Caracterização funcional de um fator de transcrição hipotético no fungo Neurospora crassa

• Ano de defesa: 2015
• Autor(a) principal: Imamura, Kely Braga [UNESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Estadual Paulista (Unesp)
• Palavras-chave em Português: Genetica microbiana; Morte celular; Engenharia genetica; Neurospora crassa; Stress oxidativo; Microbial genetics
• Link de acesso: http://hdl.handle.net/11449/135929

The fungus Neurospora crassa has been widely used as a model organism for the study of some aspects of biology in eukaryotes. The sequencing of its genome has enabled functionally analyze various transcription factors and therefore, assign function to hypothetical proteins. In this study, we investigated the functional role of the ORF NCU01629 product, a transcription factor that belongs to the zinc-finger protein family without functional homologues in fungi database. In vitro analysis of DNA-protein interaction, allowed the identification of its DNA binding motif and, as a consequence, the most likely genes regulated by this transcription factor. The genes were classified by FunCat. The results revealed the involvement of the transcription factor in multiple cellular processes including the response to oxidative stress and cell death. Analyses of radial growth were performed in Petri dishes containing agents that induce different types of stress such as osmotic, thermal and oxidative. The knockout strain showed similar growth to the wild type strain when exposed to osmotic (NaCl 0,1-1,5M and sorbitol 1-1,5M), pH (4.2 and 7.8) and heat (45°C) stresses. However, growth of the knockout strain was influenced when the strain was exposed to different ROS inducing agents, such as paraquat (10 μM), menadione (50 μM), H2O2 (2mM) and farnesol (10 μM). The knockout strain showed reduced radial growth, compared to the wild-type strain, when exposed to different concentrations of paraquat and farnesol and increased resistance to H2O2 and menadione. The expression of genes related to ROS (cat-1, cat-2, cat-3, gst-1, gst-2, sod, and nox) and apoptotic genes (bax, metascaspases, and p53) were analyzed by RT-qPCR. The results showed that the transcription factor is involved in the regulation of the oxidative stress response, controlling the expression of all genes. The gene encoding glutathione-S-transferase...