EXPRESSION OF A CORN BIFUNCTIONAL INHIBITOR OF SERINE PROTEINASES ANDINSECT ALPHA-AMYLASES IN TRANSGENIC TOBACCO PLANTS

Corn seed endosperm contains a putative defense protein (14K-CI) that inhibits both trypsin-like serine proteinases and insect alpha-amylase s. A cDNA clone encoding 14K-CI under control of the cauliflower mosai c virus 35S promoter and the nopaline synthase polyadenylation region was introduced into tobacco plants by Agrobacterium-mediated transform ation. The presence and expression of the chimeric gene in regenerated (RO) and progeny (Pi) plants was confirmed by Southern, polymerase ch ain reaction, and Northern analyses. Protein extracts of selected tran sformed plants, but not control plants, reacted positively to corn see d 14K-CI antiserum. Comparison of 14K-CI mobilities from transgenic le aves and corn seeds after denaturing polyacrylamide gel electrophoresi s indicated that recognition and cleavage of the 14K-CI signal peptide sequence occurred in tobacco leaves. Immunological assays showed that the inhibitor was expressed in amounts up to 0.05% of the total prote in in young leaves of R1 plants. Lesser accumulation was generally det ected in older leaves. Protein extracts from transgenic plants were mo re inhibitory than were control plant extracts to bovine trypsin activ ity. Four tobacco plants with a gene lacking the 14K-CI signal peptide region accumulated 3-5-fold less inhibitor than did the highest-expre ssing plant with the full cDNA clone. Use of a double 35S promoter did not enhance 14K-CI accumulation. Post-transcriptional events appear t o be a major factor in the low accumulation of 14K-CI in tobacco.