微生物学报Acta Microbiologica Sinica 48(6)：733~738; 4 June 2008
基金项目: “863 计划” (2006AA10A212); 国家自然科学基金(30571252)
任羽1，刘华梅1，宋福平1，黄大昉2，张杰1*
*通讯作者。Tel: +86-10-62815921; E-mail: jzhang@ippcaas.cn
作者简介: 任羽(1979. ), 女, 北京怀柔人, 博士研究生, 主要从事苏云金芽孢杆菌杀虫晶体蛋白分子设计研究。E-mail: renyu1020@yahoo.com.cn
（1 中国农业科学院植物保护研究所 植物病虫害生物学国家重点实验室北京 100094）
（2 中国农业科学院生物技术研究所，北京100081）
摘要：苏云金芽孢杆菌杀虫晶体蛋白Cry1Ca7 对重要的农业害虫甜菜夜蛾具有较高毒力。【目的】本文的研究目的是通过定点突变的方法获得毒力发生改变的毒蛋白，为下一步研究工作提供有价值的实验材料。【方法】利用重叠引物PCR 技术对cry1Ca7 基因进行定点突变，获得了10 种突变基因，通过生物活性测定的方法确定了各突变基因表达产物对甜菜夜蛾的杀虫活性。【结果】活性降低的突变毒蛋白有G138S，T221D，T221R，N251S，439GGT440，N306R，W376F，R522E 和 R570G，其中，位于DomainⅡ内的突变的活性依次是439GGT440<N306R<W376F；位于DomainⅢ内的突变R522E< R570G，二者的活性较Cry1Ca7 也有明显降低；只有位于结构域Ⅰ中的R148G，产生了活性提高的突变毒蛋白，其毒性较Cry1Ca7 提高了6 倍，而同样位于DomainⅠ内的突变T221D<T221R<G138S<N251S，尤其是T221D 活性完全丧失。研究结果表明，在结构域Ⅰ中的突变更容易产生活性提高的突变蛋白，而结构域Ⅱ和Ⅲ较难获得毒力提高的突变蛋白。【结论】本项研究所获得的这些活性发生不同变化的蛋白为揭示Cry 蛋白的杀虫机理提供了基础材料，而活性提高的诱变基因及其表达产物将可作为新的杀虫资源，用于抗虫遗传工程菌和转基因植物的构建。
关键词：苏云金芽孢杆菌；杀虫晶体蛋白；定点突变；cry1Ca7 基因；甜菜夜蛾
中图分类号：Q933 文献标识码：A 文章编号：0001-6209 (2008) 06-0733-06
Effect of Cry1Ca7 protein modified by site-directed mutagenesis on inhibiting Spodoptera exigua Hübner
Yu Ren1, Huamei Liu1, Fuping Song1, Dafang Huang2, Jie Zhang1*
(1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China)
(2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China)
Abstract: [Objective] To obtain the mutants with different toxicity from the wild-type Cry1Ca7. [Methods] Insecticidal crystal protein Cry1Ca7 from Bacillus thuringiensis which is highly toxic to Spodoptera exigua, an important agricultural pest in China, and we mutated this toxin by over-lapping extensive PCR method in different domains to obtain 11 chimeric mutants. [Results] The results of bioassays against Spodoptera exigua neonates showed that several conserved amino acid sites were crucial to insects. The pesticidal activities of most of mutated proteins were decreased, including Glycine138 Serine, Threonine221 Aspartic acid, Threonine221 Argine, Asparagine251 Serine, 439GlycineGlycineThreonine440, Asparagine306 Argine, Tryptophan376 Phenylalanine, Argine522 Glutamic acid and Argine570 Glycine. The activity of those mutated proteins in the DomainⅡ was 439GlycineGlycineThreonine 440 < Asparagine 306 Argine < Tryptophan 376 Phenylalanine. In the DomainⅢ, the mutant Argine522 Glutamic acid < Argine 570 Glycine, their toxicities reduced distinctly compared with Cry1Ca7. The toxicities of the mutant Argine148 Glycine in DomainⅠincreased six-fold, nevertheless the activities of the mutants Glycine138 Serine、Threonine221 Argine and Asparagine251 Serine mutant reduced totally, even the mutant of Threonine221 Aspartic acid was not toxic entirely. In [Conclusion] It is relatively easier to obtain mutant with higher toxicity in DomainⅠof Cry1Ca7 protein than these in both DomainⅡ andⅢ. We can use the improved mutant genes as the potential resources to construct novel engineering bacteria and transgenic plant, meanwhile, to perform the study of interaction mechanism between insects and Cry proteins.
Keywords: Bacillus thuringiensis; insecticidal crystal protein; Site-directed mutagenesis; cry1Ca7 gene; Spodoptera exigua
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