EXPRESSION OF MULTIPLE EUKARYOTIC GENES FROM A SINGLE PROMOTER IN NICOTIANA

We engineered an expression unit composed of three eukaryotic genes dr iven by a single plant-active promoter and demonstrated functional exp ression in planta, The individual genes were linked as translational f usions to produce a polyprotein using spacer sequences encoding specif ic heptapeptide cleavage recognition sites for NIa protease of tobacco vein mottling virus (TVMV). The NIa gene itself was included as the s econd gene of the multi-gene unit, The first and third genes, obtained from the T-R region of pTi15955, encoded enzymatic functions associat ed with the mannityl opine biosynthetic pathway, The mannityl opine co njugase gene (mas2) was the first unit of the construct and provided t he native plant-active promoter and 5' untranslated regulatory sequenc e, The third gene (mas1), encoding the mannityl opine reductase, furni shed the native 3' untranslated region, Cfs-processing of the polyprot ein by the NIa protease domain was demonstrated in vitro using rabbit reticulocyte lysate and wheat germ cell-free translation systems, Toba cco plant cells transformed with the multi-gene unit produced detectab le levels of mannopine, mannopinic acid, and their biosynthetic interm ediates, deoxyfructosyl-glutamate and deoxyfructosyl-glutamine. This i ndicates that the polygene construct results in a set of functional en zymatic activities that constitute a complete metabolic pathway.