Identification Of Cry-binding Proteins In Insect And Research Of Their Interaction With Cry Toxins

Insecticidal crystal protein?Cry?is the most important active ingredient of Bacillus thuringiensis?Bt?,which has been mostly wide used.The identification of those proteins that can recognize Cry toxins in insect pests,will provide the basis for exploring the molecular process of Cry insecticidal action,and provide new ideas for solving the key problems faced by Bt preparations and Cry toxins.From the hemolymph of the beet armyworm,Spodoptera exigua,storage protein was identified to be Cry1Ab1-binding protein,and?-1,3-glucanase,tubulin,elongation factor 1-alpha and apolipophorin were found to be Cry7Ab4-binding proteins,based on Pull-down assay and LC-MS/MS analysis.The Cry2Ab12 gene which was previously discovered in our laboratory was constructed and expressed.From the midgut juice of Plutella xylostella,methionine-rich storage protein,lipase-1,heat shock protein 70 were isolated and identified to be Cry7Ab4-binding proteins,and arylphorin,methionine-rich storage protein,apolipophorin,peroxiredoxin were found to be Cry2Ab12-binding proteins,based on Pull-down assay and LC-MS/MS analysis.Also,the correlation of these binding proteins to Cry toxins killing Plutella xylostella were investigated.The storage protein of S.exigua?SeSP?was cloned and expressed in E.coli.Furthermore,the interface between Cry1Ab1 and SeSP was predicted.Then,the possible roles of Cry1Ab1-SeSP interaction in the killing process were proposed as the following.First,Cry1Ab1 blocks those binding sites of the SeSP hexamer,making it unable to form hexamer,then SeSP can not be stably existed in the hemolymph,thus affecting the storage of nutrients in insect bodies,resulted in an irreversible effect on the epidermis formation,development and metamorphosis of insects,eventually leading to the death.Second,Cry1Ab1 binds to the three domains of SeSP,leading to reduced activity of phenoloxidase,thus exerting a pesticidal effect.In addition,how those Cry7Ab4-binding non-receptor proteins such as?-1,3-glucanase and apolipophorin correlate to the insecticidal process of Cry7Ab4against S.exigua were deeply analyzed.Finally,a hypothetical mode of action of Cry toxins against lepidoptera mediated by Cry-binding proteins from hemolymph was proposed.Dorsal and POXC have been identified in our lab to be nonreceptor Cry1Ab1-binding proteins in midgut juices of Plutella xylostella and S.exigua,respectively.Here,the purified Dorsal and Cry1Ab1 were subjected to BLI assay for their qualitative and quantitative interactions.Dorsal and Cry1Ab1 interact in 1:1ratio and with KD value 3.96×10^-99 M.The interface between Dorsal and Cry1Ab1was analyzed based on molecular docking.Six oligopeptides were artificially synthesized and their interactions with Dorsal were assayed by using BLI technology.Two of them,NFDGSFRGSAQG and NSSVSII,were confirmed to have strong binding activity to Dorsal with KD values 8.68×10^-5M and 2.64×10^-5M,respectively.The two oligopeptides correspond to the Dorsal region called p50 Domain I,which involved in the modification and nucleic acid localization,indicating that the Cry1Ab1-Dorsal interaction possibly affect the binding of Dorsal to the specific?B sequence,thereby affecting the transcription level of AMP.In addition,the qualitative interaction between Cry1Ab1 and POXC was also assayed and they interact in 1:1ratio,too.In summary,Cry1Ab1-binding proteins and Cry7Ab4-binding proteins in the hemolymph of Spodoptera exigua were isolated and identified,then the Cry1Ab1-binding protein SeSP was cloned and expressed in E.coli,the in vitro interactions between Cry toxins and their binding proteins were assayed,their interfaces were predicted,and two oligopeptides on the Cry1Ab1-Dorsal interface were confirmed.Those results provide new ideas for uncovering the overall picture that how Cry kill the insect pest,and provide a theoretical basis for dealing with resistance development and improving the activities of Cry toxins against tough insect pests.