Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana.
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.
Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and Humanities, GB Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India.
Deepesh
D
Bhatt
Saurabh C
SC
Saxena
Sourabh
S
Jain
Anoop K
AK
Dobriyal
Manoj
M
Majee
Sandeep
S
Arora
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D003681:Dehydration / Q000201:enzymology; D055864:Droughts; D031748:Eleusine / Q000201:enzymology*; D004926:Escherichia coli; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D014158:Transcription, Genetic
D010940:Plant Proteins; D060387:Ascorbate Peroxidases