A combinatorial bidirectional and bicistronic approach for coordinated multi-gene expression in corn

Transgene stacking in trait development process through genetic engineering is becoming complex with increased number of desired traits and multiple modes of action for each trait. We demonstrate here a novel gene stacking strategy by combining bidirectional promoter (BDP) and bicistronic approaches to drive coordinated expression of multi-genes in corn. A unidirectional promoter, Ubiquitin-1 (ZMUbi1), from Zea mays was first converted into a synthetic BDP, such that a single promoter can direct the expression of two genes from each end of the promoter. The BDP system was then combined with a bicistronic organization of genes at both ends of the promoter by using a Thosea asigna virus 2A auto-cleaving domain. With this gene stacking configuration, we have successfully obtained expression in transgenic corn of four transgenes; three transgenes conferring insect (cry34Ab1 and cry35Ab1) and herbicide (aad1) resistance, and a phiyfp reporter gene using a single ZMUbi1 bidirectional promoter. Gene expression analyses of transgenic corn plants confirmed better coordinated expression of the four genes compared to constructs driving each gene by independent unidirectional ZmUbi1 promoter. To our knowledge, this is the first report that demonstrates application of a single promoter for co-regulation of multiple genes in a crop plant. This stacking technology would be useful for engineering metabolic pathways both for basic and applied research.