Peter Eckes

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants.

SummaryWe have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH3. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.

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 coli and Agrobacterium tumefaciens) but permit expression in selected eukaryotic systems using the eukaryotic specific splicing mechanism. A 1014 bp maize Shrunken-1 (Sh 1) intron 1 flanked by exon1 and exon2 sequences was cloned into the N-terminal of the NPT II-coding region. Transient gene expression analysis revealed that the modified neomycin phosphotransferase II (NPT II) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, is expressed in barley protoplasts, but poorly expressed in tobacco protoplasts. In dicotyledonous cells AU-rich sequences are known to be important for efficient splicing and therefore an attempt was made to improve expression of the NPT II gene, containing the Sh 1 intron 1, in tobacco by increasing the AU content from 57% to 69%. Reverse transcriptase PCR analysis of RNA from transiently expressed NPT II transcripts from tobacco protoplasts revealed that despite the increase in AU-content, NPT II was still poorly expressed. Cryptic splice sites were identified as one possible cause for missplicing of the Sh1 intron 1 in dicots and poor levels of expression. Alternatively, cloning of the 198 bp intron 2 of the potato STLS 1 gene (81% AU) into the N-terminal part of the NPT II-coding region resulted in proper expression of NPT II in tobacco as well as in barley protoplasts and abolished marker gene expression in prokaryotes. The successful insertion of an intron into a selectable marker gene which completely abolishes gene expression in prokaryotes, without affecting expression of chimeric genes in monocotyledonous and dicotyledonous plant cells provides a suitable system to reduce the number of false-positives in transgenic plant production.