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Molecular cloning, characterization and expression analysis of CmAPX

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Abstract

The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew.

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Abbreviations

APX:

Ascorbate peroxidase

ORF:

Open reading frame

RACE:

Rapid amplification of cDNA ends

PCR:

Polymerase chain reaction

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

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Acknowledgments

This work was supported by a grant from the Provincial Natural Science Foundation of Gansu (ZS031-A25-042-D) and Provincial High Quality Seeds Project of Shandong, China.

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Authors and Affiliations

  1. College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, 271018, China

    Hong Cheng & Yumeng Huo

  2. Institute of Vegetable, Gansu Academy of Agricultural Science, Lanzhou, 730070, China

    Hong Cheng

  3. Institute of Vegetable, Shandong Academy of Agricultural Science, Jinan, 250100, China

    Qiwei He & Lixia Hou

  4. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China

    Junfen Lv

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  1. Hong Cheng
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  2. Qiwei He
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  3. Yumeng Huo
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Correspondence to Qiwei He.

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Cheng, H., He, Q., Huo, Y. et al. Molecular cloning, characterization and expression analysis of CmAPX. Mol Biol Rep 36, 1531–1537 (2009). https://doi.org/10.1007/s11033-008-9345-x

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