Enrichments of gene replacement events by Agrobacterium-mediated recombinase-mediated cassette exchange

Abstract

We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacterium-mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes (codA) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobacco Agrobacterium-mediated transformation.

Keywords: Gene replacement; Negative selection; RMCE.

Fig. 1

Molecular strategy for producing marker-free targeted transgenic plants by RMCE. When the exchange vector p2nd30 is introduced into target plants (cod23A, B), recombinase catalyzes double-crossover between the two Rs to replace the target cassette with the exchange cassette. As a result, the transgenic plants (Type I, Type II) that have an exchange cassette at the target locus are produced. The diagram shows the integrated T-DNA region containing a target cassette of target plants (cod23A, B), the T-DNA region of exchange vector p2nd30, the integrated T-DNA region containing an exchange cassette of transgenic plants (Type I, Type II), the position of probes (P1, P2, P3, P4, P5) for Southern analysis, the size (kb) of DNA fragments detected by the probes, restriction enzyme sites (E: EcoRI; EV: EcoRV; H: HindIII) and the DNA regions amplified with PCR primers (SPR3-Luc8a, Luc3-SPL3, SPR3-Luc3, Luc8a-SPL3). codA cytosine deaminase gene, npt neomycin phosphotransferase gene, gfp green-fluorescent protein gene, ipt isopentenyl transferase gene, R recombinase gene, luc firefly luciferase gene, Rs recognition site, RB and LB right and left border sequences of a T-DNA

Fig. 2

Southern analysis of T0 target plant (cod23) and transgenic plants in which the genes were exchanged from two target lines (cod23A, B). Genomic DNA was digested with three restriction enzymes (E: EcoRI; EV: EcoRV; H: HindIII) and hybridized with five probes (P1, P2, P3, P4, P5), respectively