The Transformation Of TMOF And The Ecdysteroid Receptor Genes In Tobacco

Transgenic plant is the effective way to control pests. Trypsin-modulating oostatic factor(TMOF) can inhibite the activity of trypsin in the insect midgut dramatically.And the insect ecdysone receptor genes EcR and USP are the main target genes in the regulation of development.In our paper, we reconstructed the vector of TMOF and the ecdysone receptor genes respectively, then transformed in tobacco.The reconstructed vector plasmids were identified by PCR reaction.To analysis of the inhibite ability of TMOF analogues genes to the insect, we have reconstructed the monovalent plant expression vectors PCAMBIA1301 containing the TMOF homologous genes Aea-TMOF1,Aea-TMOF2 and Neb-TMOF.We also reconstructed the bivalent plant expression vectors PCAMBIA1301 containing Aea-TMOF2+Neb-TMOF. All the vector were drived by the constitutive promoter CaMV35S.And the recombinant plasmids were identified by PCR reaction.The plant-mediated RNAi can silence the particular genes in insects. We choosed the Helicoverpa armigera,and the ecdysone receptor genes EcR and USP as our research objects.We reconstructed the binary plasmid which can be transcribed into double stranded RNA of EcR and USP in plant cells. And the recombinant plasmids were identified by PCR reaction.The native intron-derived miRNAs can induce RNA interference.We designed an artificial intron containing ds-EcR, mimicking the biogenesis of the native intronic miRNA.Then we reconstructed the bivalent plant expression vectors PCAMBIA1301 containing Aea-TMOF2+ds-EcR. The recombinant plasmids were identified by PCR reaction and restriction endonuclease digestion.Finally, we accomplished tobacco genetic transformation mediated by Agrobacterium tunefaciens.Six recombinant plasmids were transformed into Agrobacterium tunefaciens LBA4404 by the freeze-thaw method,and then they had tobacco leaf discs infected.Using PCR and GUS methods,we proved that the six recombinant plasmids of plant expression vectors were integrated into the tobacco genome.Our work provided a platform for further study in the development of insect-resistant transgenic tobacco and the molecular breeding of tobacco.