EXPRESSION OF INTRON MODIFIED NPT-II GENES IN MONOCOTYLEDONOUS AND DICOTYLEDONOUS PLANT-CELLS

Intron sequences from monocotyledonous and dicotyledonous origin were used to abolish marker gene expression in prokaryotes (Escherichia col i and Agrobacterium tumefaciens) but permit expression in selected euk aryotic systems using the eukaryotic specific splicing mechanism. A 10 14 bp maize Shrunken-1 (Sh I) intron 1 flanked by exon 1 and exon 2 se quences was cloned into the N-terminal of the NPT II-coding region. Tr ansient gene expression analysis revealed that the modified neomycin p hosphotransferase II (NPT II) gene, driven by the cauliflower mosaic v irus (CaMV) 35S promoter, is expressed in barley protoplasts, but poor ly expressed in tobacco protoplasts. In dicotyledonous cells AU-rich s equences are known to be important for efficient splicing and therefor e an attempt was made to improve expression of the NPT II gene, contai ning the Sh 1 intron 1, in tobacco by increasing the AU content from 5 7% to 69%. Reverse transcriptase PCR analysis of RNA from transiently expressed NPT II transcripts from tobacco protoplasts revealed that de spite the increase in AU-content, NPT II was still poorly expressed. C ryptic splice sites were identified as one possible cause for missplic ing of the Sh I intron 1 in dicots and poor levels of expression. Alte rnatively, cloning of the 198 bp intron 2 of the potato STLS I gene (8 1% AU) into the N-terminal part of the NPT II-coding region resulted i n proper expression of NPT II in tobacco as well as in barley protopla sts and abolished marker gene expression in prokaryotes. The successfu l insertion of an intron into a selectable marker gene which completel y abolishes gene expression in prokaryotes, without affecting expressi on of chimeric genes in monocotyledonous and dicotyledonous plant cell s provides a suitable system to reduce the number of false-positives i n transgenic plant production.