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Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis)

机译：提高针对棉铃象鼻虫（Anthonomus grandis）的Cry8Ka毒素活性

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Background The 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 . Results Bioassays 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. Conclusions The 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 .
机译：背景技术棉铃象鼻虫（Anthonomus grandis）在美洲，特别是在巴西是一种严重的害虫。由于棉铃象鼻虫的内生生活方式，因此使用化学或生物防治方法对棉铃象鼻虫无效。因此，使用生物技术工具生产抗虫转基因植物是减少棉铃象鼻虫对棉株的损害的重要策略。本研究的重点是鉴定对棉铃象鼻虫具有改善的毒性的新型分子。通过DNA改组和噬菌体展示筛选进行体外定向分子进化，以增强苏云金芽孢杆菌Cry8Ka1蛋白变异体的杀虫活性。结果用大孢曲霉幼虫进行的生物测定表明，筛选出的突变型Cry8Ka5毒素的LC 50 比野生型Cry8Ka1毒素高3.15倍。 Cry8Ka1和Cry8Ka5突变体的同源性建模表明，这两种蛋白都保留了典型的三结构域Cry家族结构。突变的残基大部分位于环中，似乎不太可能干扰分子稳定性。结论本研究中获得的Cry8Ka5突变体毒性的改善将导致转基因棉花事件的产生，从而具有更大的潜力控制大曲霉。

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