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Isolation and Characterization of the First Putative Peroxidase Gene from Oilseed Rape (Brassica napus) which is Highly Homologous to HRPC

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Bioscience Reports

Abstract

A new gene, designated as BnPrx (GenBank Accession No. DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification of cDNA Ends (RACE). The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide. The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca2+ sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted 3-D structure of BnPrx is very similar to that of HRPC. Assisted by genomic walking technology, the genomic DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN, MeJR, were found in its promoter region. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H2O2. The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HRPC.

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Abbreviations

3-D:

Three dimension

UPM:

Universal primer mix

NUP:

Nested universal primer

AP:

Adaptor primer

RACE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse transcription polymerase chain reaction

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Acknowledgments

We thank Ms. Gloria Jorgensen for helpful discussion and revision of the manuscript. This work was founded by China National “863” High-Tech Program, China Ministry of Education, Shanghai Science and Technology Committee and Shanghai Agriculture Committee.

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai , 200433, China

    Weisheng Wu, Jie Lu, Yamin Wei, Juan Lin, Shuwen Cao, **aofen Sun & Kexuan Tang

  2. Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai, 200030, China

    Kexuan Tang

  3. Mianyang Teacher’s College, Mianyang, 621000, China

    ** Wang

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  1. Weisheng Wu
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Wu, W., Lu, J., Wei, Y. et al. Isolation and Characterization of the First Putative Peroxidase Gene from Oilseed Rape (Brassica napus) which is Highly Homologous to HRPC . Biosci Rep 26, 263–280 (2006). https://doi.org/10.1007/s10540-006-9021-0

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