Key Laboratory of Microbial Molecular Biology of Hunan Province, College of Life Science, Hunan Normal University, Changsha, 410081, People's Republic of China.
Curr Microbiol. 2011 Feb;62(2):358-65. doi: 10.1007/s00284-010-9714-2. Epub 2010 Jul 29.
Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16-β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.
苏云金芽孢杆菌（Bt）产生的 Cry1Ac 杀虫晶体蛋白已成为控制鳞翅目昆虫的重要天然生物制剂。在本研究中，采用易错 PCR、交错延伸过程（StEP）改组与 Red/ET 同源重组相结合的方法，对苏云金芽孢杆菌 4.0718 的 cry1Ac 毒素基因进行了修饰，以研究 δ-内毒素 Cry1Ac 的杀虫活性。通过昆虫生物测定筛选出的 Cry1Ac 毒素突变体（命名为 T524N）对甜菜夜蛾幼虫表现出增强的杀虫活性，而对棉铃虫幼虫的原始杀虫活性仍得以保留。与原始 Cry1Ac 毒素相比，突变毒素 T524N 在位置 524 处发生了一个氨基酸取代，它可以在无结晶菌株 Cry-B 中积累，并形成比原始 Cry1Ac 毒素更多但稍小的双锥晶体。突变和原始 Cry1Ac 蛋白理论分子模型的分析表明，突变 T524N 位于 Cry1Ac 毒素结构域 III 的β16-β17 环中，发生在第四保守区，该区域富含精氨酸，形成一个高度疏水性表面，涉及与受体分子的相互作用。本研究首次表明，单个突变 T524N 在杀虫活性中起着重要作用。这一发现为 Cry1Ac 毒素结构域 III 在杀虫活性中的结构功能提供了生物学证据，这可能会深入了解有毒蛋白与受体大分子的相互作用。
