Overexpression of homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) enhances α-tocopherol content in transgenic tobacco

Abstract

Photosynthetic organisms synthesize the amphipathic antioxidants called tocopherols which are essential components of the human diet. To increase the α-tocopherol (vitamin E) content, Arabidopsis genes encoding homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) were constitutively expressed individually and in combination (HPT:TC) in tobacco plant by Agrobacterium mediated transformation. The transgene was confirmed by polymerase chain reaction (PCR), transgene expression was studied by reverse transcriptase (RT)-PCR, integration of the transgene in the plant genome was confirmed by Southern blot, and α-tocopherol content was quantified using high performance liquid chromatography (HPLC). The α-tocopherol content in transgenic tobacco plants expressing HPT, TC, and HPT:TC was increased by 5.4-, 4.0-, and 7.1-fold, respectively, when compared to the wild type (WT). These results indicate that, the HPT and TC activities are critical for enhancing the vitamin E content in tobacco plants.

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Abbreviations

BAP:

benzylaminopurine

CTAB:

cetyltrimethylammonium bromide

DPPH:

1,1-diphenyl-2-picrylhydrazyl

FRAP:

ferric reducing antioxidant power

HPLC:

high performance liquid chromatography

HPT:

homogentisate phytyltransferase

IAA:

indoleacetic acid

MS:

Murashige and Skoog

RT-PCR:

reverse transcriptase — polymerase chain reaction

TC:

tocopherol cyclase

WT:

wild type

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Correspondence to R. Sathishkumar.

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Acknowledgements: The authors are very thankful to Defence Research and Developmental Organization — Centre for Life Sciences, Bharathiar University for financial assistance and support.

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Harish, M.C., Dachinamoorthy, P., Balamurugan, S. et al. Overexpression of homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) enhances α-tocopherol content in transgenic tobacco. Biol Plant 57, 395–400 (2013). https://doi.org/10.1007/s10535-012-0298-5

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Additional key words

  • Agrobacterium mediated transformation
  • RT-PCR
  • Southern blot