Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence

Abstract

A synthetic transcribed, untranslated leader sequence from alfalfa mosaic virus RNA4 (AMV leader) has been assessed for its in vivo properties as a cis-active ‘translational activator’ in transient expression assays in protoplasts of Nicotiana tabacum and Picea glauca, as well as in stable expression in transformed Nicotiana tabacum. Levels of GUS enzyme activity produced by chimeric genes with or without the AMV leader sequence, in combination with either a CaMV 35S promoter or a duplicated-enhancer CaMV 35S promoter construct were assessed. In transient assay systems, the presence of a synthetic 40-base leader sequence lead to a 20-fold elevation in GUS activity when the constructs contained a native cauliflower mosaic virus (CaMV) 35S promoter, whereas a 4-fold elevated expression lebel was observed in constructs containing a duplicated-enhancer 35S promoter. Furthermore, elevated expression in chimeric constructs was influenced by the sequence context for translation initiation of the marker gene. In transgenic tobacco plants the mean values for steady-state expression of GUS-containing 35S/AMV constructs were elevated about 8-fold relative to plasmids containing the native 35S promoter alone. A quantitative PCR approach was used to assess relative transcript levels in plants expressing GUS from AMV-containing chimeric constructs. The results showed that elevated expression attributable to the AMV leader sequence was independent of abundance of the corresponding AMV- gus transcript, suggesting a post-transcriptional mechanism of action in vivo. Further, we describe the construction of general-purpose constitutive high expression plant promoter cassettes which incorporate the AMV translational enhancer sequence, as well a duplicated-enhancer 35S promoter in an optimized translational context.