Abstract
BackgroundThe cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis.ResultsBioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability.ConclusionsThe improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.
Highlights
The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, in Brazil
None of the commercially available genetically modified (GM) cotton events are effective against the cotton boll weevil, which is the most harmful cotton insect-pest in Latin America
The DNA shuffling product was visualised as a single band of around 2000 bp that represented a population of cry8Ka1 variant genes (Figure 1)
Summary
The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, in Brazil. Since 1996, several insectresistant GM cotton events have been used commercially throughout the world, including Bollgard® (expressing the cry1Ac protein) and Bollgard II® (expressing the cry1Ac + cry2Ab proteins) from Monsanto, Widestrike® (expressing the cry1Ac + cry1F proteins) from Dow Agrosciences and VipCot® (expressing Vip3A protein) from Syngenta/Deltapines This technology has been used to control lepidopterans, allowing an increase in productivity, reductions in production costs, human intoxication and environmental damage due to a reduction in chemical pesticide application [9]. In the intracellular signalling model [15], the binding of toxins to the cadherin-like receptor triggers an Mg2+-dependent cAMP signalling pathway that promotes cell death. In both models, the affinity for cadherin receptors has been reported as the main step that determines specificity. Molecular strategies that involve structural and biochemical studies of Cry toxins, as well as the isolation and characterisation of new cry genes, are necessary to help elucidate the mechanisms of action of Cry toxins and to select molecules that have the potential for improved toxicity and specificity [14]
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