Radish phospholipid hydroperoxide glutathione peroxidase gene structure and upstream regulatory sequence analysis

A novel radish RsPHGPx cDNA, which encodes a functional phospholipid hydroperoxide glutathione peroxidase (PHGPx) protein, was identified in the previous work. In the study genomic organization and the upstream regulatory sequence analysis of this gene was presented. Southern blot analysis showed that RsPHGPx gene existed in radish genome in manner of single copy. Moreover, a 3.3 kb genomic DNA fragment of RsPHGPx gene was isolated by combination of common PCR and genome-walking method. Sequence analysis on this genomic fragment demonstrated that RsPHGPx gene consists of seven exons separated by six introns, and suggested that a short 5'-flanking sequence immediately before the exon I should be the putative RsPHGPx promoter region, which is proceeded by the upstream neighboring biotin synthase gene. Cis-acting elements search showed that the putative promoter contains elements responsive to hormones (eg. E-Box and W-Box), abiotic stresses (eg. MYB and MYC binding sites), and light (Box H and I-Box), etc. Northern blot analysis indicated that the expression of RsPHGPx was subjected to up-regulation of chilling and down-regulation of ABA and successive illumination (in etiolated seedlings), implying the regulatory roles of some predicted elements. However the up-regulation effect of herbicide paraquat, which can induce oxidative stress, suggested the presence of some unknown elements in the promoter region. This is the first report on gene structure and upstream regulatory sequence analysis in reported plant PHGPx genes, which will be a prerequisite to understand regulatory mechanism of PHGPx gene expression in plants.