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
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