Construction of high-efficiency transformation vector with multiple insect-resistant genes and expression in tobacco

A high-efficiency plant transgenic vector expressing multiple genes has been modified from expression vector pCAMBIA1302. Briefly, multiple cloning sites of a cloning vector pUCm-T were constructed into plant transgenic vector pCAMBIA1302 backbone using Bsp120 I and Spe I restriction digest site. Two Bt genes, cry1Ac and cry3A, were then constructed into the modified transgenic vector in different orders as p1870-35S::cry1Ac-Coy::cry3A and p1870-Coy::cry3A-35S::cry1Ac, respectively. Transgenic tobacco plants were generated using Agrobacterium-mediated transformation method. Polymerase chain reaction (PCR) results showed that both the exogenous genes were integrated into the genome of tobacco. Fluorescence qRT-PCR detected both transgenes and further ELISA assay validated the expressed Bt proteins. Transgenic lines showed enhanced resistance to larvae of Helicoverpa armigera Hubner in the further feeding assays. In conclusion, our modified transgenic vector was suitable for multiple gene expression and would greatly facilitate our future research.