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2C-methyl- d- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene

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Abstract

Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-d-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5′ diphospho)-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100 μM) down-regulated the expression of SrMDS at 4 h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.

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Acknowledgments

Authors thank Council of Scientific and Industrial Research (CSIR) for funding under the mission mode project entitled “Exploration and acquisition of specific and targeted neutraceuticals with a back drop of nutrigenomic stevia, tea, and potato species; CMM0014”. Mutant strain EB370 was kindly provided by Dr. Eric Brown, McMaster University, Canada. HK gratefully acknowledges the Junior/Senior Research Fellowship awarded by Indian Council of Medical Research (ICMR). The technical assistance provided by Digvijay Singh Naruka in sequencing is duly acknowledged. The manuscript represents IHBT communication number 818.

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  1. Hitesh Kumar

    Present address: Department of Botany, SCVB Government College, Palampur, 176061, Himachal Pradesh, India

Authors and Affiliations

  1. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (Council of Scientific and Industrial Research), Palampur, 176061, Himachal Pradesh, India

    Hitesh Kumar & Sanjay Kumar

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

    Kashmir Singh

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Correspondence to Sanjay Kumar.

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11033_2012_1998_MOESM1_ESM.ppt

Agarose gel showing PCR amplification of SrMDS using colony lysate of putative positive colonies of EB370 transformant (lanes, L1–L7). DNA size markers (M) are shown on the left side of panel. Presence of the cloned insert was verified using a vector pQE30 UA (Qiagen, Germany) specific primer pair (as detailed in the “Materials and methods” section) that added additional 378 bp to the amplicon. Since size of cloned coding region of SrMDS was 696 bp, PCR with the said primer pair yielded an amplicon of 1,074 bp. The directional cloning of the insert was also confirmed by sequencing

11033_2012_1998_MOESM2_ESM.ppt

SDS–polyacrylamide gel showing expression of recombinant SrMDS. Expression of SrMDS was induced by inclusion of IPTG for 12 h (lane, “I”). Un-induced culture (lane, “U”) served as control. Presence of recombinant SrMDS (marked by arrow) was confirmed by the appearance of a band of 24.8 kD in the induced sample. Protein markers (lane, “M”; Fermentas, USA) were loaded as shown

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Kumar, H., Singh, K. & Kumar, S. 2C-methyl- d- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene. Mol Biol Rep 39, 10971–10978 (2012). https://doi.org/10.1007/s11033-012-1998-9

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