Construction Of Expression Vector Bt And ARA Anti-insect Bivalent Genes And Research On Transformation Of Rape

Bacillus thuringiesis gene and Amaranthus retroflexus agglutinin gene were ligated into bivalent plant expression vector, which encode two lepidopter-resistance and coleopterous insect-resistance proteins. Then the new plant expression vectorws transformed into rapeseed by the Agrobacterium-mediated methodThe ARA gene and the Bt gene were ligated into prokaryotic(pET-28a) expression vector, After IPTG induction, the Bt gene expressed a fusion protein with the molecular weight of 73.7kDa and the ARA gene expressed a fusion protein with the molecular weight of 36kDa by SDS-PAGE electrophoresis.The result is consentaneous compared with theoritical computation. Along with the inducing time was prolong , the expression of protein was gradually risingtrend . These results show that the ARA gene and the Bt gene could express in microorganism, but also the reading frame is intact, these genes expression was integral.The ARA gene and the Bt gene were ligated into plant expression vector(pBI-121). These recombinant plasmids and pBI-Bt-BA were confirmed by restriction enzyme and PCR analysis and the results showed that these recombinant plasmids were constructed successfully.The ? gene is CaMV 35S enhancers expressional elements.The BSP gene is phloem-specific promoter.These recombinant plasmids pBI-Bt-BA were confirmed by restriction enzyme and PCR analysis and the results showed that these recombinant plasmids were constructed successfully.The new plant expression vectorws transformed into rapeseed by the Agrobacterium-mediated method and laser microbeam puncture method to obtain transgenic rape. The bivalent gene was induced into rapeseed, we had received transgenic rapeseed. which was detected by PCR analysis, Agarose gel electrophoresis of DNA revealed the size of gene fragment is about1.8 kb and 915bp. Plant Genome DNA was detected by Southern blotting,The results showed that there was 1or 2 hybrid zones in transgenic plant, there was not hybrid zones in non-transgenic plant and as expected there was 2 hybrid zones in positive control,which proved that Bt-BA genes were successfully transformed into tobacco. Transgenic rape with tolerance to the tested pest were demonstrated by bioassays, but also the reading frame is intact.