FLP/FRT recombination from yeast: application of a two gene cassette scheme as an inducible system in plants

Abstract

Phytosensors are plants that are genetically engineered for sensing and reporting the presence of a specific contaminant, including agriculturally important biological agents. Phytosensors are constructed by transforming plants to contain specific biotic- or abiotic-inducible promoters fused to a reporter gene. When such transgenic plants encounter the target biotic or abiotic agent, the specific inducible promoter is triggered and subsequently drives the expression of the reporter gene, which produces a signal for detection. However, several systems lack robustness, rapid induction and promoter strength. Here, we tested the FLP/FRT recombination system in a construct containing a two gene cassette organization and examined its potential in transgenic Arabidopsis and tobacco plants using a β-glucuronidase (GUS) reporter. In this model system, a heat-shock inducible promoter was employed to control the expression of the FLP recombinase gene. Upon heat induction and subsequent active FLP-mediated excision event, the GUS gene was placed in close proximity to the 35S promoter resulting in an active GUS reporter expression. Our results demonstrate that the two gene cassette scheme of inducible FLP/FRT recombination system is functional in tobacco and Arabidopsis, providing additional insights into its possible application in phytosensing such as creating strong readout capabilities.

Keywords: Arabidopsis; FLP/FRT; GUS reporter; heat shock; phytosensing; site-specific recombination; tobacco.

Figure 1.

Schematic representation of the FLP/FRT site-specific recombination system in phytosensing. Upon induction, the FLP recombinase protein produced will recognize the FRT sites and the region between these two FRT recognition sites will be excised and the reporter gene GUS is brought under the influence of the CaMV 35S promoter resulting in plant-wide expression of GUS. FLP—flipping DNA recombinase, FRT—FLP recombination target; GUS—β-glucuronidase.

Figure 2.

Schematic diagram of the biolistic and binary vectors. (a) The source plasmid of the recombination cassette. (b) Region between the left border (LB) and right border (RB) in the binary vector pBIN-HSP-FLP-GUS-Hyg containing the recombination cassette. 35S—CaMV 35S promoter, FRT—FLP recombination target, NptII—kanamycin resistance gene, T35S—CaMV 35S terminator, HSP—heat shock promoter, FLP—flipping DNA recombinase, Stls1—intron from potato, OCS—octopine synthase terminator, GUS—β-glucuronidase, NOS—nopaline synthase terminator.

Figure 3.

Histochemical analysis of GUS expression in leaf tissues of tobacco (A) and Arabidopsis (B) plants exposed to heat-shock at 42 °C for 6 h. HSP-FLP-GUS (transgenic containing FLP/FRT recombination system); HSP-GUS (transgenic control: heat-shock promoter driving GUS expression); 35S-GUS (transgenic control: CaMV 35S promoter driving GUS expression); wild type (non-transgenic control).

Figure 4.

PCR results (representative) from heat-induced and un-induced tobacco and Arabidopsis plants. PCR fragments of size 4,165 bp and 931 bp is expected from un-induced and induced plants, respectively. Similar results were obtained by PCR analysis of several independent lines of tobacco and Arabidopsis. M—HI-LO DNA marker, 1—Tobacco un-induced, 2—Tobacco induced, 3—Arabidopsis un-induced, 4—Arabidopsis induced.