants involved the induced resistance. On the other hand, the fruiting body extracts, obtained from different isolates of both mushrooms, protected locally passion fruit plants inoculated mechanically with the Passion fruit woodiness virus (PWV) by reducing viral infectivity, what was proven through tests carried out with Chenopodium quinoa, a PWV local lesion host. However, there was no systemic protection in passion fruit plants against the virus in the experiments involving mechanical inoculation, reducing the possibilities of the mushroom use for the PWV control in the field. In a general way, the results showed that the mushrooms L. edodes and A. blazei have substances that activate the plant defense mechanisms and they show some potential in the control of vegetable diseases, depending upon the nature of the pathogen.

Au, Chun Hang.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 124-146).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

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

by Chan Ngar Lei Annie.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1990.
Bibliography: leaves 90-102.
ACKNOWLEDGEMENTS --- p.i
ABSTRACT --- p.ii
TABLE OF CONTENTS --- p.iv
LIST OF FIGURES --- p.vii
LIST OF TABLES --- p.ix
Chapter 1. --- INTRODUCTION --- p.1
Chapter 2. --- LITERATURE REVIEW --- p.5
Chapter 2.1 --- Economic and Biotechnological Significance of Lentinus edodes --- p.5
Chapter 2.1.1 --- General Review --- p.5
Chapter 2.1.2 --- Nutritional and Medicinal Values --- p.7
Chapter 2.1.3 --- Lignocellulose Degradation and Utilization --- p.9
Chapter 2.2 --- Biological Background --- p.11
Chapter 2.2.1 --- Life Cycle --- p.11
Chapter 2.2.2 --- Patterns of Sexuality --- p.12
Chapter 2.3 --- Genetic Improvement of Lentinus edodes --- p.16
Chapter 2.3.1 --- Introduction --- p.16
Chapter 2.3.2 --- Mutagenic Agents --- p.17
Chapter 2.3.3 --- Genetic Markers and Strain Improvement --- p.20
Chapter 3. --- MATERIALS AND METHODS --- p.24
Chapter 3.1 --- Biological Materials --- p.24
Chapter 3.2 --- Media --- p.25
Chapter 3.2.1 --- Complete Medium (CM) --- p.25
Chapter 3.2.2 --- Complete Medium with Yeast Extract (CM with Y.E.) --- p.25
Chapter 3.2.3 --- Complete Fruiting Medium (CF) --- p.25
Chapter 3.2.4 --- Complete Migration Medium (CMM) --- p.25
Chapter 3.2.5 --- Minimal Medium (MM) --- p.25
Chapter 3.2.6 --- Carboxymethyl Cellulose - Leatham Medium (CMC - Leatham) --- p.26
Chapter 3.2.7 --- Potato Dextrose Agar (PDA) --- p.26
Chapter 3.3 --- Anti-metabolites for Screening of Resistant Mutants --- p.27
Chapter 3.4 --- Characterization of Monokaryons --- p.28
Chapter 3.4.1 --- Isolation of Monosporous Mycelia --- p.28
Chapter 3.4.2 --- Assessment of Mycelial Growth --- p.28
Chapter 3.4.3 --- Determination of Mating Type of Monosporous Mycelia --- p.28
Chapter 3.5 --- Mutagenesis and Isolation of Mutants --- p.29
Chapter 3.5.1 --- Effects of Homogenization on Growth --- p.29
Chapter 3.5.2 --- Determination of the Ultraviolet Irradiation Killing Curve --- p.29
Chapter 3.5.3 --- Isolation of High Temperature Tolerant Strains --- p.30
Chapter 3.5.4 --- Isolation of Auxotrophic Mutants --- p.30
Chapter 3.5.5 --- Isolation of Anti-metabolite Resistant Mutants --- p.33
Chapter 3.6 --- Characterization of Anti-metabolite Resistant Mutants --- p.33
Chapter 3.6.1 --- "Measurements of Growth Rate, Anti-metabolites Resistance and Osmotic Sensitivity" --- p.33
Chapter 3.6.2 --- Determination of Dominance Relationships --- p.34
Chapter 3.6.3 --- Detection of Extracellular Enzymes --- p.34
Chapter 4. --- RESULTS --- p.36
Chapter 4.1 --- Radial Growth Rate of Monosporous Cultures --- p.36
Chapter 4.2 --- Mating Reactions in Lentinus edodes --- p.36
Chapter 4.3 --- Effects of Homogenization on Growth of L. edodes --- p.46
Chapter 4.4 --- Determination of Ultraviolet (UV) Killing Curve --- p.46
Chapter 4.5 --- Selection of High Temperature Tolerant Strains --- p.50
Chapter 4.6 --- Auxotrophic Mutants Isolation and Identification --- p.50
Chapter 4.7 --- Selection of Anti-metabolite Resistant Mutants --- p.52
Chapter 4.8 --- Characterization of Cycloheximide Resistant Mutants V --- p.63
Chapter 4.8.1 --- "Growth Rate, Anti-metabolite Resistance and Osmotic Sensitivity of Cycloheximide Resistant Mutants" --- p.63
Chapter 4.8.2 --- Dominance Tests for Cycloheximide Resistant Strains --- p.66
Chapter 4.8.3 --- Detection of Extracellular Enzymes --- p.73
Chapter 5. --- DISCUSSION --- p.78
Chapter 5.1 --- Mating Reactions in Lentinus edodes --- p.78
Chapter 5.2 --- Isolation of Auxotrophic and Anti-metabolites Resistant Mutants --- p.81
Chapter 5.3 --- Characterization of Cycloheximide Resistant Mutants of Lentinus edodes --- p.84
CONCLUSION --- p.88
REFERENCES --- p.90

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

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

by Xie Weijun.
Publication date from spine.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1995.
Includes bibliographical references (leaves 107-122).
ABSTRACT --- p.i
ACKNOWLEDGMENTS --- p.iii
TABLE OF CONTENTS --- p.iv
LIST OF FIGURES --- p.vii
LIST OF TABLES --- p.x
Chapter 1. --- INTRODUCTION --- p.1
Chapter 2. --- LITERATURE REVIEW --- p.4
Chapter 2.1 --- Economic and biotechnological significance of Lentinula edodes --- p.4
Chapter 2.2 --- Biological background --- p.8
Chapter 2.2.1 --- Life cycle --- p.8
Chapter 2.2.2 --- Morphological changes during fruit body development --- p.9
Chapter 2.2.3 --- Biochemistry of fruit body development --- p.13
Chapter 2.2.4 --- Molecular studies --- p.17
Chapter 2.3 --- RNA AP-PCR --- p.23
Chapter 3. --- MATERIALS AND METHODS --- p.26
Chapter 3.1 --- Biological materials --- p.26
Chapter 3.2 --- Media --- p.26
Chapter 3.3 --- RNA AP-PCR with RNAs isolated from four different developmental stages --- p.27
Chapter 3.3.1 --- Isolation of total RNAs --- p.27
Chapter 3.3.2 --- RNA AP-PCR --- p.30
Chapter 3.3.3 --- PCR reamplification --- p.33
Chapter 3.4 --- PCR cloning and screening --- p.33
Chapter 3.4.1 --- PCR product purification --- p.34
Chapter 3.4.2 --- Cloning --- p.34
Chapter 3.4.3 --- Transformation --- p.36
Chapter 3.4.4 --- Screening --- p.36
Chapter 3.4.4.1 --- Restriction enzyme digestion of plasmid DNA --- p.36
Chapter 3.4.4.2 --- PCR Screening --- p.38
Chapter 3.5 --- DNA sequencing --- p.38
Chapter 3.5.1 --- Preparation of double-stranded template --- p.38
Chapter 3.5.2 --- Sequencing reaction using double-stranded templates --- p.39
Chapter 3.5.3 --- Electrophoresis --- p.40
Chapter 3.5.4 --- Autoradiography and analysis --- p.41
Chapter 3.6 --- Dot-blot analysis --- p.41
Chapter 3.6.1 --- Dot-blot --- p.41
Chapter 3.6.2 --- Probe preparation --- p.42
Chapter 3.6.3 --- Prehybridization and hybridization --- p.43
Chapter 3.6.4 --- Autoradiography and Analysis --- p.43
Chapter 3.7 --- Study on one clone: the gene encoding ubiquitin --- p.44
Chapter 3.7.1 --- "Design of primers:UBR5A, UBR3A and Polyub5A, polyub3A" --- p.44
Chapter 3.7.2 --- Isolation of DNA from mycelium and fruit body --- p.46
Chapter 3.7.3 --- Specific PCR with Polyub primers --- p.46
Chapter 3.7.4 --- RT-PCR --- p.47
Chapter 4. --- RESULTS --- p.48
Chapter 4.1 --- Total RNA isolation --- p.48
Chapter 4.2 --- RNA AP-PCR --- p.52
Chapter 4.3 --- PCR reamplification --- p.58
Chapter 4.4 --- Molecular cloning of RAP products --- p.58
Chapter 4.5 --- Screening --- p.61
Chapter 4.6 --- DNA sequencing --- p.65
Chapter 4.7 --- Sequence analyses --- p.80
Chapter 4.8 --- Dot blot --- p.84
Chapter 4.9 --- Further study on pMrG290a --- p.84
Chapter 5. --- DISCUSSION --- p.92
Chapter 5.1 --- RNA AP-PCR --- p.92
Chapter 5.2 --- PCR cloning --- p.94
Chapter 5.3 --- Nucleotide sequencing --- p.95
Chapter 5.4 --- Dot blot --- p.96
Chapter 5.5 --- Effect of polyphenol in RNA or DNA samples to the efficiency of reverse transcription and PCR --- p.98
Chapter 5.6 --- Ubiquitin and fruit body development --- p.99
Chapter 5.7 --- "Mitochondrial biogenesis, bioenergetics and fruit body development" --- p.102
CONCLUSION --- p.105
REFERENCES --- p.107

Wong, Man Chun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 141-146).
Abstracts in English and Chinese.
Abstract --- p.iii
摘要 --- p.v
Acknowledgments --- p.vii
Table of contents --- p.viii
List of tables --- p.xi
List of figures --- p.xii
List of appendix --- p.xv
Abbreviations --- p.xvi
Chapter Chapter 1 --- Literature review --- p.1
Chapter 1.1 --- Background of Lentinula edodes --- p.1
Chapter 1.2 --- Life cycle and mating system of Lentinula edodes --- p.1
Chapter 1.3 --- Breeding and strain improvement --- p.5
Chapter 1.4 --- Application of molecular markers --- p.6
Chapter 1.5 --- Objectives and long term significance --- p.9
Chapter Chapter 2 --- Genome survey sequencing and preliminary analysis --- p.11
Chapter 2.1 --- Introduction --- p.11
Chapter 2.1.1 --- Genome sequencing of basidiomycetes --- p.11
Chapter 2.1.2 --- Polymerase chain reaction-single strand conformational polymorphism --- p.12
Chapter 2.1.3 --- Sequencing chemistry --- p.13
Chapter 2.2 --- Materials and methods --- p.15
Chapter 2.2.1 --- Strain and DNA extraction --- p.15
Chapter 2.2.2 --- PCR-SSCP analysis --- p.15
Chapter 2.2.3 --- Shotgun sequencing and sequence assembly --- p.17
Chapter 2.2.4 --- Comparison with 5 basidiomycetes --- p.17
Chapter 2.3 --- Results --- p.19
Chapter 2.3.1 --- PCR-SSCP --- p.19
Chapter 2.3.2 --- Shotgun sequencing and assembly --- p.21
Chapter 2.3.3 --- Comparison with 5 basidiomycetes --- p.22
Chapter 2.4 --- Discussion --- p.30
Chapter Chapter 3 --- Cloning of A mating-type locus of Lentinula edodes --- p.33
Chapter 3.1 --- Introduction --- p.33
Chapter 3.2 --- Materials and methods --- p.35
Chapter 3.2.1 --- Genome sequencing and assembly --- p.35
Chapter 3.2.2 --- Genomic screening of A-mating type genes --- p.35
Chapter 3.2.3 --- Gap filling and sequence confirmation --- p.36
Chapter 3.2.4 --- Alignment of overlapping sequences to give contiguous sequence --- p.37
Chapter 3.2.5 --- Open reading frame prediction and protein homolog search --- p.37
Chapter 3.2.6 --- Conserved domain search --- p.37
Chapter 3.2.7 --- Testing for polymorphism --- p.38
Chapter 3.3 --- Results --- p.39
Chapter 3.3.1 --- Genomic screening of A-mating type genes --- p.39
Chapter 3.3.2 --- Gap filling and sequence confirmation --- p.45
Chapter 3.3.3 --- Protein homologs and putative protein domains --- p.48
Chapter 3.3.4 --- Polymorphism of A mating-type genes --- p.53
Chapter 3.4 --- Discussion --- p.55
Chapter 3.4.1 --- Genome mining of the A mating-type locus of L. edodes --- p.55
Chapter 3.4.2 --- Genomic structure of the A mating-type region in L. edodes --- p.55
Chapter 3.4.3 --- Functional protein domains in A mating-type genes --- p.56
Chapter 3.4.4 --- Polymorphism of A mating- type locus --- p.58
Chapter 3.4.5 --- Conclusion and future perspectives --- p.59
Chapter Chapter 4 --- Simple sequence repeat (SSR) markers development --- p.60
Chapter 4.1 --- Introduction --- p.60
Chapter 4.2 --- Materials and methods --- p.62
Chapter 4.2.1 --- Strains --- p.62
Chapter 4.2.2 --- Datasets for SSRs mining --- p.63
Chapter 4.2.3 --- in silico detection of SSR motifs and primer design --- p.63
Chapter 4.2.4 --- SSR amplification --- p.64
Chapter 4.2.5 --- Cloning and sequencing of PCR products --- p.64
Chapter 4.2.6 --- Testing for polymorphism --- p.65
Chapter 4.3 --- Results --- p.66
Chapter 4.3.1 --- in silico detection of SSR motifs and primer design --- p.66
Chapter 4.3.2 --- SSR amplification --- p.69
Chapter 4.3.3 --- SSR polymorphism --- p.83
Chapter 4.4 --- Discussion --- p.86
Chapter 4.4.1 --- Efficiency of in silico detection of SSR motifs and primer design --- p.86
Chapter 4.4.2 --- Effectiveness and polymorphism of SSR primer pairs --- p.89
Chapter 4.4.3 --- Conclusion and future perspectives --- p.90
Chapter Chapter 5 --- High-throughput sequencing of AP-PCR amplicons for SCAR markers development and phylogenetic analysis --- p.91
Chapter 5.1 --- Introduction --- p.91
Chapter 5.2 --- Materials and methods --- p.94
Chapter 5.2.1 --- Strains --- p.94
Chapter 5.2.2 --- AP-PCR analysis --- p.94
Chapter 5.2.3 --- Re-amplification of AP-PCR amplicons --- p.96
Chapter 5.2.4 --- GS-FLX sequencing --- p.96
Chapter 5.2.5 --- Strain-specific sequences identification --- p.97
Chapter 5.2.6 --- SCAR marker analysis --- p.97
Chapter 5.2.7 --- Phylogenetic analysis --- p.99
Chapter 5.3 --- Results --- p.100
Chapter 5.3.1 --- AP-PCR analysis --- p.100
Chapter 5.3.2 --- Re-amplification of AP-PCR amplicons --- p.100
Chapter 5.3.3 --- GS-FLX sequencing and strain-specific sequence identification --- p.103
Chapter 5.3.4 --- SCAR marker analysis --- p.106
Chapter 5.3.5 --- Phylogenetic analysis --- p.108
Chapter 5.4 --- Discussion --- p.111
Chapter 5.4.1 --- Sensitivity of band detection --- p.111
Chapter 5.4.2 --- SCAR marker development --- p.111
Chapter 5.4.3 --- Phylogenetic analysis --- p.113
Chapter 5.4.4 --- Conclusion --- p.114
Chapter Chapter 6 --- Concluding remarks --- p.115
Chapter 6.1 --- Project summary --- p.115
Chapter 6.2 --- Future perspectives --- p.119
Appendix --- p.121
References --- p.141

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

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

Chum Wing Yan Winnie.
"August 2006."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (p. 190-223).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.

碩士
國立屏東科技大學
食品科學系
89
Abstract This shiitake mushroom sorting machine was equipped with a programmable logic controller, a set of sorting mechanism, and a set of color machine vision system. The machine could classify the sizes, the defects and colors of shiitake mushrooms for the purpose of reducing the labor cost and grading automation. In this study, Averaged gray level of B values were used first to detect the normal color on the gill of shiitake mushroom, then used A/P values to analyze if the shiitake mushrooms were broken or not. If not, then, mushrooms were measured by their areas and were classified by their sizes during the sorting process. A fiber optics sensor first detected the shiitake mushrooms. Signal was sent out to catch images. After that, the computer analyzed the images and wrote commands into PLC. Finally, as the shiitake mushrooms arrived at the corresponding exit, PLC would initial the sorting mechanism and gave the correct grading of shiitake mushrooms. Experimental results showed that, compared with the labor grading, accuracy rate of sorting by the machine were up to 97.6%. It took 4.8 seconds of the whole process for the sorted shiitake mushrooms from the beginning of image catching to the final grading among which image processing took only 0.3 to 0.7 seconds.

Shiitake (Lentinula edodes (Berkeley) Pegler) produces an edible mushroom that has been cultivated for centuries in China, Korea, Japan, Singapore, Thailand and other Asian countries. Shiitake mushrooms grow naturally on decaying wood of hardwood trees and have traditionally been grown on short lengths of freshly-cut logs. Until now, there has been no serious exploration of the potential for Australian forest owners to utilise small logs of native or plantation forest species for shiitake mushroom production, such as eucalypt (Eucalyptus spp.).
Logs of six tree species were harvested from farm forestry plantations in Victoria and inoculated with shiitake infected dowels imported from the United States. Over the course of the next 18 months the logs were soaked four times to initiate fruiting. The fresh mushrooms were harvested and weighed to allow a comparison between log species and size. A sample of the mushrooms from each log species produced in the 2nd and 3rd fruiting were tested for their protein and fibre content.
Quercus robur was the most productive species. Over the course of the trial (four frutings) the oak logs produced almost 1 kilogram of fresh mushrooms per log which was significantly more than E. cladocalyx (527 g/log) and Alnus glutinosa (465 g/log) and Eucalyptus nitens (389 g/log) which were all, in turn, significantly more productive than Populus sp. (140 g/log) and Acacia melanoxyon (98 g/log). Larger logs produced more fruit although this may have been related to the greater number of inoculations. The protein and fibre content of mushrooms produced from shining gum logs was slightly lower than that from the oak logs but greater than that from alder. Sugar gum mushrooms had the lowest protein content.
The research suggests that there is potential to use eucalypt logs thinned from young fast-grown farm plantations as the basis for a log-based shiitake industry although more work is required to test the marketability of eucalypt grown shiitake and the economic viability of small scale production units.

by Sham Lok To.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 143-171).
Abstracts in English and Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgement --- p.iv
Abbreviations --- p.v
Table of contents --- p.vi
List of figures --- p.ix
List of tables --- p.xi
Chapter Chapter One --- Literature Review --- p.1
Chapter 1.1 --- Introduction --- p.1
Chapter 1.1.1 --- About L. edodes --- p.3
Chapter 1.1.2 --- Nutritional and medicinal values of L. edodes --- p.4
Chapter 1.1.3 --- Life cycle of L. edodes --- p.6
Chapter 1.1.4 --- Environmental factors affecting fruiting body formation in L. edodes --- p.6
Chapter 1.2 --- Molecular mechanisms of fruiting body development in L. edodes --- p.8
Chapter 1.2.1 --- Expression profiling and identification of differentially expressed genes during fruiting --- p.8
Chapter 1.2.2 --- Changing in membrane structure --- p.11
Chapter 1.2.3 --- The signal transduction cascade --- p.12
Chapter 1.3 --- Transformation in L. edodes and in other fungi --- p.14
Chapter 1.3.1 --- Transformation of L. edodes --- p.14
Chapter 1.3.2 --- Transformation in other fungi --- p.17
Chapter 1.4 --- Bioinformatics tools for comparative promoter analysis --- p.22
Chapter 1.5 --- Objectives and significance --- p.26
Chapter Chapter Two --- Promoter analysis of differentially expressed genes (DEGs) in the fruiting body development in L. edodes --- p.27
Chapter 2.1 --- Introduction --- p.27
Chapter 2.2 --- Materials and methods --- p.29
Chapter 2.2.1 --- Strains and cultivation conditions --- p.29
Chapter 2.2.2 --- Genome walking of the 5' flanking region of the DEGs --- p.29
Chapter 2.2.3 --- Annealing Control Primed (ACP) PCR --- p.31
Chapter 2.2.4 --- Construction of genomic DNA library --- p.36
Chapter 2.2.5 --- Nested PCR to amplify the target sequences --- p.37
Chapter 2.2.6 --- Cloning and sequencing of the 5' flanking region --- p.38
Chapter 2.2.7 --- Determination of transcription start site by the Neural Network algorithm --- p.39
Chapter 2.2.8 --- Identification of putative transcription factor binding sites --- p.40
Chapter 2.3 --- Results --- p.41
Chapter 2.3.1 --- Construction of adaptor linked template for genome walking --- p.41
Chapter 2.3.2 --- Sequence analysis and quality control --- p.41
Chapter 2.3.3 --- Comparison of various methods in genome walking --- p.42
Chapter 2.3.4 --- Promoter analysis --- p.42
Chapter 2.4 --- Discussion --- p.58
Chapter Chapter Three --- In-silico analysis of transcription factor binding sites and identification transcription factors expressed in L. edodes --- p.64
Chapter 3.1 --- Introduction --- p.64
Chapter 3.2 --- Material and methods --- p.67
Chapter 3.2.1 --- Sequence manipulation and extraction of homologous ESTs from C. cinereus --- p.67
Chapter 3.2.2 --- Extraction of 5' flanking region of the corresponding ESTs and promoter prediction --- p.67
Chapter 3.2.3 --- Positional cloning of mating type factor A --- p.68
Chapter 3.3 --- Results --- p.70
Chapter 3.3.1 --- Sequence extraction and manipulation --- p.70
Chapter 3.3.2 --- In-silico analysis of transcription factor binding sites in C. cinereus . --- p.70
Chapter 3.3.3 --- Comparison of putative TFBS between L. edodes and C. cinereus --- p.71
Chapter 3.3.4 --- Identification of transcription factors in L. edodes by positional cloning --- p.71
Chapter 3.4 --- Discussion --- p.85
Chapter Chapter Four --- Identification，expression profiling and promoter analysis of hydrophobin genes --- p.91
Chapter 4.1 --- Introduction --- p.91
Chapter 4.2 --- Material and methods --- p.92
Chapter 4.2.1 --- Clustering and grouping of the hydrophobin ESTs --- p.92
Chapter 4.2.2 --- Identification of the consensus sequences of the hydrophobin groups --- p.93
Chapter 4.2.3 --- RNA Sources and Preparation --- p.93
Chapter 4.2.4 --- Expression profiling of hydrophobin genes by RT-PCR --- p.95
Chapter 4.2.5 --- Promoter cloning and analysis of hydrophobin genes --- p.95
Chapter 4.3 --- Results --- p.97
Chapter 4.3.1 --- Isolation and characterization of four newly found hydrophobin genes --- p.97
Chapter 4.3.2 --- Expression levels of hydrophobins --- p.100
Chapter 4.3.3 --- Promoter sequencing of the hydrophobins --- p.103
Chapter 4.4 --- Discussion --- p.103
Chapter Chapter Five --- Transformation of L. edodes --- p.110
Chapter 5.1 --- Introduction --- p.110
Chapter 5.2 --- Materials and methods --- p.112
Chapter 5.2.1 --- Vectors and primers design --- p.112
Chapter 5.2.2 --- Maxi-preparation of plasmids --- p.112
Chapter 5.2.3 --- Cultural condition and optimization of protoplasts release --- p.114
Chapter 5.2.4 --- PEG mediated transformation --- p.115
Chapter 5.2.5 --- Electroporation mediated transformation --- p.116
Chapter 5.2.6 --- PCR screening of regenerated transformant --- p.116
Chapter 5.2.7 --- Particle bombardment --- p.117
Chapter 5.3 --- Results --- p.121
Chapter 5.4 --- Discussion --- p.128
Chapter Chapter Six --- General discussions --- p.132
References --- p.143

by Lai, Shiu Hong.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1993.
Includes bibliographical references (leaves 129-147).
TITLE PAGE --- p.I
THESIS COMMITTEE --- p.II
ABSTRACT --- p.III
ACKNOWLEDGMENTS --- p.V
ABBREVIATIONS --- p.VI
TABLE OF CONTENTS --- p.VII
LIST OF TABLES --- p.XI
LIST OF FIGURES --- p.XII
Chapter Chapter 1. --- Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) Analysis of Lent inula edodes
Chapter 1. --- Introduction
Chapter 1.1 --- General Introduction --- p.1
Chapter 1.2 --- Purpose of Study --- p.4
Chapter 2. --- Literature Review
Chapter 2.1 --- Biology of Lentinula edodes
Chapter 2.1.1 --- "Overview," --- p.6
Chapter 2.1.2 --- Life Cycle of Lentinula edodes --- p.6
Chapter 2.1.3 --- Dedikaryotization (Monokaryotization) --- p.12
Chapter 2.2 --- Genome Analysis of the Mushroom --- p.13
Chapter 2.3 --- Genetic Markers of Lentinula edodes
Chapter 2.3.1 --- Overview --- p.15
Chapter 2.3.2 --- Auxotrophic Markers --- p.16
Chapter 2.3.3 --- Biochemical Markers --- p.17
Chapter 2.3.4 --- Molecular Markers
Chapter 2.3.4.1 --- RFLPs --- p.19
Chapter 2.3.4.2 --- PCR-Based Markers --- p.20
Chapter 2.4 --- Polymerase Chain Reaction (PCR)
Chapter 2.4.1 --- The principle of PCR --- p.22
Chapter 2.4.2 --- Applications of PCR on Mushroom Studies --- p.26
Chapter 2.5 --- Arbitrarily Primed Polymerase Chain Reaction
Chapter 2.5.1 --- Principle of AP-PCR --- p.27
Chapter 2.5.2 --- Applications of AP-PCR on Mushroom Studies --- p.29
Chapter 2.6 --- Genetic Linkage Analysis
Chapter 2.6.1 --- Overview --- p.31
Chapter 2.6.2 --- The LOD Score Method --- p.34
Chapter 3. --- Materials and Methods
Chapter 3.1 --- Mushroom Strains and Culture Media --- p.36
Chapter 3.2 --- Culture Method --- p.36
Chapter 3.3 --- Solutions --- p.36
Chapter 3.4 --- Primers --- p.38
Chapter 3.5 --- Isolation of DNA from Lentinula edodes
Chapter 3.5.1 --- Mini-Preparation of Fungal DNA from L. edodes for PCR amplification --- p.42
Chapter 3.5.2 --- Cesium Chloride Method: Mini-Preparation of Fungal DNA for PCR amplification --- p.43
Chapter 3.6 --- Quantitative Measurements of DNA --- p.44
Chapter 3.7 --- Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) for the Amplification of Genomic DNA of L. edodes --- p.45
Chapter 3.8 --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.46
Chapter 3.9 --- Analysis of DNA Samples with Polyacrylamide Gel Electrophoresis (PAGE) --- p.47
Chapter 3.10 --- Silver Staining --- p.48
Chapter 3.11 --- Single Stranded Conformation Polymorphism (SSCP) Analysis of Polymorphic DNA Fragments
Chapter 3.11.1 --- Elution and Amplification of DNA --- p.49
Chapter 3.11.2 --- PCR-SSCP --- p.50
Chapter 3.12 --- Segregation and Linkage Analysis
Chapter 3.12.1 --- Chi-Square Test --- p.51
Chapter 3.12.2 --- The LOD Score Method --- p.52
Chapter 4. --- Results
Chapter 4.1 --- DNA Extraction --- p.54
Chapter 4.2 --- AP-PCR Amplified Fragments and Fragment Number --- p.58
Chapter 4.3 --- Dedikaryotization Demonstration --- p.60
Chapter 4.4 --- Identification of Polymorphic Genetic Markers --- p.64
Chapter 4.4.1 --- AP-PCR Fingerprints from Single Primer --- p.66
Chapter 4.4.2 --- AP-PCR Fingerprints Using Two Primers --- p.76
Chapter 4.5 --- Segregation of Polymorphic Markers in Single Spore Isolates (SSIs) --- p.81
Chapter 4.6 --- Single Stranded Conformation Polymorphism (SSCP) of Identified Polymorphic DNA Fragments --- p.86
Chapter 4.7 --- Linkage Analysis of the Identified AP-PCR Markers --- p.89
Chapter 5. --- Discussions
Chapter 5.1 --- DNA Extraction --- p.92
Chapter 5.2 --- Arbitrary Primers --- p.93
Chapter 5.3 --- Dedikaryotization Demonstration --- p.95
Chapter 5.4 --- Identification of Polymorphic Genetic Markers --- p.96
Chapter 5.5 --- AP-PCR Analysis of a Mushroom --- p.97
Chapter 5.6 --- Mendelian Segregation Pattern of the Polymorphic Markers --- p.99
Chapter 6. --- Conclusion and Further Studies --- p.101
Chapter 2. Electrophoretic Karyotype Analysis of Lentinula edodes
Chapter 7. --- Introduction --- p.106
Chapter 8. --- Literature Review
Chapter 8.1 --- Overview --- p.108
Chapter 8.2 --- Protoplasts --- p.109
Chapter 8.3 --- Pulsed Field Gel Electrophoresis (PFGE)
Chapter 8.3.1 --- Principle --- p.110
Chapter 8.3.2 --- Applications of PFGE in Studies of Fungi --- p.112
Chapter 9. --- Materials and Methods
Chapter 9.1 --- Strains and Culture Media --- p.114
Chapter 9.2 --- Solutions --- p.114
Chapter 9.3 --- Production of Lentinula edodes Protoplast --- p.115
Chapter 9.4 --- Electrophoretic Conditions --- p.116
Chapter 9.4.1 --- Condition for Saccharomyces cerevisiae chromosomes --- p.117
Chapter 9.4.2 --- Condition for Candida albicans chromosomes --- p.117
Chapter 9.4.3 --- Condition for Schizosaccharomyces pombe chromosomes --- p.118
Chapter 10. --- Results
Chapter 10.1 --- Protoplast Production of Lentinula edodes
Chapter 10.1.1 --- Effects of Age of Mycelium on Protoplast Yield --- p.119
Chapter 10.1.2 --- Effects of Various Osmotic Stabilizers on Protoplast Yield --- p.121
Chapter 10.1.3 --- Effects of Two Lytic Enzymes on Protoplast Yield --- p.123
Chapter 10.1.4 --- The Optimal Condition --- p.123
Chapter 10.2 --- Electrophoretic Karyotype of L. edodes --- p.124
Chapter 11. --- Discussions
Chapter 11.1 --- Protoplast Production of Lentinula edodes --- p.126
Chapter 11.2 --- Electrophoretic Karyotype --- p.127
REFERENCES

碩士
國立中興大學
生物產業機電工程學系所
96
Mushrooms are a traditional Chinese medicine and also commonly used as food. This study was divided into two sections. First of all, the response surface methodology (RSM) was used to analyze the effect of mushroom powder level (8%、10%、15%、20%、22%) and screw speed (150rpm、172rpm、225rpm、278rpm、300rpm) on the physical properties such as (radial expansion ratio, longitudinal expansion, bulk density, hardness, max. shear force) and chemical properties such as (water solubility index and water absorbability index) attributes of a corn-based mushroom extrudates. A Box-Behnken design was used to develop models for the objective responses. Individual contour plots of the different responses were overlaid, and regions meeting the optimum were identified at the mushroom level of 14.7% and the screw speed of 207.8 rpm, respectively. Secondly, extrudates used for antioxidant properties tests (scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals, chelating ability on ferrous ions, reducing power, and antioxidant activity determined by the conjugated diene method) were prepared according to the optimum mushroom level of 14.7% and the screw speed of 207.8 rpm. Pure mushroom powder, pure corn powder extrudates, Vitamin C, butylated hydroxyanisole (BHA) and vitamin E (a-tocopherol) were used for comparison. At IC50, scavenging abilities on DPPH radicals were 9.859±0.522 mg/m, 17.187±0.335 mg/ml and 32.520±0.798 mg/ml for mushroom powder, mushroom extrudates and corn powder extrudates, respectively; At IC50, chelating abilities of mushroom powder, mushroom extrudates and corn powder extrudates on ferrous ions were 0.848±0.046 mg/ml, 14.961±0.889 mg/ml and 51.608±3.220 mg/ml, respectively; A higher absorbance indicates a higher reducing power. The reducing power of mushroom powder was the best, followed by the mushroom extrudates and corn powder extrudates, respectively; At IC50, antioxidant activity of mushroom powder, mushroom extrudates and corn powder extrudates determined by the conjugated diene method were 0.443±0.055 mg/ml, 1.847±0.891 mg/ml and 6.412±0.756 mg/ml, respectively; After incubation for 15 h, the antioxidant activity for each treatment was slightly decreased. At IC50, antioxidant activity of mushroom powder, mushroom extrudates and corn powder extrudates determined by the conjugated diene method were 0.566±0.062 mg/ml, 1.920±0.937 mg/ml and 8.043±0.588 mg/ml, respectively. Overall, according to the results of these four antioxidant properties, the mushroom powder exhibited the best antioxidant abilities than the mushroom extrudates and corn powder extrudates, respectively.

碩士
國立中興大學
食品科學系
91
Abstract This research used silver-ear, shiitake-stipe and winter-mushroom adding to flour in three ratios (2%, 5% and 10%) to made of silver-ear steamed buns, shiitake steamed buns and winter-mushroom steamed buns. The control was made by original recipe. Crude fiber content among three various steamed buns in proximate composition is higher than control(0.91). Crude fiber content in silver-ear steamed buns (1.52< 1.70 < 1.76), shiitake-stipe steamed buns (2.19 < 2.51 < 3.12) and winter-mushroom steamed buns (1.38 < 1.81 < 2.34) increased with increase of adding ratios. The specific volume in the control was 4.0, and in three various steamed buns were decrease with increase of adding ratios (silver-ear steamed buns 3.54 > 3.31 > 2.66, shiitake-stipe steamed buns 4.23 > 4.20 > 3.69, winter-mushroom 3.45 > 3.28 > 2.98) Based on the texture profile analysis, the surface hardness and fracturability among three various steamed buns increased with increase of storage time (days 0, 3, 6, 9 and 12), otherwise, the adhesiveness , cohesiveness , springiness , chewiness and gumminess decreased with increase of storage. The surface lightness of control and three various steamed buns decreased with increase storage time. As assessed by sensory evaluation, we found that the overall preference decreased with increase storage time, its also decreased while adding ratios in silver-ear steamed, shiitake-stipe steamed buns and winter-mushroom. The only special phenomenon was that there was the same acceptability between control and shiitake-stipe steamed buns with different adding ratios. The scanning electron micrographs indicate that the structure of these three various steamed buns became finer during storage time. Further more, the X-ray diffraction patterns showed more sharp peak apparently during storage time, and the diffraction patterns were from V-type turned to B-type. Keyword: mushroom, steamed buns, physico-chemical properties, sensory evaluation.

by Chu Kin Kan Astley.
Thesis submitted in: December 2000.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves xi-xx (3rd gp.)).
Abstracts in English and Chinese.
Abstract (English) --- p.i
Abstract (Chinese) --- p.iii
Acknowledgments --- p.iv
Table of Contents --- p.v
List of Tables --- p.ix
List of Figures --- p.x
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- Popularity of Shiitake Mushroom --- p.1
Chapter 1.2 --- Inheritance of Genetic Materials in L.edodes --- p.1
Chapter 1.3 --- Genetic Markers and Linkage Maps of L.edodes --- p.2
Chapter 1.4 --- Aims of Study --- p.5
Chapter Chapter 2 --- Mapping of Sequence Tagged Sites (STSs) and Expressed Sequence Tags (ESTs) on the Linkage Map of L.edodes by PCR-Single Strand Conformational Polymorphism (SSCP) Test
Chapter 2.1 --- Literature Review --- p.7
Chapter 2.1.1 --- Construction of Genetic Linkage Map --- p.7
Chapter 2.1.2 --- Logarithm of the Odds (LOD) Score --- p.8
Chapter 2.1.3 --- MAPMAKER Program --- p.10
Chapter 2.1.4 --- Sequence Tagged Site (STS) and Expressed Sequence Tag (EST) --- p.11
Chapter 2.1.5 --- Polymerase Chain Reaction - Single Strand Conformational Polymorphism (PCR-SSCP) --- p.13
Chapter 2.2 --- Material and Methods --- p.18
Chapter 2.2.1. --- Detection of the STS and EST markers with PCR-SSCP test --- p.18
Chapter 2.2.1.1 --- Biological Material and Growth Conditions --- p.18
Chapter 2.2.1.2 --- DNA Samples Preparation --- p.18
Chapter 2.2.1.3 --- PCR Primers Designation --- p.20
Chapter 2.2.1.4 --- PCR Amplification --- p.21
Chapter 2.2.1.5 --- SSCP Test --- p.21
Chapter 2.2.1.6 --- Silver Staining of the Polyacrylamide Gel --- p.22
Chapter 2.2.2. --- Mapping of the STS and EST Markers --- p.22
Chapter 2.2.2.1 --- Biological Material and Growth Conditions --- p.22
Chapter 2.2.2.2 --- SSI DNA Preparation --- p.22
Chapter 2.2.2.3 --- PCR-SSCP Test among SSIs --- p.24
Chapter 2.2.2.4 --- Chi Square (X2) Test --- p.24
Chapter 2.2.2.5 --- LOD Score Test and Mapping of the Markers --- p.26
Chapter 2.3 --- Results --- p.27
Chapter 2.3.1 --- Detection of the STS and EST Markers from PCR-SSCP Test --- p.27
Chapter 2.3.1.1 --- DNA Sample Preparation --- p.27
Chapter 2.3.1.2 --- Primers Designed for STS and EST Amplification --- p.27
Chapter 2.3.1.3 --- Size Differences between Experimental and Expected PCR Products --- p.38
Chapter 2.3.1.4 --- Markers of PCR Polymorphism (PCRP) --- p.38
Chapter 2.3.1.5 --- Markers of PCR-SSCP and PCR Length Polymorphism (PCR-LP) --- p.42
Chapter 2.3.2 --- Mapping of the STS/EST Markers --- p.49
Chapter 2.3.2.1 --- DNA Templates of SSIs --- p.49
Chapter 2.3.2.2 --- Polymorphism Profiles of STSs and ESTs among the L54-SSIs --- p.49
Chapter 2.3.2.3 --- Chi-square Test --- p.55
Chapter 2.3.2.4 --- Repeated EST Markers --- p.58
Chapter 2.3.2.5 --- Anonymous Expressed Sequence Tag - EST31 --- p.58
Chapter 2.3.2.6 --- Linkage Analysis and Mapping of Markers --- p.59
Chapter 2.3.2.6.1 --- Linkage Relationships between the16 STS/EST Markers --- p.59
Chapter 2.3.2.6.2 --- Mapping of the STS/EST Markers onto the RAPD Linkage Map --- p.62
Chapter 2.4 --- Discussion --- p.69
Chapter 2.4.1 --- DNA Template Preparation --- p.69
Chapter 2.4.2 --- Size Difference Between Expected and Experimental PCR Product --- p.69
Chapter 2.4.3 --- PCR Polymorphism (PCRP) --- p.70
Chapter 2.4.4 --- PCR-LP --- p.70
Chapter 2.4.5 --- PCR-SSCP --- p.71
Chapter 2.4.5.1 --- Primer Designed for PCR-SSCP --- p.71
Chapter 2.4.5.2 --- Markers Producing Efficiency of PCR-SSCP Test --- p.72
Chapter 2.4.6 --- Linkage Map of L.edodes --- p.73
Chapter 2.4.6.1 --- Map Distance --- p.73
Chapter 2.4.6.2 --- Linkage Groups --- p.74
Chapter 2.4.6.3 --- Map Markers --- p.75
Chapter Chapter 3 --- Mapping of Agronomic Features of L.edodes
Chapter 3.1 --- Literature Review --- p.77
Chapter 3.1.1 --- Aroma Feature of L.edodes --- p.77
Chapter 3.1.1.1 --- Volatile Compounds in Shiitake (L.edodes) Mushroom --- p.77
Chapter 3.1.1.2 --- Fragrance Signature of Shiitake Mycelium --- p.79
Chapter 3.1.2 --- Mapping of Quantitative Trait Loci (QTL) --- p.84
Chapter 3.1.2.1 --- Complex Traits --- p.84
Chapter 3.1.2.2 --- Quantitative Traits Locus (QTL) --- p.85
Chapter 3.1.2.3 --- Maximum-likelihood Estimate in QTL mapping --- p.86
Chapter 3.1.2.4 --- MAPMAKER/QTL --- p.87
Chapter 3.2 --- Material and Methods --- p.88
Chapter 3.2.1 --- Aroma feature of Mycelium --- p.88
Chapter 3.2.1.1 --- Preliminary Screening of Volatiles in the SSI Mycelia of L.edodes --- p.88
Chapter 3.2.1.1.1 --- Biological Material and Growth Conditions --- p.88
Chapter 3.2.1.1.2 --- Volatile Extraction from SSI Mycelia --- p.88
Chapter 3.2.1.1.3 --- Screening of Volatile Compounds with GC-MS --- p.89
Chapter 3.2.1.2 --- Quantification of the Target Aromatic Volatile in the Mycelia of SSI and Parents --- p.90
Chapter 3.2.1.2.1 --- Sample Preparations --- p.90
Chapter 3.2.1.2.2 --- Quantification of the Target Volatile --- p.90
Chapter 3.2.2 --- Measurement of Mycelial Growth --- p.91
Chapter 3.2.3 --- Observation of Pigment Secretion during Mycelial Growth --- p.91
Chapter 3.2.4 --- Locating Putative QTL on the Genetic Map of L.edodes --- p.92
Chapter 3.3 --- Results --- p.93
Chapter 3.3.1 --- Aroma Feature --- p.93
Chapter 3.3.1.1 --- Preliminary Screening of Volatiles in Mycelia of L.edodes --- p.93
Chapter 3.3.1.2 --- Quantification of l-octen-3-ol in SSI Mycelia --- p.103
Chapter 3.3.1.2.1 --- Sample Preparation --- p.103
Chapter 3.3.1.2.2 --- l-Octen-3-ol contents in SSI Mycelia --- p.103
Chapter 3.3.1.3 --- Mapping of QTL for l-octen-3-ol level on the genetic map --- p.106
Chapter 3.3.2 --- Mycelial Growth Rate (MGR) --- p.111
Chapter 3.3.2.1 --- Measurement of Mycelial Growth Rate --- p.111
Chapter 3.3.2.2 --- Mapping of QTL for MGR on the genetic map --- p.111
Chapter 3.3.3 --- Pigment Secretion form SSI mycelia --- p.116
Chapter 3.4 --- Discussion --- p.118
Chapter 3.4.1 --- Significance of the QTLs --- p.118
Chapter 3.4.2 --- QTL for Aroma Feature --- p.119
Chapter 3.4.2.1 --- Trait of Aroma: l-octen-3-ol level --- p.119
Chapter 3.4.2.2 --- QTL of l-octen-3-ol level --- p.120
Chapter 3.4.3 --- Mycelial Growth Rate (MGR) --- p.123
Chapter 3.4.4 --- Pigment Secretion --- p.125
Chapter Chapter 4 --- General Discussion and Conclusions --- p.127
Chapter 4.1 --- Future Works --- p.127
Chapter 4.1.1 --- Mapping of L.edodes Genes --- p.127
Chapter 4.1.2 --- Characterizing and Mapping of Agronomic Traits --- p.128
Chapter 4.2 --- Conclusions --- p.128
Referencesxi

by XU hai-Lou.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1997.
Includes bibliographical references (p. 184-198).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.

碩士
實踐大學
食品營養研究所
92
Mushrooms have been known as a medicine in traditional Chinese medicine. From previously studies, there are many result showed that the mushrooms extracts could enhanced immunity response both on in vivo and in vitro model. The aim of this study, we try to investigate whether long term oral administration Ganoderma lucidum, Antrodia camphorata, Shiitake mushroom, and Phellinus linteus can effect the immunity for young mice. Besides, we also would like to explore the inhibition of those mushrooms extract on hepatitis B virus (HBV) with HepES-2 cells, and do those extracts have antioxidant activity. In animal model, the 3 to 4 weeks old male BLAB/c mice were divided into 4 groups oral administered 4 kinds of mushrooms G. lucidum, A. camphorata, S. mushroom and P. linteus at different dose age, 0 mg /Kg bw /day, 20 mg /Kg bw /day, 100 mg /Kg bw /day, and 250 mg /Kg bw /day, respectively for 8 weeks. MTT assay was used for detection of the lymphocyte proliferation rate. The flow cytometry analysis was used to estimate T, B, and NK cell number. The ELISA assay was used to determine cytokine (IL-2, IL-4, IFN- γ) secretion. The result showed that long term oral administration G. lucidum in BLAB/c mice could increased T, NK cell numbers (by using flow cytometry), IL-2 and IL-4 secretion (by using ELISA) when compared with control group. The lymphocyte proliferation rate was no significant difference in MTT assay, but T, NK cell numbers (by using flow cytometry) and IFN-γsecretion (by using ELISA) had significant increase with long term oral administration of A. camphorata when compared with control group in BLAB/c mice . On the other hand, long term oral administration S. mushroom in BLAB/c mice, the lymphocyte proliferation rate had significant increase in MTT assay, T, B, NK cell numbers (by using flow cytometry), IL-2, IL-4 and IFN-γsecretion (by using ELISA) also had significant increase when compared with control group. The lymphocyte proliferation rate was significant increase in MTT assay, T, B cell numbers (by using flow cytometry), IL-2, IL-4 and IFN-γsecretion (by using ELISA) also had significant increase with long term oral administration of P. linteus when compared with control group in BLAB/c mice. In this study, we conclude that long term oral administration G. lucidum and A. camphorata might enhanced T and NK cell activation, proliferation after antigen stimulation, and long term oral administration S. mushroom and P. linteus might enhanced T、B and NK cell activation, proliferation after antigen stimulation. Thus, long term oral administration mushrooms in mice could enhance the immunity of health host when they exposed in antigen. HepES-2 cells, which derived from HepG2 cells, transfected with HBV genome, and HepES-2 cells has been examined with constant HBeAg and HBsAg expression. The hot water extract of those mushrooms were used to treat with HepES-2 cells, and MTT assay was used to detect the cytotoxcity of those mushroom extracts. HepES-2 cells were plated at a density of 5×104 cells with suitable concentration of mushrooms extract, which caused less than 15 % HepES-2 cells death. The secretion of HBeAg and HBsAg from HepES-2 cells were determined via ELISA assay. DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay was used for evaluation of antioxidant activity of those mushroom extracts. The result showed that G. lucidum, A. camphorata, and S. mushroom extracts had significant cytotoxcity for HepES-2 cells start from the concentration of 125μg/ml treatment. Compared with those in sense control, all mushrooms have significant decrease secretion of HBeAg from HepES-2 cells, but only G. lucidum and S. mushroom have significant decrease secretion of HBsAg from HepES-2 cells. Besides, those extract of mushrooms scavenged the stable free radical DPPH resulting in a increase of does-dependently (250 ~ 2000μg/ml). Thus G. lucidum and S. mushroom extract possesses a significant anti-HBV activity and perhaps can develop into adjuvant or therapeutic drug of HBV infection patients in the future.

Being the most abundant carbon-containing terrestrial biopolymer, lignocellulose serves as one of the best candidate feedstocks for biofuel production. The current cost-ineffective method for lignocellulose pretreatment is one of the major barriers that hinder the development of biofuel production. This leads to an exploration in the potential application of lignocellulolytic enzymes in this biorefinery process. Taking advantage of the strong activity of ligninolytic enzymes in L. edodes, we aimed at cloning and heterologously expressing these enzymes. The present project applied a yeast expression system, Pichia pastoris, as a laboratory-scale platform for heterologous expression of one of our target ligninolytic enzymes, manganese peroxidase (MnP). We successfully cloned and expressed recombinant MnP. Its enzymatic activity was the highest when grown in the presence of hemoglobin. Our long-term goal is to establish a platform for the large-scale production of recombinant lignocellulotyic enzymes at low-cost, which would strengthen their application in biofuel production.
The shitake mushroom, Lentinula edodes, is one of the most commonly consumed edible mushrooms in Asian countries. It is a saprophyte that naturally colonizes dead wood. As a member of wood-decaying white rot basidiomycete, L. edodes is able to depolymerize lignin and hydrolyze wood polysaccharides. However, the enzymatic mechanism for its lignocellulolytic system is poorly understood. Examination on the L. edodes genome and transcriptome revealed a unique lignocellulolytic system. L. edodes has a diverse enzymatic arsenal for lignin degradation. The enzymes include laccase, manganese peroxidase, cellobiose dehydrogenase and various lignin degrading auxiliary enzymes. When compared to another white rot fungus Phanerochaete chrysosporium, L. edodes possesses more hemicellulase- and pectinase-coding genes, and fewer genes encoding cellulases, suggesting that it preferentially attacks non-cellulosic polysaccharides. The transcription analysis on genes related to antioxidative mechanisms also offers insights to the oxidative stress encountered by mycelium during the free radical-mediated lignin degradation.
Kwok, Sze Wai.
Adviser: Hoi-Shan Kwan.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 141-160).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

by Wong Wing Lei.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 118-129).
Abstracts in English and Chinese.
Abstract (English) --- p.i
Abstract (Chinese) --- p.iii
Acknowledgements --- p.iv
Abbreviations --- p.v
Table of Contents --- p.vi
List of Figures --- p.xi
List of Tables --- p.xiiii
Chapter Chapter One --- Literature Review
Chapter 1.1 --- Introduction --- p.1
Chapter 1.2 --- Life cycle of L. edodes --- p.3
Chapter 1.3 --- Environmental Stimuli for primoridum initiation --- p.6
Chapter 1.4 --- Two component system (TCS) --- p.7
Chapter 1.4.1 --- Modular structure of TCS --- p.7
Chapter 1.4.2 --- Overview of the difference of TCS from other signaling cascades --- p.8
Chapter 1.4.3 --- Functional roles of TCS in different organisms --- p.9
Chapter 1.4.4 --- Molecular studies on histidine kinases --- p.16
Chapter 1.5 --- Rationale and Summary --- p.18
Chapter Chapter Two --- Screening of Differentially Expressed Genes
Chapter 2.1 --- Introduction --- p.20
Chapter 2.2 --- Materials and Methods --- p.24
Chapter 2.2.1 --- Reverse Dot Blot Hybridization --- p.24
Chapter 2.2.1.1 --- Construction of primordial cDNA library --- p.24
Chapter 2.2.1.2 --- PCR amplification of cDNA clones --- p.24
Chapter 2.2.1.3 --- Membrane preparation --- p.24
Chapter 2.2.1.4 --- cDNA probe preparation --- p.25
Chapter 2.2.1.4.1 --- RNA extraction --- p.25
Chapter 2.2.1.4.2 --- RAP-PCR --- p.26
Chapter 2.2.1.4.3 --- Purification of RAP-PCR product --- p.27
Chapter 2.2.1.4.4 --- Labeling of probe --- p.27
Chapter 2.2.1.5 --- Stringent washing and signal detection --- p.27
Chapter 2.2.2 --- Confirmation of the expression level of differentially expressed genes by Reverse transcription PCR (RT-PCR) --- p.28
Chapter 2.2.2.1 --- Primer Design --- p.28
Chapter 2.2.2.2 --- First strand total cDNA synthesis --- p.28
Chapter 2.2.2.3 --- Reverse transcription PCR --- p.29
Chapter 2.3 --- Results --- p.30
Chapter 2.3.1 --- Screening of differentially expressed genes --- p.30
Chapter 2.3.2 --- Sequence analysis --- p.30
Chapter 2.3.3 --- Confirmations of differential expression of candidate genes by RT-PCR --- p.34
Chapter 2.4 --- Discussion --- p.39
Chapter 2.4.1 --- Putative roles of differentially expressed genes --- p.39
Chapter 2.4.2 --- Confirmation of differential expression --- p.40
Chapter Chapter Three --- Characterization and Full Length Sequence Analysis of Le.nikl clone
Chapter 3.1 --- Introduction --- p.42
Chapter 3.2 --- Materials and Methods --- p.43
Chapter 3.2.1 --- Full length sequence of partial Le.nikl cDNA clone by primer walking
Chapter 3.2.1.1 --- Primer Design --- p.43
Chapter 3.2.2 --- Confirmations of differentially expressed Le.nikl by Northern blot analyses --- p.44
Chapter 3.2.2.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.44
Chapter 3.2.2.2 --- Northern blotting --- p.45
Chapter 3.2.2.3 --- Probe preparation --- p.46
Chapter 3.2.2.4 --- "Hybridization, Stringency washes, Signal detection" --- p.46
Chapter 3.2.3 --- Cloning of 5' end of Le.nikl --- p.47
Chapter 3.2.3.1 --- Amplification of 5' partial end of Le.nikl from primordium cDNA library --- p.47
Chapter 3.2.3.2 --- Polishing the PCR products --- p.48
Chapter 3.2.3.3 --- Cloning of PCR products --- p.48
Chapter 3.2.3.4 --- PCR screening of the transformants --- p.49
Chapter 3.2.3.5 --- Sequencing analysis --- p.49
Chapter 3.2.3.6 --- PCR for confirmation of the 5' sequence originated from Le.nikl gene instead of genes from the same gene family --- p.49
Chapter 3.3 --- Results --- p.51
Chapter 3.3.1 --- Northern analysis of Le.nik1 --- p.51
Chapter 3.3.2 --- Sequence analysis of 2.75kb Le.nikl
Chapter 3.4 --- Discussion --- p.63
Chapter Chapter Four --- Protein Interaction Study of Response Regulator of Le.NIK1 by Yeast two hybrid
Chapter 4.1 --- Introduction --- p.65
Chapter 4.2 --- Materials and Methods --- p.68
Chapter 4.2.1 --- "Yeast strains, media, yeast vectors" --- p.68
Chapter 4.2.2 --- Bait construction --- p.68
Chapter 4.2.2.1 --- "Cloning of bait insert Le.nik1-rec into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.68
Chapter 4.2.2.1.1 --- Design of cloning primer --- p.70
Chapter 4.2.2.1.2 --- First strand total cDNA synthesis --- p.70
Chapter 4.2.2.1.3 --- PCR amplification of bait insert --- p.70
Chapter 4.2.2.2 --- Small-scale transformation of GBKT7-Le.nik1-rec plasmid --- p.71
Chapter 4.2.2.2.1 --- Preparation of yeast competent cells --- p.71
Chapter 4.2.2.2.2 --- Transformation of the GBKT7-Le.nik1-rec plasmid into the yeast strain AH109 --- p.72
Chapter 4.2.2.2.3 --- Test for the Self-Activation of DNA-BD fusion --- p.72
Chapter 4.2.2.2.4 --- Verification of bait protein expression --- p.72
Chapter 4.2.2.2.4.1 --- Yeast protein extraction by TCA method --- p.72
Chapter 4.2.2.2.4.2 --- Western blot analyses --- p.73
Chapter 4.2.2.2.4.2.1 --- SDS-PAGE --- p.73
Chapter 4.2.2.2.4.2.2 --- Western blotting --- p.75
Chapter 4.2.2.2.4.2.3 --- Immunodetection --- p.75
Chapter 4.2.2.2.4.2.4 --- ECL detection --- p.75
Chapter 4.2.2.2.5 --- Test for the toxicity of DNA-BD fusion --- p.76
Chapter 4.2.3 --- Prey construction -Total primordium cDNA synthesis --- p.76
Chapter 4.2.3.1 --- First-strand cDNA synthesis --- p.76
Chapter 4.2.3.2 --- ds cDNA amplification --- p.77
Chapter 4.2.3.3 --- ds cDNA purification --- p.77
Chapter 4.2.4 --- Yeast two hybrid screening assay --- p.77
Chapter 4.2.4.1 --- Yeast competent cells preparation --- p.77
Chapter 4.2.4.2 --- Screening by co-transformation --- p.78
Chapter 4.2.4.2.1 --- for positive and negative control --- p.78
Chapter 4.2.4.2.2 --- for prey ds DNA and pGBKT7-Le.nik1-rec --- p.78
Chapter 4.2.4.3 --- Selection for transformants expressing interacting proteins --- p.78
Chapter 4.2.5 --- β-galactosidase analysis- colony lift filter assay --- p.79
Chapter 4.2.6 --- PCR screening of the prey --- p.80
Chapter 4.2.7 --- DNA Sequencing of the prey insert --- p.80
Chapter 4.2.8 --- Plasmid Isolation --- p.81
Chapter 4.2.8.1 --- Isolation and sequencing of yeast plasmid --- p.81
Chapter 4.2.9 --- Confirmations of protein interaction and self-activation test of AD-prey insert by small-scale co-transformation --- p.82
Chapter 4.2.10 --- Northern Blot analyses of prey genes --- p.82
Chapter 4.3 --- Results --- p.83
Chapter 4.3.1 --- DNA-BD fusion construction --- p.83
Chapter 4.3.2 --- AD fusion library construction --- p.88
Chapter 4.3.3 --- Yeast two hybrid screening assay by co-transformation --- p.88
Chapter 4.4 --- Discussion --- p.96
Chapter Chapter Five --- Effect of Different Mannitol Osmolarity on Le.nikl Transcriptional Expression
Chapter 5.1 --- Introduction --- p.101
Chapter 5.2 --- Materials and Methods --- p.102
Chapter 5.2.1 --- "Mycelium inoculum preparation, cultivation mediums and cultivation conditions" --- p.102
Chapter 5.2.2 --- RNA extraction --- p.102
Chapter 5.2.3 --- Northern analysis --- p.102
Chapter 5.3 --- Results --- p.103
Chapter 5.4 --- Discussion --- p.112
Chapter 5.4.1 --- Effect of high osmolarity on mushroom growth --- p.112
Chapter 5.4.2 --- Effect of high osmolarity on gene expression --- p.113
Chapter Chapter Six --- General Discussion --- p.114
References --- p.118

In this study, a novel compound was isolated and purified from the solid culture medium (potato dextrose agar) of shiitake 1358 strain through series of methods, such as ethanol precipitation, macroporous resin column separation, semi-preparative high performance liquid chromatography separation and preparative thin-layer chromatography separation. Analyzing spectra from fourier transform infra-red spectroscopy, gas chromatography-mass spectrometry, 1-dimension and 2-dimension nuclear magnetic resonance, the chemical structure of the novel compound was determined and named as 4-amino-5,6-dihydrobenzo[d]oxonine-2,7(1H,4H)-dione. It could inhibit the proliferation of HL-60 leukemia cells significantly and with an IC50 of 1.56 mug/ml (7.123 mumol/L) in the 72-hour treatment. From the results, it is suggested that this compound could activate the G2 phase checkpoint control of the cell cycle to arrest the cell cycle in G2 phase. In addition, it could suppress the replicative DNA synthesis to inhibit the proliferation of HL-60 leukemia cells. The more important is that this compound can induce the apoptosis of HL-60 leukemia cells significantly through intrinsic and extrinsic apoptotic pathways. The compound could induce intrinsic and extrinsic apoptosis through the regulation of the apoptosis-related proteins, such as Fas ligand, Bax, Bcl-2, Caspase 8, Caspase 9, and Caspase 3. For intrinsic pathway, the compound might upregulate Bax, downregulated Bcl-2, activated the Caspase 9, subsequently activated Capase 3, and ultimately led to cell death. For extrinsic pathway, the compound upregulated the Fas ligand, cleaved and activated Procaspase 8 to active Caspase 8, further cleaved and activated Procaspase 3 to active Caspase 3 to commit the cells to apoptosis.
Leukemia is a malignant cancer that involves the bone marrow and blood circulation systems. Leukemia results in the uncontrolled growth of abnormal (leukemic) white blood cells and may also invade other organs, including the liver, spleen, lymph nodes, testes, and brain. In 2007, about 44,240 new cases of leukemia were diagnosed and 21,790 patients died from all types of leukemias in USA.
Shiitake was first cultivated in China more than 800 years ago. It is the second most commonly cultivated edible mushrooms in the world nowadays. For a long time, shiitake has been valued for its unique taste and flavor and as a medicinal invigorant. According to ancient Chinese medicinal theory, consumption of shiitake was in favor of long life and good health. In China and Japan, shiitake has been used as both a food and a medicinal herb for thousands of years. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan. Currently, most researches concentrate on the anticancer activities of the extracts from the fruiting body of shiitake, especially polysaccharides. Report about the anti-cancer effects of other components from the shiitake mushroom is scarce. The objectives of this investigations were: (1) to study the anticancer activities of brownish substances obtained during the solid medium culture of shiitake on specific cancer cell unes, especially HL60 cancer cell line; (2) to isolate and characterize the active compound(s) in the brown mushroom exudates; and (3) to propose the possible mechanism of actions, especially the function of the bcl-2 family genes and proteins.
by Guo, Yuming.
Adviser: Chung Hale Yin.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3314.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 188-199).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.