Transgenic crops have been used in agriculture to control insect pests. Although the most common strategy is to produce an insecticidal Bacillus thuringiensis delta-endotoxin from an introduced gene, the use of other insecticidal proteins, stacked genes or fusion proteins is also important for preventing development of pest resistance. Earlier it was found that the seeds of pea plants expressing a gene for alpha-amylase inhibitors (aAI) from the common bean (Phaseolus vulgaris) were protected from damage by bruchids through inhibiting the insect alpha-amylase enzymes. We have now designed a strategy to improve aAI molecules to confer higher and specific activity against target insects. Our studies focused on the phage-display screening of a-amylase inhibitors by using a 10⁷ recombinant a-amylase inhibitors library produced by in vitro recombination (DNA shuffling) of homologous aAI1 and aAI2 genes. Several different mutants were selected using affinity for a-amylase enzymes from both Zabrotes subfasciatus and Anthonomus grandis insect pests. The different mutants were sub-cloned in plant expression vectors and introduced into Arabidopsis thaliana plants. This represents an important step to guarantee the correct folding needed for the activity of these inhibitor variants. The transformed plants were used to evaluate, in vitro and in vivo, toxicity against those insect pests by bioassay. Our data indicate variability among the toxicity of the recombinant proteins. We interpret the results as showing that the novel aAI genes have potential for use in gene constructions, such as pyramiding of genes encoding for different insecticidal molecules, as an alternative method to preventing the development of pest resistance, and for conferring greater levels of pest control. In additional, this study documents that DNA shuffling and phage-display combined techniques are important strategies to generate large number of mutants in vitro and to identify new molecules with improved activities towards insects of different orders.

Supported by Embrapa, CNPq. CAPES