Construction Of Plant Expression Vector Harbouring Cry1Ac And Vip3A Bivalent Insectividal Genes And Transformation Into Rice

Rice(Oryza sativa) is one of the world’s most imporant food crops,the decrease of rice by pest bring people huge economic losses.traditional prevention using chemical pesticides cause pollution to the environment and kill all targeted insect or not.newly Microbial Insecticides are safty but low effect and much expensive.At present,cultivating anti-insect rice by transgenic technology is one of the important approach to increase rice’s yield.It was proved that pyramiding two (or more) toxins into one variety appears to be the best way to increase insecticidal activity and delay resistance,but the toxins should have different binding sites and action mechanism. Bacillus thuringiensis (Bt) insecticidal crystal protein(ICP) Cry1Ac is toxic especially to lepidopteran insects,such as Helicoverpa armigera、Beet armyworm、Yellow stem borer(Scirpophaga incertulas). Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3A is widely toxic for lepidopteran insects,even some anti-ICP insects. Vip3A protein is called second generation insecticides,the binding site of which is different from that of the first generation Cry-like protein in target insect.In this stuy,we transform crylAc and vip3A from Bacillus thuringiensis (Bt) into Taipei 309(Oryza sativa cv.Taipei 309) by Agrobacterium tumefaciems EHA 105,which lay the foundation for the transgenic rice.The main results are as follows: 1.we construct a plasmid pCMUASV harboring cry1Ac and vip3A by a newly-developed techniques of Red/ET recombination,termed as "Triple recombineering",which include crylAc regulated by ubiqutin promoter and vip3A regulated by CaMV35S promoter.2.Embryogenic calli were initiated from scutellar tissues of unmature seed embryo of japonica rice Taipei309, which were transformed by agrobacterium transformation.After selection,regeneration,rooting,we got ten transgenic seedling.3.molecular analysis for these transgenic seedling.It was proved that both cry1Ac and vip3A were integrated into genome of rice,and positive rate reach to 100%.