Effects Of Site-Directed Mutagenesis Ofβ19-β20Loop Of Cry1Ac From Bacillus Thuringiensis On The Structure And Function

The crystals of Bacillus thuringiensis are widely used as an agricultural pest insecticide. These crystal proteins are swallowed by insects and act with different recepoters sequentially in the midgut. After a series of complex mechanisms, holes have formed on the epithelial cells and thus destruct the osmotic pressure of insects. Finally, the insects will die of cracking.In this study, the oligonucleotide-induced circular mutation technology has been used. The13amino acids of crystal protein of Bacillus thuringiensis Cry1Ac domain Ⅲ β19-β20loop were replaced with alanine, and constructed13single point mutants.These mutants were cultured72h in G-Tris-medium and the crystals were observed with a microscope. The protein expression was detected by SDS-PAGE. Most of strains were able to form the typical diamond crystal, except for T557A, L564A, D568A, F569A, and cry-B. Most of strains emerged in130KDa protein bands, in addition to T557A, L564A, F569A, and cry-B. The strange thing was, although the D568A couldn’t form a diamond crystal, but emerged in130KDa target protein band.Mutant and wild-type toxins were fed to newly hatched larvae of Heliothis armigera and Spodoptera exigua but the results showed a great difference. The Heliothis armigera bioassay results showed that, the T559A virulence increased by1.86times, P555A, S560A, L561A virulence were increased by12%,31%and64%. T557A, L564A, F569A, lost more than80%of the virulence, the relative wild-type virulence of the rest of the mutants were reduced to varying degrees but still significantly inhibited the growth of Heliothis armigera. Spodoptera exigua bioassay results showed that T559A relative toxicity to the wild type strain increased2.7times and the toxicity of S560A and L561A increased37%and56%respectively. Toxicity of the rest mutants were of a serious decline, and D562A, N563A, D568A, F569A completely lost their virulence.In order to further understand the relationship between protein function and structure, the T559A crystal protein whose toxicity improved the most obviously, and wild-type crystal protein were selected to be observed under an atomic force microscope. We found the T559A crystals were slightly smaller, but contours were much clearer. The edges and corners were very distinct and three-dimensional feeling was stronger than the wild type. This morphological change might mean that the mutation of this site was favorable on the crystal folding, and was contributed to the improvement of toxicity. Treat T559A, S560A, L561A and wild-type crystal proteins with trypsin separately, and we found that the three mutants had increased sensitivity to trypsin. The mutant crystal could be degraded into65KDa active fragments in a shorter time than the wild-type crystal.This paper was the first time to study the longest loop of the Cry1Ac crystal protein on domain Ⅲ. We found some key sites that affectted crystal formation and toxicity changes. The results clarified the function of domain Ⅲ loop structure and they provided us new ideas to build broad-spectrum engineered strains with enhanced virulence and to solve the increasingly serious problem of insect resistance.