Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests
Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests
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DOI：
10.3390/toxins12020099
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发表时间：
2020-02-01
期刊：
影响因子：
4.2
通讯作者：
Ferre, Juan
中科院分区：
文献类型：
--
作者：
Gomis-Cebolla, Joaquin;Ferreira dos Santos, Rafael;Ferre, Juan
细菌苏云金芽孢杆菌在营养生长阶段产生杀虫Vip 3蛋白，具有对抗几种鳞翅目害虫的活性。迄今为止，已经基于序列同一性鉴定了三种不同的Vip 3蛋白家族：Vip 3A、Vip 3B和Vip 3C。在这项研究中，我们报告了嵌合体的构建Vip 3Aa和Vip 3Ca，两个蛋白质对鳞翅目害虫具有显着的特异性差异之间的交换结构域。我们发现，某些结构域组合使蛋白质不溶或易于被胰蛋白酶降解，胰蛋白酶是最丰富的昆虫肠道蛋白酶。测试了可溶性和胰蛋白酶稳定的嵌合体，沿着亲本蛋白质Vip 3Aa和Vip 3Ca，对抗来自不同大陆的鳞翅目害虫：甜菜夜蛾、草地贪夜蛾、草地贪夜蛾、棉铃虫、甘蓝夜蛾、黎豆夜蛾和小菜蛾。Nt结构域（188个N-末端氨基酸）的交换对所得嵌合蛋白对除S. frugiperda，其中与亲本Vip 3Ca相比，具有来自Vip 3Aa的Nt结构域和来自Vip 3Ca的蛋白质的其余部分的嵌合体显示出毒性的显著增加。具有来自Vip 3Aa的C-末端结构域（从Vip 3Aa的氨基酸510到Ct）与Vip 3Ca的中心结构域（基于Vip 3Aa序列的氨基酸189-509）的嵌合体产生不能溶解的蛋白质。最后，包括Vip 3Ca的Ct结构域和Vip 3Aa的Nt和中央结构域的嵌合体是不稳定的。重要的是，对Vip 3Aa耐受但对Vip 3Ca敏感的昆虫物种，如Ostrinia damacalis，也对保持来自Vip 3Ca的Ct结构域的嵌合体敏感，这与蛋白质的Ct区域是赋予Vip 3蛋白特异性的区域的假设一致。
The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Helicoverpa armigera, Mamestra brassicae, Anticarsia gemmatalis, and Ostrinia furnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S. frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189-509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostrinia furnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.