A super twin T-DNA vector that allows independent gene expression during Agrobacterium-mediated transformation

摘要

In this study, we designed and constructed a super twin T-DNA vector (pTRIDT313-g) containing two independent T-DNA cassettes—one for the selection gene Hyg and the other for the target gene Cus—to produce marker-free transgenic lines. The resulting vector was transformed into tobacco, and polymerase chain reaction (PCR) analysis showed four types of gene combinations in the T1 and T2 generations: Cus only, Hyg only, Gus + Hyg and untransformed lines. The intermediate region from the T-DNA of the right border of Hyg to the left border of Cus in the Hyg and Cus lines was not amplified. Genome walking confirmed that the Hyg and Cus T-DNA cassettes were independently inserted in different regions of the tobacco genome. Thus, the two T-DNA cassettes were integrated randomly as independent loci into the tobacco genome. The results of reverse transcription-PCR indicated that Hyg could normally be expressed in the roots, stems, and leaves of transgenic lines, and the resistance test showed that all Hyg transgenic lines could grow in the presence of 50 mg/L hygromycin. All Gus transgenic lines showed obvious blue coloration in enzyme activity tests, indicating that the Cus gene could be normally expressed in all the lines. Therefore, the super twin T-DNA vector (pTRIDT313-g) exhibits independent integration, heredity, and normal gene function from two T-DNA cassettes. This vector could be a useful and valuable tool in the production of marker-free transgenic lines.