Construction of a bidirectional promoter and its transient expression in

Abstract

Simultaneous introduction of multiple genes into plants is a critical step in plant genetic engineering to manipulate multiple functional genes in metabolic engineering and trait stacking. It is important to construct a bidirectional promoter for transforming two or more genes into plants simultaneously. The widely used unidirectional CaMV35S promoter has been modified to a bidirectional promoter in this work by fusing a CaMV35S minimal promoter (Pmini) at its end in opposite orientation to the original promoter. To test its bi-directional transcriptional activities, two widely used histochemically visible reporter genes, gusA (?-glucuronidase) from Escherichia coli and gfp (Green Fluorescent Protein) from Aequorea victoria, were fused to the terminus of the bidirectional promoter in different orientations ending with NOS terminator sequences. The transient expression of the gusA and gfp genes were detected by histochemical staining for GUS and by fluorescence microscopy for GFP. The direction of transient expression of GUS and GFP in Agrobacterium mediated 3–4 days transformed leaf discs of Populus tomentosa, indicating that the promoter did have bidirectional transcriptional activities simultaneously in cells and tissues. It was discussed that this bidirectional promoter could possibly be applied in woody plant engineering.

Keywords

Populus tomentosa / bidirectional promoter, green fluorescent protein gene (gfp) / β-glucuronidase gene (gusA) / transient expression