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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2014.tde-25082020-132448
Document
Author
Full name
Amanda Cristina Campos Antoniêto
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2014
Supervisor
Committee
Silva, Roberto do Nascimento (President)
Goldman, Gustavo Henrique
Noronha, Eliane Ferreira
Title in Portuguese
Análise da regulação global da expressão gênica de repressão catabólica durante a formação de celulases pelo fungo Trichoderma reesei (Hypocrea jecorina)
Keywords in Portuguese
CRE1
Repressão catabólica
RNA-seq
Trichoderma reesei
Abstract in Portuguese
O ascomiceto Hypocrea jecorina (anamorfo do Trichoderma reesei) é um dos fungos celulolíticos mais bem estudados e amplamente utilizado na indústria biotecnológica, como na produção do etanol de segunda geração. O mecanismo de repressão catabólica adotado por T. reesei é mediado pelo fator de transcrição CRE1 e consiste na repressão de genes relacionados à produção de celulases quando uma fonte de carbono prontamente disponível está presente no meio. Esse trabalho tem como objetivo contribuir para o entendimento do mecanismo de repressão catabólica durante a formação de celulases, através da comparação entre a linhagem mutante de T. reesei Δcre1 com seu parental QM9414. Para isso, bibliotecas de cDNA das linhagens Δcre1 e QM9414 crescidas em 1% de celulose, 1mM de soforose e 2% de glicose foram sequenciadas por RNA-seq pela empresa LGC Genomics GmbH em Berlim/Alemanha, utilizando-se o equipamento Illumina/HiSeq2000. Os resultados do sequenciamento foram analisados pelo software de alinhamento Bowtie e pelo pacote DESeq, que faz a análise de expressão diferencial dos genes. Foram obtidas no total 264 milhões de reads que, ao serem analisadas, mostraram que 815 genes foram diferencialmente expressos no Δcre1 em relação ao QM9414 na condição celulose, 2368 em soforose e 697 em glicose, em um total de 9129 genes do genoma de T. reesei. A maioria dos genes que estavam up ou down regulados no mutante pertenciam a categorias do Gene Ontology descritas como processos metabólicos, membrana, atividade oxidorredutase, metabolismo de carboidratos e transporte. Enzimas celulolíticas, genes relacionados com o transporte de substâncias e outros fatores de transcrição foram alvo de repressão metabólica mediada por CRE1, de modo carbono-dependente. A validação dos genes diferencialmente expressos por qRT-PCR mostrou uma alta correlação entre as duas técnicas, que pode ser demonstrada por um alto coeficiente linear de Pearson (r2 = 0.94). Espera-se com esses resultados contribuir para um melhor entendimento do mecanismo de repressão catabólica em T. reesei, potencializando a aplicação desse fungo nos diversos setores biotecnológicos em que é utilizado.
Title in English
Analysis of global regulation of gene expression of carbon catabolite repression during the formation of cellulases by Trichoderma reesei (Hypocrea jecorina)
Keywords in English
Carbon catabolite repression
CRE1
RNA-seq
Trichoderma reesei
Abstract in English
The ascomycete Hypocrea jecorina (anamorph of Trichoderma reesei) is one of the most well studied cellulolytic fungi and widely used in the biotechnology industry, as in the production of second generation ethanol, pulp and paper industries, textile treatments and processing of animal feed. The carbon catabolite repression mechanism adopted by T. reesei is mediated by the transcription factor CRE1 and consists in the repression of genes related to the production of cellulase when a readily available carbon source is present in the medium. This study aims to contribute to understanding the mechanism of carbon catabolite repression during the formation of cellulases, by comparing the mutant strain of T. reesei Δcre1 with its parental, QM9414. For this, the cDNA libraries of strains QM9414 and Δcre1 grown in 1% cellulose, 1 mM sophorose and 2% glucose were sequenced by RNA-seq by LGC Genomics GmbH in Berlin/Germany, using the equipment Illumina/HiSeq2000. The results of the sequencing were analyzed by the alignment software Bowtie and DEseq package, which makes the analysis of differentially expressed genes. We obtained a total of 264 million of reads that, when analyzed, showed 815 genes differentially expressed in Δcre1 in relation to the parental QM9414 on cellulose, 2368 on sophorose and 697 on glucose, for a total of 9129 genes in the genome of T. reesei. Most of genes that were up- or down-regulated in the mutant belonged to Gene Ontology categories described as metabolic processes, membrane, oxidoreductase activity, carbohydrate metabolism and transport. Cellulolytic enzymes, genes related to the transport of substances and other transcription factors were targeted for carbon catabolite repression mediated by CRE1, in a carbon source-dependent manner. Validation of differentially expressed genes by qRT-PCR showed a high correlation between the two techniques, which can be demonstrated by a high linear coefficient of Pearson (r2 = 0.94). It is hoped that these results contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, enhancing the application of this fungus in several biotechnology sectors in which is used.
 
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Publishing Date
2020-10-19
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • C.C.ANTONIETO, A., et al. Decoding the black box of cellulase formation in hypocrea jecorina by RNA-SEQ analysis. In FEMS 2013 5th Congress of european microbiologists, Leipzig, Germany, 2013. FEMS 2013., 2013. Abstract.
  • C.C.ANTONIETO, A., et al. The transcriptional factors XYR1 and CRE1 regulate the expression of Cellulolytic and Xylanolytic genes at carbon source dependet-manner in Hipocrea jecorina (Trichoderma reesei) . In 27TH Fungal Genetics Conference, 2013. 27TH Fungal Genetics Conference., 2013. Abstract.
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