Figure 3
Cis -acting elements present in the 5'-tagged regions of tagged line ET2-17 and RT-PCR analysis of their regulation of the luc+ gene. (A) The presence of putative TATA boxes, ABRE-, and DRE-like cis-acting elements in the 5'-tagged sequences as determined by querying the PlantCARE and PLACE databases. The core sequence of the ICEr1 cis-acting element (CACATG) was manually located in sequence 17-1. Positions refer to the distance from the RB junction (bps). Primers used for RT-PCR are illustrated with dotted arrows. Drawings of 5' regions are according to scale. (B) RT-PCR analysis in different in vitro plantlet tissues at room temperature for the housekeeping actin gene (act), the codon-optimized luciferase gene (luc+) and the different 5'-tagged sequences performed at 35 amplification cycles. Actin primers flank an intron allowing discrimination between a genomic DNA product (225 bp) and the cDNA product (150 bp). Transcription of the 5'-tagged sequences 17-1, 17-2, 17-3 and 17-4 of line 17 was verified employing a sequence-specific forward primer (17-RT-1, 17-RT-2, 17-RT-3 and 17-RT-4, respectively) each time in combination with the reverse primer TAILRBLUC1. C: untransformed control plant. 17: tagged line ET2-17. Le: leaf; Ps: pseudostem; Co: corm; Ro: root, all from in vitro plants. MW: SmartLadder SF (Eurogentec, Seraing, Belgium).