Estratégias para uso de genes inseticidas em Bacillus thuringiensis: expressão heteróloga e a análises genômicas

Abstract

The demand for more sustainable methods of agricultural pest control has driven the development of bioinsecticides based on Bacillus thuringiensis (Bt), a bacterium capable of producing highly specific and biodegradable insecticidal proteins. However, the narrow spectrum of action of some toxins and the emergence of resistant insect populations highlight the need to expand the diversity of available insecticidal proteins. This dissertation evaluated complementary strategies to enhance the use of insecticidal genes in Bt by combining genomic analyses of strains for the discovery of new genes with genetic transformation aimed at heterologous expression of multiple toxins. Twenty-five Bt genomes available in the GenBank-NCBI database were analyzed, revealing that 52% of the strains carry known insecticidal genes, while 48% showed no identifiable genes, suggesting unexplored diversity. A total of 64 toxins were identified, mainly Cry proteins, with diverse chromosomal and plasmid distributions, highlighting promising strains such as IBL200, NB-176, BMP144, and C15 for the development of new bioinsecticides. In a second stage, genetic transformation of B. thuringiensis subsp. kurstaki HD-1 was performed using a polycistronic cassette containing the cyt2Aa, cry3Bb1, and cry9Ca1 genes, cloned into the pHT01 plasmid and propagated in Escherichia coli before transfer to Bt by electroporation. Protocols for competent cell preparation and electroporation were optimized, and gene integration was confirmed by PCR. The successful transformation of the HD-1 strain with multiple insecticidal genes represents an important step toward developing bioinsecticides with an expanded spectrum of action, supporting integrated pest management for tomato crops with reduced reliance on chemical insecticides.