Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from "Bacillus thuringiensis"

RESUMO

The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a 'cry' gene from "Bacillus thuringiensis", envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria "Gluconacetobacter diazothophicus" strain BR11281 and "Herbaspirillum seropedicae" strain BR11335 were used as models. The 'cry3A' gene was transferred by conjugation using a suicide plasmid and recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the 'cry' gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of (delta)-endotoxin by recombinant "H. seropedicae" strain was detected by dot blot while for "G. diazotrophicus" the Western-blot technique was used. In both cases, a specific antibody raised against the "B. thuringiensis" toxin was applied. The (delta)-endotoxin production showed by the "G. diazotrophicus" recombinant strain was dependent on the nitrogen fixing conditions since the 'cry3A' gene was fused to a 'nif' promoter. In the case of "H. seropedicae" the (delta)-endotoxin expression was not affected by the promoter ('rhi') used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests