A Bt Fusion Strategy Of Two Bt Toxins With Different Modes Of Action For Insect Resistance Management In Transgenic Corn And Rice

It has been over 20 years since the first commercialization of genetically modified crops in 1996.Today,a cumulative acrage of GM crops has been over 2 billion hectares worldwide.It was estimated that the total benefits of GM crops to the famers in this period was more US$150 billion.The first generation of Bt crops contained only one insecticidal gene.After more than 10 year's utilzation,insect resistance to Bt have been developled both in lab and farmland globally.Recently in China,cotton bollworm and pink bollworm were found to have gained some level of resistance in Xinjiang where cultivated transgenic cotton expressing Cry 1 Ac were widely planted.Therefore,Bt resistance management strateges should be implemented in the new generation of GM crops to be developed.Traditionally,high-dose/refuge strategy and pyramids strategy are two major methods for the management of insect resistance.Pyramids strategy stacked two or more toxins in a single crop cultivar by hybridization of two or more single Bt gene events or by molecular stacking,which two or more Bt expression cassettes were constructred into into one T-DNA.In these cases,unbalanced expression of different toxins and gene silencing would be potentially a problem.To over come these issues,we designed a Bt fusion strategy of two Bt toxins with different mode of action.Transgenic corn and rice introduced with this fusion gene would express the entire fusion protein stably and simultaneously.In this study,we fused CrylAb and Vip3da to generate fusion proteins with a linker between the two peptides.CrylAb was a widely used toxin in commercial transgenic crops.Vip3da was developed by our group with high activity against several Lepidopteran pests.A short peptide linker consist of 8 non-polarity amino acids was used as linker to connect CrylAb and Vip3DA.A longer linker derived from a section of nucleotide connecting two domains of maize chitinase was also utilized to link crylAb and vip3da.Bioassay revealed that E.coli expressed fusion protein had high activities against fall armyworm,cotton bollworm and black cutworm,which suggested that this novel fusion proteins maintain the specific activity of both Cry 1 Ab and Vip3da.We constructed T-DNA plasmids carrying the crylAb and vip3da fusion gene using different promoters to transform corn and rice.Results revealed that the entire fusion protein could been detected in transgenic corn and rice events,and they all showed expected bioactivities against several Lepidopteran pests.Finally,we picked two transgenic corn lines SL21 and SL34,and three transgenic rice lines AIL,SL6 and SL14 for detailed analysis of insect resistance and for molecular chacterization.Molecular characters such as expression quantities,border sequences and T-DNA copy numbers had been measured and determined.All of them could been candidates for further commercialization and these data would provide references.