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Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum

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Abstract

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

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Acknowledgments

DPW thanks University Grant Commission, India for fellowships. This work is supported by funding by Department of Biotechnology, India.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Dhammaprakash Pandhari Wankhede, Dipul Kumar Biswas & Alok Krishna Sinha

  2. CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

    Subramani Rajkumar

  3. National Bureau of Plant Genetic Resource, New Delhi, India

    Subramani Rajkumar

Authors
  1. Dhammaprakash Pandhari Wankhede
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  2. Dipul Kumar Biswas
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  3. Subramani Rajkumar
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  4. Alok Krishna Sinha
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Correspondence to Alok Krishna Sinha.

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Handling Editor: Klaus Harter

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Supplementary Fig. 1

Heterologous expression of GST fused PhPLR. GST-PhPLR was induced in E. coli BL21 cell with IPTG (TIFF 2931 kb)

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Wankhede, D.P., Biswas, D.K., Rajkumar, S. et al. Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum . Protoplasma 250, 1239–1249 (2013). https://doi.org/10.1007/s00709-013-0505-z

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  • DOI: https://doi.org/10.1007/s00709-013-0505-z

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