Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC
Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC
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斜纹夜蛾特异性苏云金芽孢杆菌毒素 CryIC 杀虫片段的定位
DOI：
10.1007/s004380050290
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
A. Zilberstein
中科院分区：
文献类型：
--
作者：
N. Strizhov;M. Keller;Z. Konez;A. Regev;B. Sneh;Jozef Schell;Csaba Koncz;A. Zilberstein
摘要 苏云金芽孢杆菌的杀虫CryI原毒素通过昆虫中肠中的蛋白水解作用被激活。 CryI 蛋白中蛋白水解切割位点的保守性促进了转基因植物中活性毒素的表达，以获得免受各种昆虫侵害的保护。然而，迄今为止，CryIC 毒素的工程设计未能对代表全球常见害虫的斜纹夜蛾属粘虫产生适用的抗性。为了提高重组CryIC毒素的产量，我们通过比较分析三个cryIC基因建立了CryIC共有序列，并测试了截短的CryIC毒素在大肠杆菌和体外的稳定性和蛋白酶敏感性。与之前的数据相反，胰蛋白酶抗性 CryIC 核心毒素的边界被映射到氨基酸残基 I28 和 R627。截短的 CryIC 蛋白的蛋白水解表明，斜纹夜蛾中肠蛋白酶可以进一步将 CryIC 毒素的 C 末端缩短为残基 A615。然而，CryIC C 端截断为残基 L614，以及导致氨基酸替换 I610T 的突变，消除了 CryIC 毒素对北潜蝇幼虫的杀虫活性，以及其对胰蛋白酶和斜纹夜蛾中肠蛋白酶的抗性。由于携带蛋白水解加工的 N 末端的 CryIC 毒素不能在细菌中稳定表达，因此我们的数据表明，与其他 CryI 蛋白相比，需要位于氨基酸位置 1 和 627 之间的杀虫片段才能稳定生产重组 CryIC 毒素。
Abstract Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI poteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.
DOI：
10.1073/pnas.90.19.9041
发表时间：
1993-10-01
影响因子：
11.1
作者：
CHEN, XJ;LEE, MK;DEAN, DH
通讯作者：
DEAN, DH