Peptide Selection And Modification Of Bacillus Thuringiensis Cry Against Empoasca Vitis(Gothe)

Bacillus thuringiensis(Bt)is the most widely employed and investigated microbial pesticides.Cry toxin proteins(δ-Endotoxins)prevent efficiently damages of Lepidoptera,Coleoptera,Diptera and plant pathogenic nematodes.However,there is limited knowledge about Bt against Empoasca vitis(Gothe),a kind of sap-sucking insect.As a large group of Hemiptera,E.vitis control is still mainly relying on chemical pesticides.Given the significant damage to tea agricultural production and pest high resistance outbreak,it urgently needs to develop new Cry molecules for this pest management.So,in order to obtain the effective Bt Cry toxic to E.vitis,molecular modification was carried out in this study.(1)Two proteins(RGATPMS and GYSSFNRG,named R and G)were screened from a phage-displayed random peptide library.They were made up from 7 amino acids,and might bind effectively to the gut of E.vitis.Peptide-green fluorescent protein(P-GFP)fusion proteins were prepared and then validated their binding activity to the BBMV receptor by pull-down,western blot and fluorescence microscopy observation.Results were shown that these two proteins hold strong binding activity to E.vitis gut membrane.It provides good peptides for subsequent Cry domain loop replacement.(2)The two selected peptides were replaced respectively to Cry1Ab domain Ⅱ 3 loops that can recognize possible receptors on the gut membrane of E.vitis by genetic fragments replacement.Six novel Cry proteins(Cry1Ab-1R,Cry1 Ab-2R,Cry1Ab-3R,Cry1 Ab-1G,Cry1 Ab-2G,Cry 1 Ab-3G)have been successfully expressed and purified.Results of structure prediction and activity validation showed that modified proteins were stable on model projections,binding more strongly to E.vitis BBMV.Toxicology tests indicated the lethal speed of new Cry proteins were improved in varied degree.This not only expands insecticidal specificity of Bt Cry,but also provides important theoretical and applicative value for newly toxic and broad-spectrum biopesticides.