Isolation and functional characterization of a waterlogging-induced promoter from maize

Using thermal asymmetric interlaced polymerase chain reaction (TAIL PCR), promoter sequences of 1,444 bp (HM241145) and 1,249 bp (HM241146) from the Zea mays zinc finger (zmzf) protein gene were isolated from the maize inbred lines Mo17 and Hz32, respectively. Sequence analysis demonstrated that the Mo17 zmzf promoter contained multiple cis-regulatory elements responding to anaerobic conditions. These included two GC-motifs, five anaerobic response elements (AREs), one GT-motif, and four G-boxes. Sequence alignment revealed that there was a 195 bp DNA deletion (-258 to -452 bp) in the Hz32 zmzf promoter compared with the Mo17 promoter. The deleted fragment contained three AREs. According to quantitative real time PCR analysis, the expression of the uidA gene driven by the Mo17 zmzf promoter increased after day 1 waterlogging treatment, peaked after day 2 and decreased at days 4-8. Staining for β-glucuronidase (GUS) was observed in the roots of Mo17 zmzf transgenic lines under waterlogged conditions, but not in the leaves. GUS expression was not observed in the roots and leaves of Hz32 zmzf transgenic lines, even under submerged conditions, indicating that the Hz32 zmzf promoter was non-functional because of the deletion of three AREs. We propose that the Mo17 zmzf promoter was inducible by waterlogging, highly active, and root-specific, and might be useful for the genetic engineering of waterlogging tolerance.