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

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

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

Made available in DSpace on 2014-06-11T19:24:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-01-29Bitstream added on 2014-06-13T20:52:29Z : No. of bitstreams: 1 regina_m_me_botfca.pdf: 1560024 bytes, checksum: 420f87684566823c84c615c10ed00381 (MD5) / 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.

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.

Hui Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 166-172). / Abstracts in English and Chinese. / Abstract --- p.iii / Acknowledgments --- p.vi / Abbreviations --- p.vii / Table of Contents --- p.viii / Table of Figures --- p.xiii / Table of Tables --- p.xviii / Chapter Chapter 1. --- Literature Reviews / Chapter 1.1 --- Functional Genomics and Its Developments --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- "Transcriptomics, Proteomics and Metabolomics" --- p.1 / Chapter 1.1.3 --- Gene-perturbing Strategies --- p.3 / Chapter 1.1.4 --- Applications of Functional Genomics --- p.4 / Chapter 1.2 --- Serial Analysis of Gene Expression (SAGE) and Generation of Longer cDNA Fragments from SAGE tags for Gene Identification (GLGI) --- p.6 / Chapter 1.2.1 --- Introduction --- p.6 / Chapter 1.2.2 --- Principles and Methods of SAGE --- p.6 / Chapter 1.2.3 --- Data Analysis --- Bioinformatics --- p.9 / Chapter 1.2.4 --- Applications of SAGE --- p.9 / Chapter 1.2.5 --- Modifications of SAGE --- p.10 / Chapter 1.2.6 --- Principles and Methods of GLGI --- p.11 / Chapter 1.2.7 --- Applications and Improvements of GLGI --- p.14 / Chapter 1.3 --- Transformation --- p.15 / Chapter 1.3.1 --- Introduction --- p.15 / Chapter 1.3.2 --- Different Methods of Transformation --- p.15 / Chapter 1.3.2.1 --- General Transformation Strategy --- p.15 / Chapter 1.3.2.2 --- Polyethylene Glycol (PEG)-mediated Transformation --- p.16 / Chapter 1.3.2.3 --- Restriction Enzyme Mediated Integration (REMI) --- p.16 / Chapter 1.3.2.4 --- Electroporation --- p.17 / Chapter 1.3.2.5 --- Particle Bombardment --- p.17 / Chapter 1.3.3 --- The Future Needs of Transformation --- p.18 / Chapter 1.4 --- RNA Silencing --- p.20 / Chapter 1.4.1 --- Introduction --- p.20 / Chapter 1.4.2 --- Major Components and Principles of RNAi --- p.21 / Chapter 1.4.3 --- Applications of RNA Silencing --- p.23 / Chapter 1.5 --- The Target Organism Lentinula edodes --- p.25 / Chapter 1.5.1 --- Introduction --- p.25 / Chapter 1.5.2 --- The Life Cycle of L. edodes --- p.26 / Chapter 1.5.3 --- Biochemical and Molecular Studies on L. edodes --- p.27 / Chapter 1.5.4 --- Prospectus --- p.29 / Chapter Chapter 2. --- Development of Methods for Studying Gene Function in Lentinula edodes / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.32 / Chapter 2.2.1 --- Cultivation of Lentinula edodes --- p.32 / Chapter 2.2.2 --- Proplast Release and Regeneration --- p.32 / Chapter 2.2.3 --- Preparation of Plasmid DNA --- p.33 / Chapter 2.2.4 --- Selectable Marker …Bialaphos --- p.35 / Chapter 2.2.5 --- Transformation --- p.35 / Chapter 2.2.5.1 --- Electroporation --- p.35 / Chapter 2.2.5.2 --- PEG-mediated Transformation --- p.36 / Chapter 2.3 --- Results --- p.37 / Chapter 2.3.1 --- Cultivation of Lentinula edodes --- p.37 / Chapter 2.3.2 --- Proplast Release and Regeneration --- p.37 / Chapter 2.3.3 --- Preparation of Plasmid DNA --- p.43 / Chapter 2.3.4 --- Selectable Marker--- Bialaphos --- p.43 / Chapter 2.3.5 --- Transformation --- p.46 / Chapter 2.3.5.1 --- Electroporation --- p.46 / Chapter 2.3.5.2 --- PEG-mediated Transformation --- p.46 / Chapter 2.4 --- Discussions and Conclusions --- p.57 / Chapter Chapter 3. --- Identification of Interested Genes in Expression Profile of SAGE using GLGI Method. / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.1.1 --- Results of SAGE Analysis --- p.61 / Chapter 3.1.2 --- Use of GLGI Method for Extension of SAGE Tags --- p.63 / Chapter 3.1.3 --- 5´ة Extension of GLGI (5'GLGI) --- p.65 / Chapter 3.1.3.1 --- Introduction --- p.65 / Chapter 3.1.3.2 --- "Overall strategy of 5, GLGI Method" --- p.67 / Chapter 3.1.3.3 --- Two-Steps PCR Method --- p.69 / Chapter 3.2 --- Generation of Longer cDNA Fragments from SAGE tags for Gene Identification (GLGI) --- p.71 / Chapter 3.2.1 --- Materials and Methods (GLGI Analysis) --- p.71 / Chapter 3.2.1.1 --- Total RNA Extraction --- p.71 / Chapter 3.2.1.2 --- Messenger RNA (mRNA) Extraction --- p.72 / Chapter 3.2.1.3 --- Preparation of 3´ة cDNA for GLGI --- p.73 / Chapter 3.2.1.4 --- NIaIII digestion of double strand cDNA --- p.74 / Chapter 3.2.1.5 --- PCR amplification of the 3'-cDNAs (Optional) --- p.77 / Chapter 3.2.1.6 --- GLGI Amplification of The Target Template --- p.80 / Chapter 3.2.1.7 --- DNA Cloning (Optional) --- p.82 / Chapter 3.2.1.8 --- Sequencing of GLGI PCR products --- p.85 / Chapter 3.2.2 --- 5' Materials and Methods (5' GLGI Analysis) --- p.86 / Chapter 3.2.2.1 --- Preparation of unique antisense primers --- p.86 / Chapter 3.2.2.2 --- 5' extension of GLGI products --- p.87 / Chapter 3.2.2.3 --- DNA Cloning (Optional) --- p.89 / Chapter 3.2.2.4 --- Sequencing of 5' GLGI PCR products --- p.89 / Chapter 3.2.3 --- Results (GLGI Analysis) --- p.90 / Chapter 3.2.3.1 --- Total RNA Extraction --- p.90 / Chapter 3.2.3.2 --- Messenger RNA Extraction --- p.90 / Chapter 3.2.3.3 --- Preparation of 3' cDNA for GLGI --- p.90 / Chapter 3.2.3.4 --- NIaIII digestion of double strand cDNA --- p.94 / Chapter 3.2.3.5 --- GLGI Amplification of The Target Template --- p.94 / Chapter 3.2.3.6 --- Sequencing of GLGI PCR products --- p.103 / Chapter 3.2.4 --- Results (5' GLGI Analysis) --- p.111 / Chapter 3.2.4.1 --- 5' extension of GLGI products --- p.111 / Chapter 3.2.4.2 --- Sequencing of 5´ة GLGI PCR products --- p.116 / Chapter 3.3 --- Discussions and Conclusions --- p.126 / Chapter 3.3.1 --- GLGI amplification of the target template --- p.126 / Chapter 3.3.2 --- 5' extension of GLGI products --- p.129 / Chapter 3.3.3 --- Two-Steps PCR Method --- p.130 / Chapter 3.3.4 --- Sequencing results of GLGI method and 5' GLGI method --- p.131 / Chapter Chapter 4. --- Identification of Unknown EST Using PCR Method With cDNA Library / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.2 --- Materials and Methods --- p.134 / Chapter 4.2.1 --- Extension of 5' end of EST sequence by PCR method --- p.134 / Chapter 4.2.2 --- Purification of PCR products --- p.136 / Chapter 4.2.3 --- Sequencing of Extended EST products --- p.136 / Chapter 4.3 --- Results --- p.137 / Chapter 4.3.1 --- Extension of 5' end of EST sequence by PCR method --- p.137 / Chapter 4.3.2 --- Sequencing of Extended EST products --- p.137 / Chapter 4.4 --- Discussions and Conclusions --- p.147 / Chapter Chapter 5. --- General Discussions --- p.151 / Appendix I --- p.156 / Reference --- p.166

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2015. / Made available in DSpace on 2016-03-15T04:02:22Z (GMT). No. of bitstreams: 1 334747.pdf: 9033118 bytes, checksum: 027254caae7a74df7d45fa337ad31361 (MD5) Previous issue date: 2015 / O mercado de cogumelos comestíveis se expande rapidamente, demandando um aumento na sua produção. Para isso, é fundamental o fornecimento de inoculantes de qualidade, que na falta representa um gargalo importante na produção. Os grãos de cereais utilizados tradicionalmente na produção de inoculantes, fornecem nutrientes para o crescimento miceliano do fungo e o prepara para a rápida colonização do substrato. Esses são produzidos usando-se resíduos agrícolas, reciclando-se assim o material lignocelulósico. Alguns fungos comestíveis estão entre os únicos organismos capazes de degradar lignina, transformando-a em biomassa de valor nutricional. O objetivo deste trabalho foi desenvolver uma alternativa biotecnológica para o processo de fermentação em estado sólido, tradicionalmente utilizado na produção dos inoculantes. Um inoculante em alginato de cálcio foi desenvolvido utilizando o processo de fermentação submersa com aditivos nutrientes a fim de simular as condições do inoculante em grãos, induzindo a produção de enzimas lignocelulolíticas (peroxidases e ß-glicosidases). Foi possível cultivar Lentinula edodes e Pleurotus ostreatus var. florida em condições submersas com aeração em frascos estáticos utilizando farinha de trigo integral e serragem de eucalipto como aditivos e a biomassa obtida foi encapsulada em alginato de cálcio. Os inoculantes em alginato aditivados de L. edodes colonizaram o substrato mais rapidamente do que o inoculante em alginato não aditivado, mas não foram observadas diferenças de colonização entre os inoculantes em alginato de P. ostreatus var. florida. A viabilidade dos inoculantes em alginato foi mantida em 100 % por pelo menos seis meses, ultrapassando em quatro meses a viabilidade dos inoculantes tradicionais. Um biorreator airlift foi utilizado com sucesso no cultivo de P. ostreatus var. florida utilizando farelo de trigo como aditivo. Uma grande quantidade de biomassa foi obtida, assim como uma pequena produção de exopolissacarídeos. Atividade para as duas enzimas estudadas foi detectada. Este trabalho possibilitou a produção de um inoculante em alginato de cálcio de qualidade em menor tempo que o inoculante tradicional, com alta viabilidade, utilizando aditivos para preparar o metabolismo fúngico e com grande potencial para produção e uso comercial. O uso de um biorreator airlift se mostrou eficiente na produção de quantidades desejáveis de biomassa para a produção de inoculante.<br> / Abstract : The edible mushroom market is expanding rapidly, demanding an increased production. Ergo, it is necessary a regular supply of high quality grain spawn, making it the bottleneck of the process. The cereal grains used in spawn making provide nutrients for mycelium growth and prepare the fungus for the rapid colonization of the substrate. Mushrooms are grown on agro industrial residues, thus recycling the lignocellulosic materials. Some edible mushrooms are amongst the only organisms capable of degrading lignin, transforming this recalcitrant material in a valuable biomass. The aim of this work was to develop a biotechnological alternative to the solid-state fermentation, traditionally used to produce spawn. An alginate-based inoculant was developed using submerged fermentation using additives so to mimic the conditions in the grain spawn, inducing the fungi to produce lignocellulolytic enzymes, namely peroxidases and ß-glucosidases. It was possible to grow both Lentinula edodes and Pleurotus ostreatus var. florida in submerged conditions provided with aeration in static flasks using whole-wheat flour and eucalypt sawdust as additives. The biomass obtained was encapsulated in calcium alginate. The alginate-based inoculants were capable of colonizing the substrate for mushroom production. L. edodes additivated alginate inoculants colonized the substrate faster than the non-additivated alginate inoculant, but no differences in colonization were observed between P. ostreatus var. florida alginate inoculants. The inoculants maintained a 100 % viability for at least six months, surpassing in four months that for the traditional grain spawn. An airlift bioreactor was used to successfully cultivate P. ostreatus var. florida using wheat bran as additive. A high yield of biomass was obtained as well as a small production of exopolysaccharides. Activity for both enzymes studied was detected. This work made possible to produce an alginate inoculant of quality in less time than the grain spawn, with high viability, using additives to prepare the fungus metabolism with great potential for production and commercial use. The use of an airlift bioreactor proved to be efficient in producing desirable amounts of biomass for inoculant production.

Orientador: Jose Gilberto Jardine / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-23T08:39:14Z (GMT). No. of bitstreams: 1 Rodrigues_RobertaBelandrino_M.pdf: 4230796 bytes, checksum: 8fa9785a293103c7cf89e36fd0aaf70d (MD5) Previous issue date: 1998 / Resumo: O objetivo deste trabalho foi desenvolver uma tecnologia para obtenção de um concentrado natural de aroma e sabor de cogumelo shiitake (Lentinula edodes Sing), utilizando as tecnologias de ultrafiltração e osmose inversa conjugadas. Foi realizada a extração contínua em contracorrente dos compostos aquossolúveis, entre eles, os responsáveis pelo aroma e sabor característico do cogumelo shiitake e posterior separação desses compostos por diferença de peso molecular utilizando a tecnologia de ultrafiltração. O processo de ultrafiltração foi realizado em módulo Lab Unit M-20 DOS, utilizando membranas de polissulfona com peso molecular de corte de 40.000 daltons (GR 61 PP), a pressão transmembrânica de 5 bar. O permeado da ultrafiltração foi concentrado pelo processo de osmose inversa. Este foi realizado no mesmo módulo da ultrafiltração, utilizando membranas de filme composto (HR95), a pressão transmembrânica de 40 bar. Esta tecnologia produziu um concentrado final com 10° brix, de aroma característico intenso e de grande aceitabilidade através da análise sensorial. Os resultados das determinações químicas e físicas, em particular a cromatografia gasosa, demonstraram que não houve perda dos compostos voláteis durante o processo de ultrafiltração e osmose inversa. Pode-se concluir que é possível a preparação de concentrado de sabor e aroma de cogumelo shiitake a partir da extração aquosa em contra corrente, e concentração pelo processo conjugado de ultrafiltração e osmose inversa / Abstract: The objective of this work was to develop the technology to obtain a natural concentrate of the aroma and taste of Shiitake mushroom (Lentinula edodes Sing.), using conjugated ultrafiltration and reverse osmosis technologies. A continuous counter-current extraction of the water soluble compounds was carried out, which among included those responsible for the characteristic aroma and taste of the Shiitake mushroom, and subsequently, the compounds were separated by molecular weight using ultrafiltration. The process of ultrafiltration was effected using a Lab Unit M-20 DOS module, and polysulfone membranes with a cut-off of 20.000 daltons (GR 61 PP), and transmembrane pressure of 5 bar. The permeate of the ultrafiltration was concentrated by reverse osmosis. This process was carried out in the same ultrafiltration module, using thin film composite membranes (HR 95), with a transmembrane pressure of 40 bar. This produced a final concentrate of 100 Brix, with a characteristic and strong aroma and taste, and considerable acceptability as measured by sensory evaluation. The results of the chemical and physical determinations, especially, gas chromatography, showed that there was no loss of volatile compounds as a result of the ultrafiltration and reverse osmosis processes. It was concluded that it is possible to prepare an aroma and taste concentrate of Shiitake mushroom by aqueous counter-current extraction, followed by concentration using a conjugated process of ultrafiltration and reverse osmosis / Mestrado / Mestre em Tecnologia de Alimentos

Orientador: Fábio Minoru Yamaji / Resumo: Os atuais padrões mundiais de consumo energético estão baseados na utilização de combustíveis fósseis, os quais, além de serem não renováveis, geram diversos problemas ambientais. Nos últimos anos houve um aumento na busca por fontes energéticas alternativas e mais sustentáveis, como as biomassas provenientes de resíduos agroindustriais, as quais podem apresentar grande potencial energético por área e baixos impactos ambientais. Com o intuito de minimizar os problemas ambientais relacionados à geração e descarte de resíduos provenientes das atividades de cultivo de cogumelos, esse estudo teve como objetivo avaliar o potencial bioenergético do resíduo de cultivo de shiitake (RCS) seco e por meio de sua conversão termoquímica pelo processo de pirólise em três temperaturas diferentes (350°C, 450°C e 550°C). Mediante às análises, o biocarvão de 350°C (BC350) foi considerado com o maior potencial energético devido à menor quantidade de cinzas e maiores quantidades de materiais voláteis, carbono fixo, poder calorífico, rendimento gravimétrico e fator de rendimento gravimétrico. As análises químicas e físicas revelaram que o RCS seco apresenta uma natureza bastante complexa, característica comumente encontrada em materiais de origem lignocelulósica. As análises termogravimétricas foram realizadas em três rampas de aquecimento, 10°C/min, 15°C/min e 20°C/min, em atmosfera oxidante e inerte. Foram identificadas etapas de conversão referentes à perda de água e degradação de hemicelulo... (Resumo completo, clicar acesso eletrônico abaixo) / Current global patterns of energy consumption are based on the use of fossil fuels, which are not only non-renewable resources, but also generate vast environmental problems. Recent years have witnessed an increasing search for alternative, more sustainable, energy sources, such as biomasses originated from agroindustrial residues, a material that may present great energetic potential per area and minor environmental impacts. Aiming at minimizing environmental impacts related with the generation and discard of mushroom cultivation residues, the present study intended to evaluate the bioenergetic potential of the spent shiitake substrate (SSS), both in its dry form and after its thermochemical conversion by a pyrolysis process in 3 different temperatures (350°C, 450°C e 550°C). Further analyses founded the consideration that the 350°C biochar (BC350) was that with the greatest energetic potential, due to its reduced ash amounts and higher volatile amounts, fixed carbon, heat value, gravimetric yield and gravimetric yield factor. Chemical and physical analyses revealed that the dry SSS possesses a highly complex nature, characteristics commonly found in lignocellulosic materials. Thermogravimetric analyses were performed in 3 heating rates, 10°C/min, 15°C/min and 20°C/min in oxidizing and inert atmospheres. Conversion steps related with water loss and degradation of hemicellulose, cellulose and lignin have been identified. The kinetic study was carried out by non-isothermal and non-isoconversional Kissinger method. The activation energies obtained were of 110.3 kJ mol-1 and 136 kJ mol-1 for the SSS and BC350, respectively and are within the range found for other biomasses. The SSS presented features that allow the conclusion that such residue possesses great potential for bioenergy generation applications, potential which may be improved by means ... (Complete abstract click electronic acess below) / Mestre

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