Using a modified TAIL-PCR technique, the 5' -flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here: using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29 degrees C instead of 44 degrees C as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles and reducing five low-stringency cycles; and using single primers for the third round of product identification. Isolated 5' -flanking region was fused to the GUS gene, and tested for expression in Arabidopsis plants. Histochemical analysis of the transgenic plants showed the report gene was driven by isolated 5'-flanking region. Modified TAIL-PCR technique could isolate rapidly the promoter of any gene from organisms with large genomes.
Base Sequence ; Genes, Plant ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics ; Triticum ; genetics ; metabolism