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Fig. 3 | BMC Plant Biology

Fig. 3

From: A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers

Fig. 3

Simultaneous modification of two different chromosomal loci by a single-step cotransformation with BSD and CAT selectable markers in C. merolae. (a) Schematic diagram of the insertion of the mVENUS transgene with the CAT selectable marker and that of the MITmSCARLET transgene with the BSD selectable marker into two different chromosomal loci by homologous recombination. (1) Schematic diagram of the insertion of the MITmSCARLET transgene with the BSD selectable marker as in Fig. 1a. (2) Schematic diagram of the mVENUS expression cassette and the CAT selectable marker. To constitutively express mVENUS, mVENUS orf was conjugated with the C. merolae CPCC promoter and ubiquitin 3′-utr. To constitutively express the CAT selectable marker, CAT orf was conjugated with the C. merolae APCC promoter and β-tubulin 3′-utr. (3) Schematic diagram of the insertion of the mVENUS transgene with the CAT selectable marker into a chromosomal neutral locus. The mVENUS transgene was connected with the CAT selectable marker and integrated into the chromosomal region at CMK046C upstream. The first line indicates the introduced linear DNA, the second line indicates the genomic structure of the WT chromosome, and the third line indicates the expected genomic structure of the transformant (CAT-mV). (b) Selection of BS- and CP-resistant transformants in MA2 liquid medium supplemented with BS and CP. After PEG-mediated transformation of the two constructs, cells were recovered in a drug-free MA2 liquid medium for 2 days in the light. Cells were transferred to MA2 liquid medium supplemented with 1 mg/mL BS and 0.2 mg/mL CP and incubated for 14 days in the light in a 24-well plate. WT cells were also cultured as a negative control. (c) Colony PCR analyses of BS- and CP-resistant clones. The primer positions are indicated in (a). The transformants in the liquid culture above were spread onto a drug-free MA2 gellan gum plate to generate colonies of transformant clones before the PCR analyses. For detection of the BSD marker insertion either by off-target and on-target insertion (the primer set, nos. 15/16), the predicted size of the PCR product was 0.4 kb. In the PCR amplifying CMD184C-CMD186C loci (the primer set, nos. 11/12), the predicted size of the PCR product of on-target insertion of the BSD-MITmS construct was 6.3 kb and the size for off-target insertion or the WT chromosome was 3.3 kb. In the PCR amplifying the CMK046C locus and its upstream (the primer set, nos. 13/14), the predicted size of the PCR product of on-target insertion of the CAT-mV construct was 7.5 kb and the size for off-target insertion or the WT chromosome was 4.3 kb. The exact positions and sequences of the primers are indicated in Supplementary Table S1. Full length unprocessed gel images of Fig. 3c are shown in Supplementary Fig. S1b. (d) Fluorescent micrographs showing mSCARLET fluorescence in the mitochondrion (MITmSCARLET, orange) and mVENUS (green) fluorescence in the cytosol in the BSD-MITmS/CAT-mV transformant clone. PC, phase-contrast; red, autofluorescence of the chloroplast. Bar, 5 μm

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