Effects of the sequence and orientation of an expression cassette in tobacco transformed by dual Bt genes

Abstract

This study investigated the effects of the sequence arrangement and orientation of a target gene expression cassette in vectors on expression levels to determine the optimal combination for highly efficient multi-gene expression. Five plant transformation vectors were constructed using dual Bt genes, Cry1Ac and Cry3A, which differed in the sequence arrangement and orientation of the target gene expression cassette. Through an Agrobacterium-mediated method, 5 vectors were used for the genetic transformation of tobacco to obtain transgenic lines. Fluorescence quantitative PCR showed that the target genes were expressed at the transcriptional level, which did not differ significantly among the different vectors. However, an enzyme-linked immunosorbent assay showed that there were significant differences in the toxin expression levels of the different vectors. In vectors N12 and N19, the Cry1Ac gene, located upstream, showed lower average expression than the Cry3A gene, located downstream. Similarly, in vectors N13 and N18, the Cry3A gene, located upstream, had lower expression than the downstream Cry1Ac gene. For vector N21, with the expression cassette containing the Cry1Ac gene located upstream in a trans-arrangement and that of the Cry3A gene located downstream in a cis-arrangement, the Cry1Ac and Cry3A toxin levels were the highest, at 7.41 and 13.24μg·g^-1, respectively. The insect resistance of transgenic lines transformed by the different vectors was related to the Bt toxin level. Resistance to H. armigera, Lepidoptera, and Cry1Ac toxin level were positively correlated; resistance to A. germari larvae, Coleoptera, and Cry3A toxin content were also positively correlated. This study showed that the sequence arrangement of 2 expression cassettes with target genes may be the key to the target gene expression. Two expression cassettes in the same orientation had little influence on gene expression; however, when the 2 expression cassettes were in the reverse arrangement, the expression of both of the target genes was promoted to a certain extent.

Keywords: Bt genes; Expression cassette arrangement sequence and orientation; Gene expression; Plant transformation vector.