Dicistronic binary vector system—A versatile tool for gene expression studies in cell cultures and plants

Abstract

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase ( gus) gene followed by an internal ribosome entry site and a firefly luciferase ( luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (At nhx1). Comparative studies with At nhx1 transgenic luc expressing tobacco cell cultures and pea plants ( Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.