A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants

Tobacco plants expressing constitutive chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity were obtained by transformation with a chimeric CAT gene driven by the cauliflower mosaic virus 19S promoter. Plants expressing different levels of CAT activity were retransformed with vectors containing CAT sequences transcriptionally fused in the antisense orientation between the coding region of the hygromycin-resistance gene and the 3′ end of the nopaline synthase gene. Several plants regenerated on high concentrations of hygromycin exhibited a loss of CAT activity, whereas plants retransformed with a vector conferring hygromycin resistance but lacking the antisense CAT sequence showed no reduction in CAT activity. RNA blot analysis revealed a strong correlation between the degree of CAT gene inactivation and the levels of stable antisense transcripts accumulated. The possibility that CAT gene inactivation was due to transferred DNA instability was discounted since a kanamycin-resistance gene contiguous with the CAT gene was expressed normally, and DNA blot analysis indicated no loss or rearrangements of the transferred DNA fragments. Thus, the imposed selection pressure enabled the selection of plants expressing high levels of stable bifunctional antisense transcripts that inhibited the activity of the targeted gene.