A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.
A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.
Romero-Gómez
S
S
Biotechnology Group, Facultad de Química, Universidad Autónoma de Querétaro, CU Cerro de las Campanas s/n, Querétaro, Mexico.
Duarte-Vázquez
M A
MA
García-Almendárez
B E
BE
Mayorga-Martínez
L
L
Cervantes-Avilés
O
O
Regalado
C
C
D000595:Amino Acid Sequence; D029688:Brassica napus / Q000201:enzymology*; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D005784:Gene Amplification; D007526:Isoelectric Point; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D009195:Peroxidase / Q000235:genetics* / Q000302:isolation & purification; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification; D018517:Plant Roots / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D009195:Peroxidase