Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybe an seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened w ith a peroxidase-specific probe. The probe was generated by 3' rapid amplif ication of cDNA ends with Soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme l igand). Positive clones were recovered by PCR using the degenerate peroxida se-specific primer and the vector primer T-7 flanking the cloning site. Fou r cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 132 6, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature p roteins of 303, 303, 292, and 292 amino acids, respectively. The four predi cted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% a mino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity . GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and ac tive forms of the two denatured proteins were recovered after in vitro fold ing in a medium containing hemin, urea acid Ca2+. GmEpa1 and GmEpa2 message s were detected in developing seed and root, while GmEpb1 and GmEpb2 messag es were present in root, leaf, stem and seed pod. These cDNAs and cDNA-spec ific primers will allow investigations into peroxidase's role in developmen t, stress response and in other physiological processes. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.