Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease
Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease
复制标题
甜菜夜蛾中肠蛋白酶对苏云金芽孢杆菌 Vip3Aa 蛋白的蛋白水解激活
DOI：
10.1016/j.ijbiomac.2017.09.101
复制
发表时间：
2018-02-01
影响因子：
8.2
通讯作者：
Chen, Qing-Xi
中科院分区：
文献类型：
--
作者：
Zhang, Ling;Pan, Zhi-Zhen;Chen, Qing-Xi
昆虫中肠蛋白酶对Vip 3Aa的蛋白水解对于它们对靶标昆虫的毒性是必不可少的。在本研究中，Vip 3Aa的蛋白水解甜菜夜蛾中肠蛋白酶（MJ）进行了评估。胰蛋白酶被证实参与Vip 3Aa的活化，并鉴定了三个潜在的切割位点（Lys（195）、Lys（197）和Lys（198））。设计并构建了4个Vip 3Aa突变体（KKK 195, 197, 198 AAA、KK 197, 198 AA、KK 195, 198 AA和KK 195, 197 AA），它们分别由Lys（195，197，198）、Lys（197，198）、Lys（195，198）和Lys（195，197）替换为Ala。蛋白酶解试验表明，突变体KK 197198 AA、KK 195198 AA和KK 195197 AA能被胰蛋白酶或MJ加工成66 kDa的活化毒素，而突变体KKK 195197198 AAA不能被胰蛋白酶切割，对MJ不敏感。生物测定表明突变体KK 197198 AA、KK 195198 AA和KK 195197 AA对S.突变体KKK 195197198 AAA对甜菜夜蛾的LC_（50）与野生型Vip 3Aa相似，而突变体KKK 195197198 AAA对甜菜夜蛾的LC_（50）与野生型Vip 3Aa相似，突变体KKK 195197198 AAA对甜菜夜蛾的LC_（50）与野生型Vip 3Aa相似。甜菜红色素含量高于野生型。这些结果表明，MJ的蛋白水解与Vip 3Aa的杀虫活性有关。exigua.胰蛋白酶在Vip 3Aa激活毒素的形成过程中起重要作用。我们的研究特征的Vip 3Aa的蛋白水解加工，并提供了新的见解激活这种新的Bt毒素。(C)2017爱思唯尔B. V.保留所有权利。
Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys(195), Lys(197) and Lys(198)) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys(195,197,198), Lys(197,198), Lys(195,198) and Lys(195,197) with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC50 of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin. (C) 2017 Elsevier B.V. All rights reserved.