IMPROVED HIGH-LEVEL CONSTITUTIVE FOREIGN GENE-EXPRESSION IN PLANTS USING AN AMV RNA4 UNTRANSLATED LEADER SEQUENCE

A synthetic transcribed, untranslated leader sequence from alfalfa mos aic virus RNA4 (AMV leader) has been assessed for its in vivo properti es as a sis-active 'translational activator' in transient expression a ssays 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 lea der sequence, in combination with either a CaMV 35S promoter or a dupl icated-enhancer CaMV 35S promoter construct were assessed. In transien t assay systems, the presence of a synthetic 40-base leader sequence l ead to a 20-fold elevation in GUS activity when the constructs contain ed a native cauliflower mosaic virus (CaMV) 35S promoter, whereas a 4- fold elevated expression level was observed in constructs containing a duplicated-enhancer 35S promoter. Furthermore, elevated expression in chimeric constructs was influenced by the sequence context for transl ation initiation of the marker gene. In transgenic tobacco plants the mean values for steady-state expression of GUS-containing 35S/AMV cons tructs were elevated about 8-fold relative to plasmids containing the native 35S promoter alone. A quantitative PCR approach was used to ass ess relative transcript levels in plants expressing GUS from AMV-conta ining 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-transcripti onal mechanism of action in vivo. Further, we describe the constructio n of general-purpose constitutive high expression plant promoter casse ttes which incorporate the AMV translational enhancer sequence, as wel l a duplicated-enhancer 35S promoter in an optimized translational con text.