Tagging low-temperature responsive promoters in banana using the luciferase reporter gene

Abstract

A genome-wide tagging strategy was followed for the isolation and characterization of novel banana promoters. Embryogenic cell suspensions were transformed with Agrobacterium tumefaciens containing a promoterless, codon-optimized luciferase (luc+) gene next to the right T-DNA border. Around 16,000 transgenic cell colonies were screened two months after transformation at room temperature for baseline luciferase activity and positive colonies were removed. The remaining cell cultures were re-screened in real-time at 26°C followed by a gradual decrease to 8°C. The baseline activation frequency was 0.98%, while the frequency of low-temperature luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies responsive to low temperature were regenerated to plantlets with the expression pattern monitored during different regeneration stages. Enhanced, repressed or stable expression patterns were observed and high luciferase activity was restricted to specific organs including the root, meristematic region and pseudostem. DNA gel blot analyses revealed the presence of multiple T-DNA inserts averaging 3.2 (range 1 to 5) in five independent lines tested. Banana DNA sequences flanking the right and left T-DNA border were cloned via TAIL-PCR and inverse PCR. However, some lines contained in the T-DNA flanks vector backbone parts, tandem repeats and sequences derived from the enhanced CaMV 35S promoter with part of the selectable marker gene neo from the tagging construct. RT-PCR analysis was performed to identify and confirm in lines with multiple inserts the sequence that activated luciferase expression. Confirmed sequences are currently fused to the uidA reporter gene and back-transformed to cell suspensions in order to confirm their promoter activity.