title: Control of gene expression in tobacco cells using a bacterial operator-repressor system.
abstract: We have investigated the efficacy of using the Escherichia coli lac operator-repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta-glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator-free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB-operator-gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl-beta-D-thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2-fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB-operator-gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15-fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor--operator complex and effectively block transcription of a downstream gene.
authors.affiliation: ICI Joint Laboratory, University of Leicester, UK.
authors.forename: R J
authors.initials: RJ
authors.lastname: Wilde
authors.forename: D
authors.initials: D
authors.lastname: Shufflebottom
authors.forename: S
authors.initials: S
authors.lastname: Cooke
authors.forename: I
authors.initials: I
authors.lastname: Jasinska
authors.forename: A
authors.initials: A
authors.lastname: Merryweather
authors.forename: R
authors.initials: R
authors.lastname: Beri
authors.forename: W J
authors.initials: WJ
authors.lastname: Brammar
authors.forename: M
authors.initials: M
authors.lastname: Bevan
authors.forename: W
authors.initials: W
authors.lastname: Schuch
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005966:Glucuronidase / Q000235:genetics; D007763:Lac Operon; D056968:Lac Repressors; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000502:physiology; D012367:RNA, Viral / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D015183:Restriction Mapping; D016385:TATA Box; D014026:Nicotiana / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D012097:Repressor Proteins; D005966:Glucuronidase