Effect Of The Interaction Between Cry1Ac Toxin And Midgut Cadherin In Spodoptera Litura On Its Insecticidal Activity

Bacillus thuringiensis(Bt)Cry1Ac toxin is an insecticidal protein that essential for control of lepidopteran pests.However,the low insecticidal activity of Cry1 Ac against parts of noctuidae pests including Spodoptera litura has adversely impacted the application of Bt insecticides and transgenic crops.Cadherin-like proteins present in the midgut of Cry1 A target insects has been identified as an essential receptor in the insecticidal mechanism.Molecular modification of Bt Cry toxin to enhance the binding activity of Cry1 Ac toxins to midgut cadherin are a feasible way to increase their insecticidal activity and expand its insecticidal spectrum.This study screened peptides with potential binding capability to the toxin binding region(TBR)of Cadherin in the midgut of S.litura,followed by the replacement of the three loops in domain II of Cry1 Ac by screened peptides respectively.The effect of these modifications on the insecticidal activity of Cry1 Ac toxin was then analyzed.According to the sequence analysis of Cadherin of S.litura,we cloned the potential Cadherin TBR sequence of the midgut of S.litura.The p ET32a-Slcad TBR recombinant vector was constructed and the TrxHis-Slcad TBR fusion protein was successfully expressed in Escherichia coli.The ～35 k Da Slcad TBR protein was purified using nickel column affinity chromatography,followed with the procedure of tag excisions.Nine peptides with potential binding acitivity to Slcad TBR were screened and isolated by phage display technology.After fusion expression of the screened peptides(PEPs)and enhanced green fluorescent protein(EGFP),binding activity of seven PEP-EGFP fusion proteins with Slcad TBR was verified by in vitro binding assays.To clarify whether the interaction between Cry1 Ac toxin molecules and the midgut cadherin could enhance its toxicity against S.litura.We used the screened peptides to replace the three loops of Cry1 Ac domain II respectively and constructed 27 p KG-Cry1Ac-PEP recombinant vectors capable of expressing the recombinant Cry1 Ac proteins.The recombinant vectors were transformed into E.coli for prokaryotic induction,expression and purification,which obtained 25 recombinant Cry1 Ac proteins.The enzyme stability analysis of the recombinant Cry1 Ac proteins showed that 23 recombinant Cry1 Ac proteins were able to form～65 k Da protein fragments stably under the action of midgut enzymes of S.litura without further hydrolysis except for GST-Cry1Ac-P1Dl2 and GST-Cry1Ac-P4Dl3.The activated unmodified Cry1 Ac toxin was biotinylated,and the binding activity of the three activated recombinant Cry1 Ac proteins of three loops replacement with peptide P5 D to the midgut of brush border membrane vesicles of S.litura was analyzed by in vitro competitive binding experiments.The results showed that the binding of Cry1 Ac to BBMV could be competed by Cry1Ac-P5Dl1 and Cry1Ac-P5Dl3,but not by Cry1Ac-P5Dl2,which proved that loop 2replacement significantly changed the interaction of Cry1 Ac toxin molecules with binding sites in BBMV.The biological activity of wildtype and UGT-KO strains with deletion of detoxification metabolism genes of S.litura was determined using 200 μg/m L recombinant Cry1 Ac protein.The results showed that compared with the unmodified Cry1 Ac toxin all recombinant Cry1 Ac proteins did not increase the toxicity against S.litura.These results indicated that the interaction between Cry1 Ac toxin molecules and the midgut cadherin of S.litura is not a critical pathway for Cry1 Ac to exert its toxic effects.In addition,we conducted a bioassay experiment in which 25 μg/m L Cry1 Ac toxin and 25 μg/m L P5D-recombinant Cry1 Ac protein were alternately fed to larvae of wild-type S.litura.Results showed that the mortality of wild-type S.litura larvae fed alternately of recombinant Cry1 Ac protein and Cry1 Ac toxin was significantly higher than that fed of water and Cry1 Ac toxin.Similar results were obtained in the bioassay of the larvae of UGT-KO strain.We speculated that although the recombinant Cry1 Ac protein failed to enhance the insecticidal activity against S.litura,it may act as a substrate to consuming the activity of gut detoxifying enzymes such as UGT to achieve the insecticidal function.