Insecticidal Activities Of Two Translational Fusion Bacillus Thuringiesis (Bt) Genes And Their Application In Transgenic Rice

The Bt (Bacillus thuringiensis) crops has been planted worldwide for control of insect pests. Recently, several Bt-resistant insect species has been discovered. For resistance management, many strategies have been reported. Gene stacking, i.e. expessing multiple toxins in a plant, can not only significantly delay the development of the insect resistance, but also confer resistance to multiple insect species to a plant. Constructing a translational fusion gene is an alternative way of using multiple toxins. And there were some researches focusing on evaluating different fusion Bt proteins expressed in either Escherichia coli or crops. At present, it was considered that the fusion Bt protein exert overlapping insecticidal activity and spectrum of the parental toxins.In this study, a fusion protein Cry1Ab-Cry9Aa deriving from the truncated CrylAb (648 amino acid residues at the N-terminus) and Cry9Aa (656 amino acid residues at the N-terminus), was expressed in E. coli. The two truncated toxins, CrylAb and Cry9Aa, include intact 3-domain structure and are sufficient for their insecticidal activities. The assay results showed that the fusion protein Cry1Ab-Cry9Aa is more active against the oriental armyworm (Mythimna separate) and the Asian corn borer (Ostrinia furnacalis) than the parental toxins, the truncated CrylAb and Cry9Aa. For cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura), the fusion protein significantly inhibited the growth of the neonate larvae and the feeding larvae in 5μg/cm2 concentration treatment were about 10% and 20% respectively in weight to the control insects. The larvae of cotton bollworm and tobacco cutworm feeding with truncated Cry1Ab or Cry9Aa almost developed normally. In addition, the assay results of the mixture treatment indicated that mixing the CrylAb and Cry9Aa cannot create any significant synergetic effect.The other fusion protein Cry1Ab-Cry9AaDel was made from the truncated Cry1Ab and Cry9AaDel, the further truncated Cry9Aa in which the 59 amino acids of the N-terminus was removed. Compared with the truncated Cry9Aa, the Cry9AaDel is inactive on the oriental armyworm and the Asian corn borer while it do not inhibit the growth of cotton bollworm and tobacco cutworm. However, the fusion protein Cry1Ab-Cry9AaDel retains similar insecticidal activity with the Cry1Ab-Cry9Aa to the four species tested. The two fusion genes were consequently introduced into a local cultivar of rice (Oryza sativa L. ssp. japonica), respectively. Western blot analysis revealed that the fusion proteins can be expressed reasonably in the transgenic rice in range from 0.2 to 2μg per gram of fresh leaves. And the fusion proteins expressed in rice were partially degraded, which was in agreement with the previous reports. These positive transgenic lines were highly resistant to four pest insects, cotton bollworm, tobacco bollworm, rice stem borer (Chilo suppressalis) and rice leaf folder (Cnaphalocrocis medinalis).In summary, the two fusion protein Cry1Ab-Cry9Aa and Cry1Ab-Cry9AaDel constructed in our study gain broader and higher insecticidal activity and can provide an excellent insect-resistance in biotech crops.