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

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Title Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana
 
Creator Bhatt, Deepesh
Saxena, Saurabh C.
Jain, Sourabh
Dobriyal, Anoop K.
Majee, Manoj
Arora, Sandeep
 
Subject Cloning Á Expression
Ascorbate peroxidase
Drought stress
 
Description Accepted date: 1 October 2012
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.
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.
 
Date 2015-11-02T07:21:33Z
2015-11-02T07:21:33Z
2013
 
Type Article
 
Identifier Mol. Biol. Rep., 40(2): 1155-1165
0301-4851
http://link.springer.com/article/10.1007%2Fs11033-012-2157-z
http://172.16.0.77:8080/jspui/handle/123456789/296
 
Language en_US
 
Publisher Springer