Prokaryotic Expression Of Aminopeptidase-N(CsAPN1) In Chilo Suppressalis Midgut And Analysis Of Binding Character Between CsAPN1 And Bt Toxins

The rice stem borer,Chilo suppressalis Walker,is a major pest of rice and causes great economic losses to rice production in China.Insect-resistant transgenic Bt rice provides a new strategy for its control,but the potential resistance risk of C.suppressalis to Bt rice wo?ld seriously threaten its sustainable application.Therefore,it is very crucial to develop precautionary insect resistance management?IRM?strategy and delay insect resistance evolution.Moreover,the definition of molec?lar resistance mechanism of C.suppressalis to Bt rice/toxin is the precondition and foundation to implement IRM strategy.Previous study showed that isozyme?CsAPN1?of aminopeptidase-N?CsAPN?from the midgut of C.suppressalis co?ld act as the putative receptor of Bt toxins.Down-reg?lated expression or mutation of receptors from insect midgut probably led to the decrease of binding ability,further result in insect Bt resistance.In previous research,we found there were two deletion mutations?lack of a total of 19 amino acids?located on the 3'end of the coding region.Additionally,the deletion mutations were proved to exist in the genome level.However,whether the mutation wo?ld lead to the decrease of binding affinity between CsAPN1 and Bt toxins and further resulted into C.suppressalis resistance to Bt toxin has not been revealed.Therefore,we conducted the construction of expression vector of CsAPN1,over-expressed the normal and deletion mutants CsAPN1 polypeptides,and measured the binding affinity between CsAPN1 peptide fragments and Bt toxins by microtiter plate assay,which would determine the effect of deletion mutation on the binding affinity of Bt toxins to CsAPN1 peptide fragement and elucideated the relationship of C.suppressalis resistance with delete mutation.The main results are as follows:1.Expression and purification of CsAPN1 peptide fragmentsCsAPN1 full-length without signal peptide was divided into four fragments,which were named as CsAPN1-F1,CsAPN1-F2,CsAPN1-F3 and CsAPN1-F4?CsAPN1-F4L or CsAPN1-F4S?,respectively.Remarkably,the fourth fragments of CsAPN1 fragments with and without deletion mutation were designated as CsAPN1-F4S and CsAPN1-F4L,respectively.These fragments were respectively constructed into pMal-c5X vector and introduced into E.coli for inducing expression and purification.The results showed that these fragments were expressed as fusion proteins with the expected molecular weight of 73.2 kDa for CsAPN1-F1,72.87 kDa for CsAPN1-F2,72.3 kDa for CsAPN1-F3,71.19 kDa for CsAPN1-F4L and 69.2 kDa for CsAPN1-F4S.After purification by affinity chromatography with maltose column,purified fusion proteins were obtained for subsequent bindng assay.2.Analysis of binding character between Bt toxins and CsAPN1 peptide fragmentsMethod of ELISA binding kinetics tests with high repeatability and reliable data was established for binding assay between CsAPN peptide fragments and Bt toxins.After ELISA binding kinetics tests,the binding affinity constant?Kd?values of Cry1Ab with CsAPN1-F1,CsAPN1-F2,CsAPN1-F3,CsAPN1-F4L and CsAPN1-F4S were 9.85±2.94 nM?85.42±72.55 nM?5.4±0.80 nM?2.78±0.16 nM and 2.70±0.14 nM,repectively.The Kd of Cry1Ab binding to CsAPN1-F2 was significantly higher than that of Cry1Ab binding to other fragments,thus its binding ability was significantly lower than that of Cry1Ab binding to other fragments.Cry1Ab could bind to both CsAPN1-F4L and CsAPN1-F4S,and no significant difference was observed for binding ability between Cry1Ab with CsAPN1-F4L and CsAPN1-F4S.We supposed that Cry1Ab binding site was not located in the deletion region of the 4^thh fragment of CsAPN1,that is,this deletion mutation could not affect the binding affinity between Cry1Ab and CsAPN1.After ELISA binding kinetics tests,the Kd of Cry1C binding to CsAPN1-F1,CsAPN1-F2,CsAPN1-F3,CsAPN1-F4L and CsAPN1-F4S were 50.48±12.07 nM?11.52±2.021 nM?19.04±5.91 nM?9.74±3.06 nM and 78.85±58.83 nM.The binding ability of Cry1C with CsAPN1-F1 fragment was significantly lower than that of Cry1C with CsAPN1-F2,CsAPN1-F3,and CsAPN1-F4L.There was no significant difference for binding ability between Cry1C to CsAPN1-F2,CsAPN1-F3 and CsAPN1-F4L.Cry1C could bind to both CsAPN1-F4Land CsAPN1-F4S,however,the binding ability of Cry1C to CsAPN1-F4S with the deletion mutation region was significantly lower than that of Cry1C to CsAPN1-F4L.It is supposed that Cry1C binding site was located in the deletion region of the 4^thh fragment of CsAPN1.This deletion mutation could not affect the binding affinity between Cry1Ab and CsAPN1.The Kd values of Cry2Aa binding to CsAPN1-F1,CsAPN1-F2,CsAPN1-F3,CsAPN1-F4L and CsAPN1-F4S were 3.26±0.31 nM,2.50±0.25 nM,1.95±0.11 nM,19.50±7.25 nM and 27.40±8.70 nM.The binding ability of Cry2Aa with CsAPN1-F4L fragment was significantly lower than that of Cry2Aa with CsAPN1-F1,CsAPN1-F2,and CsAPN1-F3.There was no significant difference for the binding affinity between Cry2Aa to CsAPN1-F1,CsAPN1-F2,and CsAPN1-F3.Though Cry2Aa could bind to CsAPN1-F4L and CsAPN1-F4S,the binding abilities between Cry2Aa to CsAPN1-F4L and CsAPN1-F4S with the deletion mutation did not differ significantly.Therefore,it was concluded that Cry2Aa binding site did not exist in the deletion mutation region of the 4^th fragment of CsAPN1.In summary,this study defined the binding character and epitode of Bt toxins with CsAPN1peptide fragment,elucidated the effect of delete mutation of CsAPN1 on the binding affinity of Bt toxins to CsAPN1 peptide,and revealed the relative resistance mechanism of C.suppressalis to Bt toxins due to the delete mutation in CsAPN1.This study will be very meaningful for elucidating the diverse insect resistance mechanism to Bt toxins,developing precautionary insect resistance management strategy and guaranteeing the sustainable utility of Bt rice in China.