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.

Gene expression during plant embryogenesis and germination: an overview.

“…Overexpression and ectopic expression of wild-type and mutant genes represent another approach. Genes can be overexpressed in appropriate time and space when driven by enhanced, homologous promoters, ectopically expressed in tissues or developmental stages in which the gene would not normally be expressed using the numerous heterologous tissue-specific promoters now available, transiently induced with small molecules such as tetracycline (Gatz et al, 1991), or repressed reversibly with the lac/lPTG system (Wilde et al, 1992). Cell and tissue ablation with dominant "killer" genes, such as diphtheria toxin or ribonuclease, driven by cell-and tissue-specific embryo promoters (e.g., Mariani et al, 1990, and references therein) will provide further insight into early embryo gene function.…”

A transcription activation system for regulated gene expression in transgenic plants

“…To overcome these limitations several attempts have been made to develop chemically regulated gene expression systems (6)(7)(8)(9)(10). Again, these are not entirely satisfactory as they are either relatively inefficient (high background and͞or only modest induction), dependent on sustained gene repression, not applicable to commonly studied plant species, or reliant on the application of chemicals at concentrations that may be toxic to plants.…”

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

“…In order to achieve an optimal expression of transgenes with minimum undesirable effects, it is highly desirable to regulate the expression of transgenes in a controllable fashion. A number of chemical inducible gene regulation systems, or gene switches, have been developed based on a diverse collection of non-plant regulatory elements that respond to a variety of chemicals (Gatz et al 1992;Wilde et al 1992;Williams et al 1992;Mett et al 1993;Rieping et al 1994;Weinmann et al 1994;Aoyama and Chua 1997;Caddick et al 1998;Bohner et al 1999;Martinez et al 1999a, b;Bruce et al 2000;Padidam et al 2003). A chemical inducible gene regulation system that specifically regulates transgene expression in response to an exogenous inducer at a particular stage of plant development or in a specific organ is very valuable when using transgenes whose constitutive over expression is detrimental or lethal to the plant.…”