Abstract:
For the isolation of promoters in plantain an Agrobacterium-mediated tagging approach was chosen. A promoterless luciferase reporter gene next to the left border of the T-DNA (pluc19) was introduced into embryogenic cells of the plantain ‘Three Hand Planty’. Five months after transformation, individual cell colonies in 24-well plates were screened in vitro for baseline luciferase activity. Positive lines showing activated luciferase expression were further screened at plantlet stage. The frequency of cultures with luciferase activity comparable to or higher than the CaMV35S promoter control was 0.15%. Cell lines were also screened at 8°C four months after transformation with a new T-DNA tagging construct containing an improved luciferase reporter gene next to the right border (pETKUL2). Screening of approximately 15,887 cell colonies revealed 155 (0.98%) with luciferase activation. Twenty three independent cell colonies (0.14%) showed responsive luciferase expression at 8°C. The isolation of flanking plant DNA sequences was accomplished by TAIL PCR; an average of four flanking sequences per line was obtained. Sequences were analyzed for conserved regions and putative transcription factor binding sites. Six out of 11 flanking T-DNA sequences of three promoter tagged lines showed vector backbone integration or T-DNA rearrangements. Five putative promoter sequences were isolated and analyzed, one of which turned out to be a putative GDP-mannose pyrophosphorylase gene.
