A Modified TAIL-PCR and Its Application in Isolating Gene Promoter of Wheat

Abstract:

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°C instead of 44°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.

Key words:TAIL-PCR, RAPD (random amplified polymorphic DNA), promoter