The Synergistic Analysis Of Helicoverpa Armigera Aminopeptidase N Toxin-binding Region Fragment To Cry1Ac Toxicity

Transgenic Bt cotton can be planted efficiently on long term basis but the potential crisis is the resistance evolution in Helicoverpa armigera. An optional strategy to cope with the resistance is the application of synergistic proteins. Both, cadherin (Cad) and aminopeptidase N (APN) are the binding receptors of Cryl A toxins to Lepidoptera pests. There were some reports showing that the toxin-binding region fragment of receptor could significantly synergist the toxicity of CrylA toxins. In present studies, the toxin-binding region fragment of aminopeptidase N of H. armigera enhanced the toxicity of CrylAc toxin to H. armigera larvae significantly. The results were described as:1. The toxin-binding fragment of APN 1 of H. armigera (APNltb) was cloned and expressed in E. coli. The molecular weight of APNltb fragment was 28 kDa, twice the predicted molecular weight. APNltb fragment was purified by using Ni-affinity purification. APNltb mainly existed as dimer by using Western blot analysis. The bioassay showed that the mortality of H armigera larvae was 44.44±6.056% with CrylAc alone; the mortality increased to 66.67±2.405%,91.66±4.167%,91.67±4.812% on addition of 5 fold,15 fold and 50 fold APNltb fragment in CrylAc toxin, respectively. It showed that APNltb fragment synergisted CrylAc toxicity to H. armigera larvae significantly. The delete mutation of toxin-binding fragment of APN1 of H. armigera (APN1tb-Del) was cloned and expressed in E. coli. The molecular weight of APN1tb was 20 kDa, also twice the predicted molecular weight. APNltb-Del fragment was purified by using Ni-affinity purification. APNltb-Del fragment existed also as dimer by using Western blot analysis. APNltb-Del fragment lost synergistic effect to CrylAc when compared to APN1tb fragment.2. APNltb fragment could bind to CrylAc with high affinity by using ELISA, while APN1tb-Del could not bind Cry1Ac. Both, APN1tb and APN1tb-Del fragment could bind to midgut brush boder membrane vesicles (BBMV) of H. armigera by using flow cytometry. The inlusion body of APN1tb (APN1tb-I) was soluted and purified. The bioassay showed that the mortality of H. armigera larvae was 48.61±6.054% with CrylAc alone; the mortality in each case increased to 98.61±1.389% on the addition of 5 fold,15 fold and 50 fold APNltb-I fragment in CrylAc, respectively. It showed that APN1tb-I fragment synergisted the Cry1Ac toxicity to H. armigera larvae significantly, and the synergistic effect of APNltb-I fragment was more significant than APN1tb fragment. Both, APN1tb and APN1tb-I fragment could bind to Cry1 Ac with high affinity by using ELISA, and the binding capability of APN1tb-I fragment was stronger than APN1tb fragment that justified the above mentioned reason.It has been concluded that APNltb fragment expressed in E. coli synergisted Cry1 Ac toxicity to H. armigera larvae significantly that strongly support our hypothesis mechanism:APN1tb could link Cry1 Ac toxin and APN receptor act as a brige. For future studies, present research can provide a method to cope with the resistance of H. armigera to the transgenic Bt cotton.