PhiC31 Integrase-Mediated Site-Specific Recombination In Barley

Molecular Analysis of the Transgenic Plants

For total DNA isolation [45], leaf segments were harvested, frozen in liquid nitrogen and stored at −80°C. Homogenization was performed using a TissueLyser™ from Qiagen (Hilden, Germany).

PCR was performed in a thermocycler (DNA-Engine™ PTC-0200, Bio-Rad, Munich, Germany), involving an initial denaturing step at 95°C for 5 minutes followed by 35 cycles (94°C for 30 s; 60°C for 30 s; 72°C for 1–2 min). The amplified fragments were run on 1–1.5% agarose gel containing 4 µg/100 ml ethidium bromide.

The positions of the primer binding sites are depicted in Figure 1. The primer sequences are given in Supplemental Figure 1. The primers were used as follows: i) to detect the gusA gene and production of the GUS probe for DNA gel blots: gusFw and gusRev; ii) to detect the GFP gene and production of the GFP probe for DNA gel blots: gfpFw and gfpRev; and iii) to show the presence of the integrase-encoding sequences of pICH14313 and pICH13130 and production of the INT probe for DNA gel blot: C31IntFw and C31IntRev.

The site-specific recombination was molecularly confirmed by PCR in which the excision footprint sequences, including the hybrid recombination product attR, were amplified with the RecFw and RecRev primers. In the case of the non-recombinant locus HW511, the PCR amplification resulted in a 1.5-kb fragment (Figure 1). If recombination occurred at the target attP and attB sites, the derivative locus HW511R was produced, and a 400-bp fragment was obtained. Detection of an excision circle was achieved using the outwards primers gfpFw2 and gfpRev2. An amplification product of 696 bp can only be synthesized in the case of a circular fragment, attL-GFP-Tnos (Figure 1).

For sequence analysis, the PCR products were subjected to direct DNA sequencing by GATC Biotech (Konstanz, Germany).

The DNA gel blots were conducted according to standard protocols [46]. The primers used to produce the probes are given in Supplemental Figure 1. The DNA fragments were separated using 0.6% agarose gels and transferred onto a nylon membrane (Biodyne B; Pall, USA). After blotting, the membranes were hybridized with [32P]-labeled DNA fragments. To estimate the copy number of the target locus HW511, total DNA was digested with HindIII and hybridized with either the GFP or GUS probe. These strategies resulted in fragments containing the homologous vector sequence along with a genomic DNA stretch of unpredictable length, which is expected to be different for every individually integrated vector sequence. These size differences allow the transgene copy numbers to be assessed. The presence of pICH13130- or pICH14313-T-DNA was also confirmed by digesting total barley DNA with HindIII, which releases a 970-bp fragment containing the integrase sequence covered by the INT probe.

To detect the recombinant locus HW511R, total plant DNA was digested with SacI. In the case of HW511R, a 2.6-kb fragment covered by the GUS probe is released, whereas the unaltered target locus results in a fragment of 3.7 kb homologous to GUS and GFP.