Single cysteine substitution in Bacillus thuringiensis Cry7Ba1 improves the crystal solubility and produces toxicity to Plutella xylostella larvae
Single cysteine substitution in Bacillus thuringiensis Cry7Ba1 improves the crystal solubility and produces toxicity to Plutella xylostella larvae
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苏云金芽孢杆菌 Cry7Ba1 中的单个半胱氨酸取代可提高晶体溶解度并对小菜蛾幼虫产生毒性
DOI：
10.1111/j.1462-2920.2011.02557.x
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发表时间：
2011-10-01
影响因子：
5.1
通讯作者：
Sun, Ming
中科院分区：
文献类型：
--
作者：
Peng, Donghai;Wang, Fenshan;Sun, Ming
许多苏云金芽孢杆菌分离株没有表现出对昆虫的毒性。在这项研究中，从“非杀虫性”苏云金芽孢杆菌菌株 YBT-978 中分离出一种新型正型晶体蛋白基因cry7Ba1。当晶体在 pH 12.5 下溶解后，Cry7Ba1 蛋白对小菜蛾幼虫表现出高毒性，这表明该蛋白的“非杀虫”特性是由于在正常昆虫中肠 pH 环境中不溶。 Cry7Ba1的C端一半被Cry1Ac或Cry1C蛋白取代后，重组蛋白包涵体在pH 9.5下可溶解，对小菜蛾幼虫表现出较高的毒性。该结果证明Cry7Ba1晶体的不溶性是由其C端半部的结构决定的。此外，通过用丝氨酸取代半胱氨酸残基构建了六个突变。溶解度研究表明，突变体 C697S、C834S 和 C854S 的晶体可以在较低 pH 值（分别为 10.5、9.5 和 11.5）下溶解。生物测定表明，突变体 C834S 的晶体对小菜蛾幼虫有毒。我们的发现表明，位于蛋白质 C 末端一半的单个半胱氨酸残基决定了一些无毒晶体蛋白质的溶解度和毒性。这项研究提供了一种从“非杀虫”苏云金芽孢杆菌菌株中分离新型杀虫晶体蛋白基因的策略。
Many Bacillus thuringiensis isolates have no demonstrated toxicity against insects. In this study, a novel holotype crystal protein gene cry7Ba1 was isolated from a 'non-insecticidal' B. thuringiensis strain YBT-978. The Cry7Ba1 protein showed high toxicity against Plutella xylostella larvae after the crystals were dissolved at pH 12.5, suggesting that the 'non-insecticidal' properties of this protein were due to insolubility in the normal insect midgut pH environment. After the C-terminal half of Cry7Ba1 was replaced by that of Cry1Ac or Cry1C proteins, the recombinant protein inclusions could be dissolved at pH 9.5, and exhibited high toxicity against P. xylostella larvae. This result proved the insolubility of Cry7Ba1 crystal was determined by the structure of its C-terminal half. Further, six mutations were constructed by substituting cysteine residues with serine. Solubility studies showed that the crystals from mutants C697S, C834S and C854S could be dissolved at lower pH (10.5, 9.5 and 11.5 respectively). Bioassays showed that crystals from mutant C834S were toxic to P. xylostella larvae. Our discoveries suggest that a single cysteine residue located in the C-terminal half of the protein determines the solubility and toxicity of some nontoxic crystal proteins. This study provides a strategy to isolate novel insecticidal crystal protein genes from 'non-insecticidal' B. thuringiensis strains.