ERI de Biotecnología y Biomedicina (BIOTECMED), Departamento de Genética, Universitat de València, 46100, Burjassot, Spain.
Bayer CropScience N.V., Ghent, Belgium.
Sci Rep. 2018 May 15;8(1):7539. doi: 10.1038/s41598-018-25346-3.
Vip3 vegetative insecticidal proteins from Bacillus thuringiensis are an important tool for crop protection against caterpillar pests in IPM strategies. While there is wide consensus on their general mode of action, the details of their mode of action are not completely elucidated and their structure remains unknown. In this work the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. From the 558 residue substitutions, 19 impaired protein expression and other 19 substitutions severely compromised the insecticidal activity against Spodoptera frugiperda. The latter 19 substitutions mainly clustered in two regions of the protein sequence (amino acids 167-272 and amino acids 689-741). Most of these substitutions also decreased the activity to Agrotis segetum. The characterisation of the sensitivity to proteases of the mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of most selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.
苏云金芽孢杆菌的 VIP3 植物源杀虫蛋白是害虫综合治理（IPM）策略中防治鳞翅目害虫的重要工具。虽然人们对其一般作用模式达成了广泛共识，但对其作用模式的细节仍未完全阐明，其结构仍然未知。在这项工作中，对 VIP3Af1 蛋白的 788 个氨基酸中的 558 个进行了丙氨酸扫描技术。在 558 个取代物中，有 19 个蛋白表达受损，另外 19 个取代物严重降低了对玉米螟的杀虫活性。后 19 个取代物主要集中在蛋白质序列的两个区域（氨基酸 167-272 和氨基酸 689-741）。这些取代物中的大多数也降低了对草地贪夜蛾的活性。对显示杀虫活性降低的突变蛋白的蛋白酶敏感性的特性研究表明，通过 SDS-PAGE 评估显示出 6 种不同的条带模式。对大多数选定突变体的固有荧光的研究仅显示出发射峰的微小位移，可能表明三级结构只有微小变化。首次提出了 VIP3Af1 的计算机模拟 3D 结构。
