Rapid isolation of promoter sequences by TAIL-PCR: the 5 '-flanking regions of Pal and Pgi genes from yams (Dioscorea)

Using a modified TAIL-PCR technique, the 5'-flanking regions of the phenyla lanine ammonia lyase (Pal) genes of a yam species, Dioscorea bulbifera, and the phosphoglucose isomerase (Pgi) gene of D. tokoro were successfully iso lated. Two novel modifications of the TAIL-PCR procedure introduced here, n amely (1) the use of a battery of random 10-mers (RAPD primers) as short ar bitrary primers, and (2) the use of a total of five nested, gene-specific p rimers, allow the rapid isolation of the 5'-flanking region of any gene fro m organisms with large genomes. Isolated 5'-flanking regions were fused to the gus gene, and tested for transient expression in tobacco BY2 cells. All the isolated 5'-flanking regions were shown to drive reporter gene express ion. Three Pal promoters responded to salicylic acid, presumably as a resul t of the binding of a MYB transcriptional activator to the multiple MREs (M yb Recognition Elements) present in these regions.