Screening Of The Peptides Binding To BBMV In Apolygus Lucorum And Modification Of Cry51Aa1 Toxin

Bt toxin,a crystal protein produced in the late growth stage of Bacillus thuringiensis(Bt),is poisonous to many pests such as lepidoptera,coleoptera and diptera,and has been widely used in pest control since the 1950 s.These Bt toxins,however,have no control efficiency on planthopper,aphid,mirid or other important sap-sucking pests.It has been an urgent issue in plant protection to develop biological pesticides for the control of hemiptera sap-sucking pests.The discovery of some new Bt toxins,such as Cry51 Aa,Cry64Ba,and Cry78 Aa,has filled the gap in this field.Cry51Aa1 toxin was isolated from the wild Bt strain F14-1by Chinese researchers and showed the toxicity both to Apolygus lucorum(Meyer-Dür)and Adelphocoris suturalis(Jakovlev).Compared with other toxins,the toxicity of Cry51Aa1 to target pests was relatively lower.In this study,short peptides that bind to brush border membrane vesicle(BBMV)in A.lucorum were screened from a random phage library using phage display technology.On this basis,Cry51Aa1 was modified in order to improve its toxicity to A.lucorum.The results can provide reference for toxin modification and pest biological control in the future.1.Screening short peptides binding to BBMV in A.lucorumShort peptides binding to brush border membrane vesicle(BBMV)in A.lucorum were screened from a random phage library using phage display technology.The same binding-peptide,CAPNNRLQC,was screened both in vitro and in vivo,and its occurrence rate was 20% in vitro and 91.6% in vivo.The binding-peptide was selectively enriched on t BBMV in A.lucorum after several rounds of biological elutriation.2.The binding of short binding-peptide CAPNNRLQC to BBMV in A.lucorumThe fusion protein,CAPNNRLQC-EGFP,was expressed in Escherichia coli strain to detect its binding property to BBMV in A.lucorum.Results showed that expression product of the fusion protein was 46 KD.CAPNNRLQC-EGFP could bind to BBMV in A.lucorum and the affinity was higher than Loop Ⅳ in domain Ⅰ of Cry51Aa1.3.The modification of Cry51Aa1 and detecting of its toxicity to A.lucorumFour Loops in domain Ⅰ of Cry51Aa1 was modified by substituting or inserting the peptide CAPNNRLQC respectively.Cry51Aa1(M)-4 and Cry51Aa1(M)-4A,the modified toxin of replacing Loop Ⅳ with CAPNNRLQC and inserting CAPNNRLQC into Loop Ⅳrespectively,were expressed successfully.The Molecular size of the expressed products was as expected.The modified toxins of substituting Loop Ⅰ,Loop Ⅲ,and Loop Ⅴ in Cry51Aa1 failed to be expressed in the Bt strain.Compared with Cry51Aa1,the toxicity of modified toxins(Cry51Aa1(M)-4 and Cry51Aa1(M)-4A)to A.lucorum was significantly reduced.This might be caused by the non-specific binding of short peptide CAPNNRLQC to BBMV in A.lucorum,which cannot mediate toxicity.