Insecticidal Activity Of Three Bt Gene In Tobacco

Bollworm(Helicoverpa armigera Hubner)belonging to Lepidoptera Noctuidae widely distributed around the world, is one of the most serious pests of cotton production. 1980 s and1990s, the main cotton-producing regions of the world bollworm outbreak in a large area,resulting in a serious cut cotton, causing huge economic losses. Transgenic Bt cotton has gained great ecological and economic benefits after over 20 years of research and production application, However, with the large area of insect resistant cotton growing, under the action of high ecological selection pressure, The resistance risk of cotton bollworm is also increasing gradually. Through the mechanism of the insecticidal protein of Bt found,The application of large area of transgenic cotton with single Bt gene is easy to cause resistance to insects.Therefore, exploration of Bt genes with different insecticidal mechanism and creation of new materials with different insecticidal mechanism of hybrid insect resistant cotton has become a hot spot in the research of genetic engineering of insect resistant cotton. Transgenic hybrid cotton with insect resistance gene can not only improves insecticidal efficiency, but also avoids or delays insect resistance effectively. The cry2Ah2, cry2Ab4 gene and vip3 Aall gene were cloned from Bacillus thuringiensis(Bacillus thuringiensis, Bt)was cloned into the three new Bt insecticidal crystal protein gene. cry2Ah2, cry2Ab4 is Insecticidal crystal protein produced in sporulation( Insecticidal crystal proteins, ICPs), Lepidoptera, Diptera,Coleoptera and other insects poisoned function. vip3Aa11 a vegetative insecticidal proteins( Vegetative insecticidal proteins, VIPs), Bollworm, Agrotis ypsilon and a series of Lepidopteran pests have a higher toxicity.In this study, Three plant expression vectors pGBI-cry2Ah2(35S::cry2Ah2::Nos), p GBIcry2Ab4(35S::cry2Ab4::Nos)and p GBI-vip3Aa11(35S::vip3Aa11::Nos)harboring cry2Ah2,cry2Ab4 and vip3 Aall gene respectively were constructed and transferred into tobacco(NC89)by Agrobacterium-mediated. 30, 33 and 35 regenerated plants were PCR positive respectively.Gene expression was done through q RT-PCR, We were selected high expression levels of transgenic plants were Southern blot experiments showed that the three genes was integrated into the tobacco genome. cry2Ah2 gene tobacco was further verified by Western blotexperiments at the protein level. Biological activity detection results show that after raised with transgenic tobacco leaves after four days, corrected mortality of bollworm can reach86.59%, 86.51% and 87.70% to achieve high level resistance, resistant plants turn vip3Aa11 insects better than turn cry2Ah2 and cry2Ab4 plants turn cry2Ah2 plant and insect gene cry2Ab4 same level. These results indicated that the three genes provided a new source of resistance genes for the study of the existing cotton.In this study, confirmed the three genes can be expressed at high levels in tobacco and with high insect resistance, It is a new candidate insect resistant gene to control the pest of bollworm, which can be transferred to cotton and other crops, for nurturing the new generation of transgenic insect-resistant crop varieties.