Research On Swapping Cry1A Protein Domains And Its Impacts On The Insecticidal Activity

Bacillus thuringiensis(Bt) is a widely distributed gram-positive bacteria. It can produce insecticidal crystal proteins(ICPs) encoded by cry or cyt genes and has specific Insecticidal activity against a variety of pests. At present, Bt was not only developed as a biological pesticide and applied for the pest control, but transgenic insect-resistant plants expressing Cry proteins have also been widely planted, which have brought huge economical and ecological benefit. Among the 112 insecticidal genes, Cry1 A is an insecticidal protein family specific to lepidoptera pests, the known cry1 A gene has 10 holotype. The cry1 Ab and cry1 Ac genes were most widely applied and the cry1 Ah gene with independent intellectual property rights was cloned by institute of plant protection, Chinese academy of agricultural sciences. cry1 Ah has higher activity against lepidoptera pests including Helicoverpa armigera, Ostrinia furnacalis and Chilo suppressalis than cry1 Ab and cry1 Ac, and has great application prospect and used for transgenic insect-resistant plants developing now. Cry1 A protein was a typical three domains(3D) toxin. The alignment of domains of insecticidal activity knowing protein showing that part of the insecticidal activity(especially Cry1 protein) can be obtained by domain III restructuring. Cry toxins’ domain swapping is a insecticidal specificity evolution mechanism. Through sequence alignment, Cry1 Ab, Cry1 Ac, Cry1 Ah have similarly domain II, Cry1 Ab and Cry1 Ac have similarly domain I; Cry1 Ac and Cry1 Ah have similarly domain III; Cry1 Ab domain III different from Cry1 Ac and Cry1 Ah, but it common in Cry1 A family; compaire with Cry1 Aa, Cry1 Ae and Cry1 Af domain III, the sequence identity was high than 97%. Cry1 Ah domain I was unique, it not differ from Cry1 Ab and Cry1 Ac, it also unique in in Cry1 A family. In this work, we compared Cry1 Ab,Cry1Ah and Cry1 Ca domain I structure, surface charge distribution, and constructed cry1 Ah and cry1Ab、cry1Ca hybrid genes, expressed the hybrid proteins, and analyzed the hybrid genes function. The results were following: Use of SWISS- MODEL automatic modeling function, Cry1Ah1 and Cry1Ab13 domain I were obtained respectively. By molecular compare, Cry1Ah1 and Cry1Ab13 have same carbon skeleton although they are with high difference in sequence. Further analysis shows that the surface charge distribution was different.By seamless cloning process, 4 hybrid genes were constructed. Ah AhCa contains Cry1 Ah domain I, II and Cry1 Ca domain III;PET-AHAHAB contains Cry1 Ah domain I, II and Cry1 Ab domain III;AhAb Ab contains Cry1 Ah domain I and Cry1 Ab domain II, III;PET-AHCACA contains Cry1 Ah domain I and Cry1 Ca domain II, III. The 4 hybrids were expressed and bio assayed. The bioassay result suggested the domain swapping effect the insecticidal activity significantly.The data suggested that the 3D-Cry protein insecticidal specificity may not only determined by domain II and domain III, it also determined by the combination of the three domain, including domain I. in 3D-Cry protein, the domains interaction with each other, micro environment in protein structure surface will changed while the domain combination is different, and, it will effects proteins characters. The further analysis the mechanism of hybrid insecticidal protein specificity changes has important significance for further reveal 3D-Cry insecticidal protein specificity evolutionary mechanism.