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Study of the biological function of the Alternative Oxidase (AOX) from Moniliophthora pernciosa (witches' broom fungus) using Saccharomyces cerevisiae.

Grant number: 11/05156-3
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): March 01, 2012
Effective date (End): November 30, 2013
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:Gisele Monteiro
Grantee:Gabriel Moretti de Almeida
Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:09/01303-1 - Characterization of unknown function ORFs involved in Saccharomyces cerevisiae antioxidant response, AP.JP

Abstract

Moniliophthora perniciosa, causal agent of witches' broom disease of cocoa, spread rapidly, causing a collapse in the regional economy of Bahia, corresponding to a total of 90% losses in cocoa production (Gildemberg et.al, 2010). Even after application of antifungal agents based on inhibition of the main chain electron transport, M. pernicious infection continues. Several authors propose that the survival of this fungus to give the electron transport chain enzyme whose main alternative is the alternative oxidase (AOX). This is because it is known that by inhibiting the mitochondrial electron transport is a high generation of reactive oxygen species (ROS). AOXp oxidase is a terminal capable of performing the mitochondrial electron transport by catalyzing the reduction of oxygen to water by diverting the electrons in the respiratory chain would be used for the synthesis of ATP. Thus, AOX-expressing cells produce less ATP, but alleviate oxidative stress. This study aims to test this hypothesis. To do this we will express the gene (AOX-Mp) in Saccharomyces cerevisiae to conduct studies of alternative pathway in preventing oxidative stress, trying to understand the molecular mechanisms involved in resistance to M. harmful to commercial fungicides. We will construct the expression vector in yeast by inserting the gene AOX and made measurements of growth rate and cell mass formation, the generation of ROS and quantify mitochondrial ATP from yeast cells expressing AOX-Mp. We will carry out further feasibility testing in the presence of the main respiratory chain inhibitors Antimycin A and azoxystrobin, the specific AOX inhibitor SHAM in the presence of hydrogen peroxide. Further isolated, mitochondria that express AOX in order to test the functionality of respiratory complexes.

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