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Functional characterization of flavanone 3-hydroxylase gene from Phyllanthus emblica (L.)

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Abstract

Phyllanthus emblica (L.) is one of the important medicinal plants possessing antioxidant, anti-tumor, hepatoprotective and immune-modulatory properties imparted by presence of high amount of flavonoid and vitamin C contents. Despite the medicinal value of P. emblica, molecular characterization of genes such as those involved in biosynthesis of flavonoids is lacking. Flavanone 3-hydroxylase (F3H) [EC 1.14.11.9] is an important regulatory enzyme of the flavonoid pathway which catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydroflavonol. Herein, we isolated and investigated F3H gene from P. emblica, expressed functional recombinant proteins in microbial expression system and studied gene expression in different developmental stages. A full length PeF3H cDNA consisting of 1,469 bp (GenBank ID KF020698) has an open reading frame (ORF) of 1,095 bp, encoding a protein of 364 amino acids. Deduced amino acid analysis confirmed the presence of conserved amino acid residues which ligate ferrous iron and participates in 2-oxoglutarate binding (R-X-S). Three-dimensional protein structure modeling predicted that F3H had a jelly roll in the enzyme core, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including F3Hs. Phylogenetic analysis revealed that PeF3H was evolutionary closer F3H sequences of Triticum aestivum and Zea mays. Expression of PeF3H in an expression vector yielded a functional protein as HPLC analysis indicated conversion of naringenin to dihydrokaempferol after incubation with purified protein. Gene expression was analyzed in different developmental stages using q RT-PCR and was found to be highest in flowers as compared to leaves and fruits. In fruits, expression of PeF3H was increasing with the growth of the fruit.

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Abbreviations

F3H:

Flavanone 3-hydroxylase

RACE:

Rapid Amplification of cDNA Ends

SMARTTM :

Switching Mechanism At 5′ End of RNA Transcript

ORF:

Open Reading Frame

BLAST:

Basic local alignment search tool

GSP:

Gene specific primers.

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Acknowledgments

Authors are thankful to Department of Science and Technology (DST) Govt. of India for their financial support to carry out this research work. Avneesh Kumar is also very thankful of Council of Scientific & Industrial Research (CSIR), India for providing scholarship during the research period.

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Authors and Affiliations

  1. Department of Biotechnology, Panjab University, Chandigarh, 160014, India

    Avneesh Kumar, Baljinder Singh & Kashmir Singh

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  1. Avneesh Kumar
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  2. Baljinder Singh
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  3. Kashmir Singh
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Correspondence to Kashmir Singh.

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Data archiving statement

The Sequence information of F3H has been submitted to NCBI Genbank database vide under accession no. KF020698

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Kumar, A., Singh, B. & Singh, K. Functional characterization of flavanone 3-hydroxylase gene from Phyllanthus emblica (L.). J. Plant Biochem. Biotechnol. 24, 453–460 (2015). https://doi.org/10.1007/s13562-014-0296-0

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