Employing in vitro directed molecular evolution for the selection of alpha-amylase variant inhibitors with activity toward cotton boll weevil enzyme

摘要

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of alpha-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, alpha-amylase inhibitors (alpha-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10~8 alpha-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil a-amylases. Twenty-six variant sequences were cloned into plant expressionvectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent alpha-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, didnot inhibit the A. grandis alpha-amylases, three alpha-AI mutants, named alpha-AlC3, alpha-AIAl 1 and alpha-AIG4 revealed high inhibitory activities against A grandis a-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new a-AI variant genes for cotton boll weevil control.