Abstract

GONGORA B, CARMENZA E. Transformation oiBeauuería bassiana strain Bb9112 with the genes from the green fluorescent protein and the proteasa pr1A of Metarhizium anisopliae. Rev. Colomb. Entomol. [online]. 2004, vol.30, n.1, pp.15-21. ISSN 0120-0488.

In order to produce an improved strain of the entomopathogen Beauveria bassiana, fungus transformation system was developed based on resistance to the herbicide glufosinate ammonium, conferred by the bar gene. B. bassiana strain Bb 9112, characterized by its resistance to UV light, was transformed with the plasmid pBarGPE1, previously cloned with the marker gene coding for green fluorescent protein (GFP). Transformed colonies were selected in a mínimal médium containing 25 µgml of the herbicide glucosinate ammonium. The expression of the protein GFF in the transformed protoplasts and the mycelium regenerated from trióse protoplasts was confirmed by UV light microscopy. Pathogenicity tests indicated no significant differences in percent pathogenicity between the transformed and nontransformed strains. In order to increase the pathogenicity of Bb9112 against the coffee berry borer, it was transformed with the plasmid pBarGPEl-pr1A containing a subtilisin - protease gene (pr1A) isolated from Metarhizium anisopliae. In the transgenic strain the presence of the pr1A gene was identified by PCR. The expression of the protein was confirmed by isoelectrofocus and enzymatic activity.

Keywords : Entomopathogen; Marker gene; Selection gene; Pathogeniciry; Coffee berry borer.