苏云金芽孢杆菌Cry1Ca7蛋白定点突变对甜菜夜蛾杀虫活性的影响

Effect of Cry1Ca7 protein modified by site-directed mutagenesis on inhibiting Spodoptera exigua Hbner

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。

[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 Glycine^138 Serine, Threonine^221 Aspartic acid, Threonine^221 Argine, Asparagine^251 Serine, ^439GlycineGlycineThreonine^440, Asparagine^306 Argine, Tryptophan^376 Phenylalanine, Argine^522 Glutamic acid and Argine^570 Glycine. The activity of those mutated proteins in the Domain Ⅱ was ^439GlycineGlycineThreonine ^440〈 Asparagine ^306 Argine 〈 Tryptophan ^376 Phenylalanine. In the Domain Ⅲ, the mutant Argine^522 Glutamic acid 〈 Argine ^570 Glycine, their toxicities reduced distinctly compared with Cry1Ca7. The toxicities of the mutant Argine^148 Glycine in Domain I increased six-fold, nevertheless the activities of the mutants Glycine^138 Serine, Threonine^221 Argine and Asparagine^251 Serine mutant reduced totally, even the mutant of Threonine^221 Aspartic acid was not toxic entirely. In [Conclusion] It is relatively easier to obtain mutant with higher toxicity in Domain Ⅰ of Cry1Ca7 protein than these in both Domain Ⅱ and Ⅲ. 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.