Molecular cloning, expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isola ted from a leaf cDNA library of Chinese cabbage. The predicted amino acid s equence of C2C-Prx has 2 conserved cysteines and several peptide domains pr esent in most of the 2Cys-Prx subfamily members. It shows the highest seque nce homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%) . Southern analysis using the cDNA insert of C2C-Prx revealed that it consi sts of a small multigene family in Chinese cabbage genome. RNA blot analysi s showed that the gene was predominantly expressed in the leaf tissue of Ch inese cabbage seedlings, but the mRNA was generally expressed in most tissu es of mature plant, except roots. The expression of C2C-Prx was slightly in duced by treatment with H2O2 (100 mu M) or Fe3+/O-2/DTT oxidation system, b ut not by ABA (50 mu M) or GA(3) (10 mu M). The C2C-Prx is encoded as a pre protein of 273 amino acids containing a putative chloroplast-targeting sign al of 65 amino acids at its N-terminus. The N-terminally truncated recombin ant protein (Delta C2C-Prx) migrates as a dimer in a non-reducing SDS-polya crylamide gel and as a monomer in a reducing condition. The Delta C2C-Prx s hows no immuno cross-reactivity to antiserum of the yeast thiol-specific an tioxidant protein, and vice versa. The Delta C2C-Prx prevents the inactivat ion of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxi dation system. In the yeast thioredoxin system containing thioredoxin reduc tase, thioredoxin, and NADPH, the Delta C2C-Prx exhibits peroxidase activit y on H2O2.