Creation Of CryNGc Transgenic Maize And Molecular Modification Of Cry3/cry8 Genes

Due to the specific toxic to pests,Bacillus thuringiensis(Bt)has been widely applied in insect control as a bio-pesticide.With the expanding application of Bt proteins,the problems of Bt pesticides have been revealed,mainly for low speed of killing and the development of insecticide resistance.As a result,studies on molecular mechanism of crystal protein pesticide have been prompted.In order to improve the resistance and expand the insecticidal spectrum of Bt genes,Bt genes were modified by means of domain swapping and molecular modification.It provided reference for the development independent intellectual Bt genes.Based on the previous study in our laboratory,a new Lepidopteran pests-resistance gene named cryNGc was constructed and transferred into the plant expression vector which carries the herbicide resistance bar gene.The gene was constructed through the domain swapping and got a new hybrid protein,named CryNGc which consists of three different domains(Domain ?,Domain ? of CrylAb and Domain ? of CrylGc).In this study,the exogenous gene cryNGc was transferred into maize immature embryos by agrobacterium-mediated method.We performed molecular identification,evaluation of resistance and other experiments by using molecular technologies(PCR,RT-PCR,PAT and BT-Cryl Ab/Ac analysis,etc.).In addition,insect resistance assessment in both laboratory and the field were conducted through the measurement of Ostrinia furnacalis resistance.The results show that the gene cryNGc was integrated into the maize genome with stable expression through the nucleic acid detection and protein detection of transgenic maize.Insect resistance assessment showed that obvious resistance of the the cryNGc transgenic maize to Ostrinia furnacalis.Subsequently,the 9 Coleopteran-specific insect-resistance genes from cry3 and cry8 were chosen to create a 52 genes mutant library,expecting to screen out a new gene which is high toxic and broad spectrum.Based on the previous experimental study,we randomly selected a recombinant mutant ACA which contains Domain I of Cry3Aa,Domain ? of Cry8Ca and Domain ? of Cry3Aa.The molecular experiments showed that the recombinant mutant ACA which was 1968 bp and encoded 656 amino acids were successfully constructed through the domain swapping.The recombinant mutant ACA was cloned into prokaryotic expression vector pET28a,the protein of this gene were expressed inE.coli BL21(DE3)strains and the molecular weight of expressed protein were 74.2 kDa.These results gave a base for further insecticidal activity bioassays.