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

Abstract

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.

Cis-regulatory elements used to control gene expression in plants

“…Plant bidirectional promoters and gene stacking can be used to avoid gene silencing in multigene cassettes and decrease the amount of transferred DNA (Shamloo-Dashtpagerdi et al 2015). Kumar et al (2015) constructed a cassette combining a bidirectional promoter obtained from ZmUbi1promoter and bicistronic gene organization. They introduced this construct to corn and obtained efficient production of four genes.…”

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

“…These include (i) smaller molecular size of the IntF2A domain, (ii) cotranslational cleavage mediated by the F2A peptide, (iii) adding only a single proline residue to the POI trailing the autoprocessing domain (in case of a secretory POI, the proline residue will be removed along with the signal peptide by the signal peptidase and hence the POI can preserve its authentic N‐terminus) and (iv) higher efficiency in releasing the downstream POI and hence more balanced co‐expression among the POIs in plants. The utility of the IntF2A approach can also be further extended, for instance, by synergistic integration with the bidirectional promoter systems (Kumar et al ., 2015) to further increase the number of transgenes that can be co‐expressed in plants.…”

Towards combinatorial transcriptional engineering

“…It provides strong transcription initiation while displaying minimum basal transcription in monocots and dicots (Liu and Stewart, 2016). The 126 bp ZmUb1 promoter provides low basal transcription and has been recently characterized for specific use in monocots (Kumar et al, 2015).…”