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Control of gene expression in tobacco cells using a bacterial operator-repressor system
- Source :
- The EMBO Journal. 11:1251-1259
- Publication Year :
- 1992
- Publisher :
- Wiley, 1992.
-
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.
- Subjects :
- Molecular Sequence Data
Restriction Mapping
lac operon
Repressor
Lac repressor
Biology
Polymerase Chain Reaction
General Biochemistry, Genetics and Molecular Biology
Bacterial Proteins
Mosaic Viruses
Tobacco
Gene expression
Escherichia coli
Lac Repressors
Promoter Regions, Genetic
Molecular Biology
Glucuronidase
Regulation of gene expression
Reporter gene
Expression vector
Base Sequence
General Immunology and Microbiology
Escherichia coli Proteins
Protoplasts
General Neuroscience
fungi
food and beverages
Promoter
Gene Expression Regulation, Bacterial
Plants, Genetically Modified
TATA Box
Molecular biology
Repressor Proteins
carbohydrates (lipids)
Plants, Toxic
Lac Operon
Oligodeoxyribonucleotides
Genes, Bacterial
RNA, Viral
bacteria
Research Article
Plasmids
Subjects
Details
- ISSN :
- 02614189
- Volume :
- 11
- Database :
- OpenAIRE
- Journal :
- The EMBO Journal
- Accession number :
- edsair.doi.dedup.....3ece4d8215864d71958e734760788004
- Full Text :
- https://doi.org/10.1002/j.1460-2075.1992.tb05169.x