Directed Modification Of Insecticidal Activity Of Cry1Ai Protein

Bacillus thutingiensis is widely used in insect control because it produces various kinds of bioactive substances that are against many insects.In this study,Cry1Ai2 and Cry1Ah1 protein,discovered by our lab,showed high toxicity against multiple Lepidoptera insects.In previous progress,the insecticidal activity against Bombyx mori and Helicoverpa armigera was found significantly different between Cry1Ai and Cry1Ah.However,a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah,namely Cry1Ai-h-loop2,showed significantly higher insecticidal activity against H.armigera(LC₅₀=64.23?g/g)while the mutant Cry1Ai-h-loop2&3,containing loop 2and loop3 from Cry1Ah simultaneously,exhibited further increasing activity against H.armigera(LC₅₀=8.61?g/g).Under these circumstances,the main contents of this study included two parts,first we characterized the key sites of loop 2 on Cry1Ai-h-loop2 by alanine site-directed mutagenesis,then the functional residues were substituted for other 18 common amino acids to analyze the alteration of toxicity against H.armigera.Next,in the process of toxic exertion against B.mori,the functions of loop2 and loop3 on domain II of Cry1Ai were analyzed by constructing loop mutants and testing toxicity against B.mori.Meanwhile,the toxicities of mutations which were constructed by substituting key loop region against B.mori and other lepidopteran pests were tested.The main results of this study were described as below:1.Gly³⁷³ and Asn³⁷⁵ were testified to be important residues on the loop 2 region of Cry1Ai-h-loop2 by alanine site-directed mutagenesis and bioassay against H.armigera.Moreover,double substituted mutant on Gly³⁷³ and Asn³⁷⁵ into alanine reduced the toxicity against H.armigera and the binding of Cry1Ai-h-loop2 with H.armigera BBMV using ELISA binding assay.2.Compared with Cry1Ai-h-loop2,the mutants that were constructed by substituting these two sites for other 18 common amino acids did not show significantly higher toxicity against H.armigera,perhaps the sequence of this loop2 was a befitting combination for toxicity.3.Loop2 on Cry1Ai was proven to be not related with insecticidal specificity against B.mori, while loop3 on Cry1Ai was testified to be important in toxicity against B.mori by constructing loop mutants and performing bioassay against B.mori.Besides,loop2 and loop3had additive effects on insecticidal activity against B.mori.More importantly,the mutant Cry1Ai-h-loop2&3,containing loop2 and loop3 from Cry1Ah simultaneously,exhibited17-fold and significantly reducing activity against B.mori.These results taken together with previously reported findings?Cry1Ai-h-loop2&3 exhibited high activity against H.armigera?indicate that we successfully constructed a mutant by directed modification with high efficacy against H.armigera and decreasing toxicity against B.mori based on the knowledge of mechanism of action.4.The binding affinity of Cry1Ai-h-loop2&3 and B.mori BBMV was reduced comparing with that of Cry1Ai through ELISA assay,which may explain the decreasing of toxicity and indicated that loop2 and loop3 participated in the interaction of toxin with B.mori BBMV.5.The toxicities of Cry1Ai and Cry1Ai-h-loop2&3 against Plutella xylostella were tested and found that Cry1Ai-h-loop2&3 still showed a certain insecticidal activity against P.xylostella.But comparing with Cry1Ai,Cry1Ai-h-loop2&3 showed a 2.2-fold and significant reduction on toxicity against P.xylostella.In this study,the key sites on Cry1Ai-h-loop2 which showed significantly higher toxicity against H.armigera were characterized,meanwhile,the key loop region on Cry1Ai against B.mori was identified and found the mutants with significantly decreased toxicity against B.mori.These researches will lay the foundation of the mechanism of action of Cry toxins and provide the new insights towards rational designs and directed modifications of Cry proteins with the features of safety,high efficiency and specificity.