Characterization of promoter tagged lines

Abstract

We have developed a fast, sensitive and high-throughput method for the identification and isolation in bananas of novel promoters and genes. Following the Agrobacterium-mediated transformation of embryogenic cell suspension cultures with a promoterless luciferase (luc) gene, candidate promoters are characterized in planta during the early stages of in vitro development. Screening for luciferase (LUC) enzymatic activity in transgenic cell colonies has been performed without induction treatments. We have previously reported that transformation of a luciferase reporter trap linked to the left T-DNA border resulted in an activation frequency of 0.03-0.07%. All tagged candidates have now been propagated for plant regeneration and molecular analysis. Southern hybridization and sequence analysis of several independent lines demonstrated the integration of 2-6 T-DNA copies in the genome with the frequent presence of the vector backbone or complex rearrangements. Therefore, TAIL-PCR products from selected tagged lines were individually cloned and sequenced. One tagged line displayed constitutive expression higher than that of the CaMV 35S promoter, which persisted in the regenerated plants, both in vitro and in the greenhouse. Detailed analysis of the LUC expression in all plant tissues will be presented. One of the T-DNA insertions in this line occurred in a metallothionein-like protein gene, demonstrating the feasibility of the technique with bananas. An improved tagging construct, which contains the codon-optimized luc+ gene fused close to the right T-DNA border, has been prepared. Transformation with this construct resulted in a 40-fold increase in activation frequency (up to 2.5% without induction) compared to the original tagging construct. Data on LUC expression in hundreds of independent tagged lines will be presented. This T-DNA trapping technology is currently being applied to isolate inducible genes and promoters.