EXPRESSION OF A REPORTER GENE IS REDUCED BY A RIBOZYME IN TRANSGENIC PLANTS

A chimeric gene encoding a ribozyme under the control of the cauliflow er mosaic Virus (CaMV) 35S promoter was introduced into transgenic tob acco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression a ssays in plant protoplasts. The ribozyme gene was transferred into tra nsgenic tobacco plants expressing an rbcS-npt chimeric gene as an indi cator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduce d npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant exp ressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achievi ng a significant reduction in target gene expression.