ANÁLISE FUNCIONAL DO GENE CODIFICADOR DE UMA N-ACETIL-Β-DGLICOSAMINIDASE (NAG1) DE TRICHODERMA HARZIANUM

Abstract

The chitinolytic system of Trichoderma spp. is composed by the enzymes chitinases and Nacetyl-β-D-glucosaminidases (NAGases). These may be relate to processes of fungal cell wall remodeling and phytopathogenic chitin degradation during biocontrol. In this work was performed the cloning of the gene encoding an N-acetyl-β-D-glucosaminidase (NAG1) and its overexpression in Trichoderma harzianum. For this, an overexpression vector containing the gene (NAG1) was construct under the control of a constitutive promoter and was performed the genetic transformation of T. harzianum via biobalistic. Three transformants were characterize in molecular analyses and enzymatic activity, confirming the insertion of the vector and the constitutive expression of the gene. Growth bioassays revealed that NAG1 overexpression in T. harzianum reduced the growth of the transformants and antagonistic activity of the phytopathogens Rhizoctonia solani and Sclerotinia sclerotirum. Fluorescence microscopy analysis revealed that transformants containing single copy of the gene showed thickening of the cell wall and decreased hyphae width. Furthermore, the expression analysis by real-time PCR revealed that the transformants exhibited a greater expression of mycoparasitism genes, both under control and in phytopathogenic conditions. Thus, it was possible to observe that the overexpression of NAG1 interfered the T. harzianum cellular wall remodeling process and in phytopathogen antagonist activity.