The Heterogenous Expressing And Functional Identification Of Cry1Ac Toxin Binding Proteins In Plutella Xyllostella And Helicoverpa Armigera

The toxicity of CrylA depends on the binding between CrylA toxins and the receptor proteins in the midgut brush border membrane vesicles(BBMV).Aminopeptidases N(APNs),Cadherin(CAD),Alkaline phosphatase(ALP)and ATP-binding cassette transporters(ABC-transporter)on the BBMV in lepidopterous larvae are the binding proteins to CrylA toixn,but their functionality as Cry1 Ac toxin-receptors is not clear in Plutella xylostella or Helicoverpa armigera.APNs,CAD,ALPs and ABC transporter were expressed in Sf9 cells using Bac-to-Bac expression system and the receptor-functionality of them was detected on two aspects of both binding to Cry 1 Ac toxin and mediating cytotoxicity.The results will reveal the mechanism of action of Bt toxin and provide useful data for resistance mechanism to Bt toxin.1.Heterogenous expressing of binding proteins to CrylAc in P.xylostella and H.armigeraThe titres of recombinant virus used for expressing of binding proteins(PxAPN1,PxAPN2,PxCAD,and PxABCC2)in P.xylostella,were 0.53,0.35,0.36,and 0.27 ×108pfu/ml,respectively.The titres of recombinant virus used for expressing of binding proteins(HaAPN1,HaAPN2,HaAPN5,and HaALP2)in H.armigera,were 0.70,1.95,1.37,and 1.90 × 108pfu/ml,respectively.PxAPN1,PxAPN2,PxCAD,PxABCC2,HaAPN1 HaAPN2、HaAPN5,and HaALP2 were expressed in membrane of Sf9 cells.The viruses used for expressing PxALP,HaABCC2,and HaALP1 were not constructed.2.CrylAc-receptor-function identification of binding proteins in P.xylostellaRecombinantly expressed PxAPN1,PxAPN2,PxCAD,and PxABCC2 in Sf9 cells bound to CrylAc in situ.The mortalities of cells expressing recombinant protein were higher than that of normal Sf9 cells when those cells were treated with a series concentration of Cry1 Ac toxin.Compared with the mortality mediated by PxAPNl,PxAPN2,or PxCAD,the mortality caused by interaction between PxABCC2 and Cry1 Ac toxin was the highest.When Sf9 cells expressing PxABCC2 were treated with 200 nM Cry1Ac toxin,the cell mortality was 86.8%.Moreover,the mortality showed dose-dependent response of Cry lAc.These results revealed that PxABCC2 mediated the toxicity of Cry1Ac and was one of Cry1 Ac-receptors in P.xylostella3.CrylAc-receptor-function identification of binding proteins in H.armigeraRecombinantly expressed HaAPN1,HaAPN2,HaAPN5,and HaALP2 in Sf9 cells bound to Cry1Ac in situ.The mortalities of cells expressing recombinant protein were higher than that of normal Sf9 cells when those cells were treated with a series concentration of Cry1Ac toxin.Compared with the mortality mediated by HaCAD(56.6%),the mortalities caused by interaction among HaAPN1,HaAPN2,HaAPN5,HaALP2 and Cry1Ac toxin were much lower(14.4%,17.2%,21.6%,and 31.1%,respectively)when Sf9 cells were treated with 200 nM Cry1Ac toxin.Moreover,the mortality showed no dose-dependent response of CrylAc.These results revealed that HaAPN1,HaAPN2,HaAPN5,and HaALP2 did not mediated the toxicity of CrylAc,was not Cry1Ac-receptors in H.armiger...