Isolation, restriction mapping, and promoter sequence analysis of an isoperoxidase gene from Korean-radish, Raphanus sativus L.

Abstract

A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named pocK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb SalI fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative TATA box, CAAT box, and TGA1b binding sequence (5′ TGACGT) are present 718 bp upstream from the start codon.