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Molecular Biology Reports

, Volume 40, Issue 2, pp 1155–1165 | Cite as

Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana

  • Deepesh Bhatt
  • Saurabh C. Saxena
  • Sourabh Jain
  • Anoop K. Dobriyal
  • Manoj Majee
  • Sandeep Arora
Article

Abstract

Eleusine coracana (finger millet) is a stress-hardy but under-utilized cereal crop that possesses an efficient antioxidant defense system. The plant is capable of enduring long durations of water deficit stress. Experiments were conducted to clone a potent stress responsive isoform of ascorbate peroxidase and validate its role under drought stress. Reverse transcriptase PCR was used to obtain the partial cDNA of apx1 gene, from a meticulously screened drought tolerant genotype of E. coracana (PR202). Using RACE strategy, the full length apx1 cDNA was cloned and sequenced. The cDNA length of the E. coracana apx1 (Ec-apx1) gene is 1,047 bp with a 750 bp ORF, encoding a 250 amino acid protein having a molecular weight of 28.5 kDa. The identity of the amino acid sequence, deduced from the cDNA, with the APX family homologs was about 74–97 %. The full-length apx1 ORF was sub-cloned in a prokaryotic expression vector pET23b. The recombinant fusion protein, Ec-apx1, had high expression level in BL21 strain of E. coli and exhibited APX enzyme activity. The structure–function relationship of the protein was deduced by modelling a three-dimensional structure of Ec-apx1, on the basis of comparative homology using SWISS-MODEL. Real time PCR analysis of Ec-apx1 expression at mRNA level showed that the transcript increased under drought stress, with maximum levels attained 5-days after imposition of stress. Our results suggest that Ec-apx1 has a distinct pattern of expression and plays a pivotal role in drought stress tolerance. Therefore, the cloned isoform of ascorbate peroxidase can be used for developing stress tolerant genotypes of important crops, through transgenic approach.

Keywords

Cloning Expression Ascorbate peroxidase Drought stress 

Abbreviations

ORF

Open reading frame

RACE

Rapid amplification of cDNA ends

Ec-apx1

Eleusine coracana cytosolic ascorbate peroxidase1

PCR

Polymerase chain reaction

Notes

Acknowledgments

The authors (SA and DB) are thankful to the Department of Biotechnology, Govt. of India, for providing financial support under the research project “Program mode support for agricultural biotechnology” at G.B. Pant University of Agriculture and Technology, Pantnagar.

Supplementary material

11033_2012_2157_MOESM1_ESM.tif (1.3 mb)
Supplementary material 1 (TIFF 1365 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Deepesh Bhatt
    • 1
  • Saurabh C. Saxena
    • 2
  • Sourabh Jain
    • 3
  • Anoop K. Dobriyal
    • 4
  • Manoj Majee
    • 2
  • Sandeep Arora
    • 1
  1. 1.Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and HumanitiesGB Pant University of Agriculture and TechnologyPantnagarIndia
  2. 2.National Institute of Plant Genome ResearchNew DelhiIndia
  3. 3.National Research Centre on Plant BiotechnologyNew DelhiIndia
  4. 4.Department of BiotechnologyHNB Garwal University (Uttarakhand)SrinagarIndia

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