A Brassica napus Bi-Directional Promoter Exhibits Differential Leaf-Expression Patterns in Soybean Transgenic Lines Generated Using Agrobacterium-Mediated Transformation

Many plant species are capable of being transformed with multiple transgenes from other species leading to trait stacking to introduce agronomically desirable characteristics. These genes are frequently controlled by identical or homologous promoters.  Homology-based gene silencing (HBGS) is likely to arise when multiple introduced transgenes are driven by the same promoters (Mol et. al., 1989). Strategies to avoid HBGS in transgenic plants frequently involve the development of various promoters that express in the desired tissue(s) spatially and temporally but have minimal sequence homology (Matzke and Matzke, 1995).  There remains a need for the development of construct design for stable expression of multiple transgenes, with the additional requirement of minimum risk for recombination or loss of transgenes through breeding. Here, we report the expression of a bi-directional promoter derived from Brassica napus (BBP) that drives two reporter genes, GUS and GFP, in the opposite directions. Agrobacterium-mediated transformation of soybean (Glycine max) was carried out with two experimental binary vectors, one where BBP regulated GUS in the forward (5’ to 3’) and GFP in the reverse (3’ to 5’) orientation, and the second where BBP regulated the reporter genes after exchanging their flanking positions. Transgenic plants generated with both plasmids displayed the expression of GUS and GFP confirming that BBP drives expression in both directions at reasonable levels. Depending on the orientation of the genes there are differences in the expression patterns within leaf tissues. Thus, bi-directional promoters can be used to effectively drive multiple transgenes for desired traits.