Abstract
The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.
Highlights
Plant genomic engineering took a big step forward after the introduction of site-specific recombinases, a group of enzymes that are capable of catalyzing reactions between two short, specific recombination sites [1,2]
The corresponding chromosomal loci of the transgenic barley plants are designated as HW511, HW511R and ICH13130 or ICH14313 for the integrase source
The target insert green fluorescent protein (GFP)-Tnos is flanked by att recognition sites for the Streptomyces phage phiC31 integrase, which itself is expressed from a second locus ICH13130 or ICH14313
Summary
Plant genomic engineering took a big step forward after the introduction of site-specific recombinases, a group of enzymes that are capable of catalyzing reactions between two short, specific recombination sites [1,2]. Recombination between directly repeated target recognition sites results in a loss of the intervening DNA [4]. This technique has been used in plant systems to remove unwanted selectable marker genes [5,6], resolve complex integration patterns [7,8,9], and activate genes by excising sequences that block the reading frame [10,11,12]. If the recognition sites are inverted, the recombination causes the sequence located in between to flip, which can be used to reconstitute a reading frame and thereby activate a plant transgene [13,14]. Site-specific recombination occurring between recognition sites in trans can result in a reciprocal translocation of two linear DNA molecules or in a targeted integration if at least one DNA molecule is circular [15,16]
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