Key messageThe choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize.AbstractViral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase (aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm2) than the OsAct1 promoter (12.6 ng/cm2) in T0 leaf tissue. When measured in T2 callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T0 leaf and T2 callus tissue.

The expression profile of a natural bi-directional promoter, derived from the Brassica napus EPSPS-A gene, was studied in transgenic soybean (Glycine max C.V. Maverick) lines. Two constructs, pDAB100331 and pDAB100333, were assembled to test the bi-directionality of the promoter. Two reporter genes, gfp and gusA, were employed and they were interchangeably placed in both constructs, one on each end of the promoter such that both proteins expressed divergently in each construct. In the T generation, GUS expression was more uniform throughout the leaf of pDAB100333 transgenic plants, where the gusA gene was expressed from the downstream or EPSPS-A end of the bi-directional promoter. Comparatively, GUS expression was more localized in the midrib and veins of the leaf of pDAB100331 transgenic plants, where the gusA gene was expressed from the upstream end of the bi-directional promoter. These observations indicated a unique expression pattern from each end of the promoter and consistently higher expression in genes expressed from the downstream end (e.g., EPSPS-A end) of the promoter in the tissues examined. The GFP expression pattern followed that of GUS when placed in the same position relative to the promoter. In the T generation, transcript analysis also showed higher expression of both gusA and gfp when those genes were located at the downstream end of the promoter. Accordingly, the pDAB100331 events exhibited a higher gfp/gusA transcript ratio, while pDAB100333 events produced a higher gusA/gfp transcript ratio consistent with the observations in T plants. These results demonstrated that the EPSPS-A gene bidirectional promoter can be effectively utilized to drive expression of two transgenes for the desired traits.