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 Hübner

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

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 DomainⅡandⅢ. 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.