Estudos Bioquímicos de proteases extracelulares e expressão de seus genes de isolados de Trichoderma spp. após crescimento em parede celular de Rhizoctonia solani

Abstract

Trichodermaspecies have been studied mainly recognized for its role in biological control of many pathogens, and proteases, glucanases and chitinases are closely involved in this process. This work aimed to study the biochemistry of extracellular proteases from Trichoderma virens (301/01), Trichoderma harzianum (494/02 and ALL 42) and Trichoderma asperellum (T00)secreted when exposed to cell wall of Rhizoctonia solani(PCRS) and analyzed the expression genes of proteases, glucanases and chitinases front PCRS to a control induction into glucose. The specific activity of proteases T harzianumwas low compared to T asperellum and T virens, showing the high capacity of mycoparasitism of this species, since the confrontation in petri dishes all isolates were effective against R. solani. Was used with bacitracin affinity chromatography for obtaining extracts of proteases and different profiles observed for isolates and pH 5.0 and 8.0. The influence of inhibitors and detergents on enzyme activity in the extract of proteases, PMSF and EDTA extracts inhibited by approximately 20%, Triton X-100 and SDS inhibited by 25%. The action of monovalent cations and divalent was evaluated revealing an increase in the enzyme activity by up to 40% and down 20%. We evaluated the affinity of the extracts on two substrates of proteases, albumin, and azocasein. The affinity for albumin was low compared to azocazeína for all isolates. In relation to temperature and pH optima found that the extracts showed optimum temperature at 50 ° C and optimum pH 8.0Toevaluate the profile of the isolated protease was performed on polyacrylamide gel chromatography SDS-PAGE. To evaluate the protease activity polyacrylamide gel electrophoresis, non-denaturing conditions were (zymogram). The analysis of gene expression of proteases was examined by RT-PCR in the isolated 494/02 42 ALL and in intervals of 6, 12, 24 and 48 hours, showing a profile varying in the expression of trypsin-like proteases, aspartate protease and serine protease. To analyze the relationship of gene expression of proteases and to the expression of genes of other enzymes involved in mycoparasitism. Also, we analyzed the gene expression of β 1-3 endoglicanase, 42 kD chitinase and N-acetylglucosaminidase (Nagase) against PCRS . The isolated T. harzianum (ALL 42)maintained higher expression profile aspartate proteases and β 1-3 endoglicanase, 42 kD chitinase and N-acetylglucosaminidase (Nagase). The Pearson coefficient was calculated for correlation between glucanases, chitinases and proteases, positive correlations were observed for isolated ALL 42 to aspartate protease and β 1-3 endoglicanase, 42 kD chitinase and N-acetylglucosaminidase (Nagase), whereas 494/02 showed no positive correlation to aspartate protease.