Figure 1.
Genetic structure of the expression vectors and the recombinant locus.
Note that only the T-DNA part of the vectors is illustrated (not drawn to scale). The integrase is expressed from the pICH13130 or pICH14313 constructs. The pHW511 vector harbors the attP and attB sequences, which serve as targets for recombination that results in the derivative locus HW511R and the released excision circle. To select transgenic plants carrying the locus ICH13130 or ICH14313, co-transformation with the vector carrying an HPT selection marker was performed (p6U, not shown). Abbreviations: phiC31, phage phiC31 recombinase coding sequence [41]; Pubi, maize ubiquitin 1 promoter; Pspm, promoter of the maize suppressor-mutator transposable element spm; intron, sequence derived from an intron of the Petunia hybrida Psk7 gene (GenBank accession number AJ224165); NLS, SV40 T antigen nuclear localization signal, amino acids PKKKRKV [31]; Tnos, nopaline synthase terminator; T35S, cauliflower mosaic virus (CaMV) 35S terminator; GFP, coding sequence for the green fluorescent protein; GUS, β-glucuronidase (gusA) gene; Pact, rice actin 1 promoter; attP and attB, Streptomyces phage phiC31 recombination sites; attR and attL, hybrid products that originate from the recombination between attP and attB; HPT, hygromycin phosphotransferase gene.
Figure 2.
Molecular and phenotypic analysis of hybrids obtained by combining transgenic plants carrying target and integrase loci.
(a–f) PCR analysis was performed using the gusFw and gusRev (lane 1), gfpFw and gfpRev (lane 2), C31IntFw and C31IntRev (lane 3), and RecFw and RecRev (lanes 4) primers on total DNA from untransformed plants (a; Wt) and total DNA from Hv10 and its descendants (b–f); (b) primary transformant (T0) containing the HW511 locus; (c) sF1, plant obtained by selfing of Hv10 containing the HW511 locus; (d) hybrid F1 carrying a phiC31 integrase and a recombined locus HW511R; (e) F2 plant that inherited the recombined locus HW511R and the integrase locus ICH14313; (f) F2 plant that inherited the recombined locus but no integrase locus. The positions of the primer binding sites are given in Figure 1. (g–l) Analysis of GUS expression in primary transgenic plants (T0) carrying the HW511 locus and no integrase (g, h, i) and a hybrid F1 plant (Hv35×14313–23) that harbors a recombined locus (j, k, l) using leaf tissue (g, j), ovary and stamen (h, k) as well as pollen (i, l).
Figure 3.
Footprint-sequence analysis of HW511R and the excision circle.
Footprint sequences of hybrid attR or attL resulting from a reaction between attB (black) and attP (white). Both recombination products share an identical 3-bp long central core, ‘TTG’, where the crossover occurs. Electropherograms were taken from sequencing analysis. PCR-fragments were sequenced by using the primers GfpFw2 and GfpRev2 or RecFw and RecRev. The positions of the primers and the adjacent vector parts are indicated.
Table 1.
Analysis of hybrid F1 plants co-transgenic for both integrase and target sequence.
Figure 4.
Molecular and phenotypic analysis of hybrid F2 plants.
(a) DNA gel blot analysis of three transgenic plants containing a single copy of the target locus (Hv10, Hv18, Hv35) and their descendants obtained by selfing (sF1) or crossing with transgenic plants carrying an integrase locus (hybrid F2). Plants that contain a recombined locus and no integrase are highlighted. For DNA preparation, 5-week-old plants were used. The designated restriction enzymes and sequence regions homologous to the hybridization probes are depicted in Figure 1. As controls, untransformed plants (Wt) are included. (b) Reporter gene assays. To monitor GFP expression, fluorescence microscopy was performed using root tips. β-glucuronidase (GUS) staining was carried out using leaf material. (c) PCR analysis.
Table 2.
Sexual transmission of recombinant loci.