Expression of a bean storage protein 'phaseolin minigene' in foreign plant tissues

Using the phaseolin gene and its cDNA counterpart we constructed a mutant phaseolin gene lacking the five introns but retaining its natural 5' and 3' plant-regulatory sequences. This mutant phaseolin gene (minigene) was inserted into the Ti-plasmid of Agrobacterium tumefaciens strain 15955 which allowed its transfer and integration into the tobacco genome. Full-length and correctly initiated phaseolin mRNA was found among the poly(A)+RNA isolated from plant callus transformed with the minigene construction by using RNA-DNA hybridization and S1 nuclease mapping techniques. The presence of phaseolin polypeptides in soluble protein extracts from transformed tobacco tissues was confirmed by immunological methods. These results demonstrate that phaseolin gene introns and intron splicing are not a necessary requirement for biogenesis of stable phaseolin mRNA and that no alternative splice site was introduced by the removal of five introns.