Expression In Tobacco Protoplasts Research Articles

Transient gene expression in tobacco protoplasts: II. Comparison of the reporter gene systems for CAT, NPT II, and GUS

The reporter genes for Chloramphenicolacetyltransferase (CAT), Neomycinphosphotransferase-(NPT)-II and β-Glucuronidase (GUS) were compared in transient gene expression experiments in tobacco mesophyll protoplasts. For this purpose, nearly identical chimeric genes controlled by the CaMV 35 S promoter were constructed. The detection level of each system was determined yielding the following order of relative sensitivity: CAT<NPT II<GUS.

Transient gene expression in tobacco protoplasts: I. Time course of CAT appearance

The early events of transient gene expression have been investigated monitoring CAT activity in tobacco protoplasts encoded by the recombinant plasmid pRT101cat. The first appearance of CAT activity was observed within 30 minutes after the outset of cultivation, and maximal values were obtained between four and 24 hours. CAT expression, at the level of RNA synthesis, could not be inhibited by cordycepin (3'deoxyadenine) added one hour after protoplast plating, whereas cycloheximide, an inhibitor of protein synthesis, showed an influence during the first four hours. This indicates a rapid decay of biologically active forms of both the DNA transferred and the CAT-mRNA synthesized within the first hours. These results suggest that in the tobacco protoplast system CAT protein stability lasts up to two weeks rather than a continuous synthesis of new enzyme.

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl2, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.