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Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content

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Abstract

Artemisinin, an endoperoxide sesquiterpene lactone, is a novel antimalarial natural product isolated from Artemisia annua L. plants. The low concentrations (0.01–1.1%) of this compound in A. annua L. plants is, however, a major constraint for commercialization of artemisinin-based combination therapies (ACTs) recommended by WHO for treating malaria caused by multidrug-resistant P. falciparum sp. In this context, in vivo yield improvement programs were undertaken by us. In the present study, HMG-Co A reductase gene (hmgr) from Catharanthus roseus (L) G. Don and amorpha-4,11-diene synthase (ads) gene from A. annua L. were over-expressed in A. annua L. plants to study their effects on artemisinin yields. The transgenic lines developed from putative transgenic regenerants were evaluated for integration and copy number of the transgenes using hptII gene probe, as it was a part of the expression cassette. The transgenic lines showed positive bands of hptII gene on Southern blots confirming the integration of transgenes. Some of the transgenic lines had single copy of the transgenes, while others had multiple copies. The expressions of hmgr and ads at the transcriptional level were also confirmed in each transgenic line employing RT-PCR assays. The HPLC analyses showed that the artemisinin contents were significantly increased in these transgenics. One of the transgenic lines, TR4, was found to contain 7.65-fold higher (1.73 mg/gDW) artemisinin than the non-transgenic plant (W). The increased artemisinin levels were found to be correlated with HMG-Co A reductase and amorpha-4,11-diene synthase enzymatic activities in the biochemical analyses.

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Abbreviations

ACT:

Artemisinin-based combination therapy

ADS:

Amorpha-4,11-diene synthase enzyme

ads :

Amorpha-4,11-diene synthase gene

BAP:

Benzene amino purine

CTAB:

Cetyl trimethylammonium bromide

HMGR:

Hydroxy methyl glutaryl coenzyme A reductase

hmgr :

Hydroxy methyl glutaryl coenzyme A gene

HPLC:

High performance liquid chromatography

MS:

Murashige and Skoog

MVA:

Mevalonic acid

NAA:

Naphthalene acetic acid

NOS:

Nopaline opine synthase

hptII:

Hygromycin phosphotransferase gene

RT-PCR:

Reverse transcriptase polymerase chain reaction

SIM:

Shoot-induction medium

SISM:

Shoot-induction selection medium

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Acknowledgments

We are thankful to Prof. S.K. Jain, Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, India, and Dr. M.A.A. Khan Scientist, NISCAIR, New Delhi, India, for editing the manuscript. The financial support from DST, Government of India and M/s Ipca Pvt. Ltd., Mumbai, India, is gratefully acknowledged. P.A. is thankful to Jamia Hamdard, New Delhi, India, for providing Junior Research Fellowship.

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  1. Centre for Transgenic Plant Development, Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, 110062, India

    Pravej Alam & M. Z. Abdin

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  1. Pravej Alam
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  2. M. Z. Abdin
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Correspondence to M. Z. Abdin.

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Communicated by P. Lakshmanan.

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Alam, P., Abdin, M.Z. Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content. Plant Cell Rep 30, 1919–1928 (2011). https://doi.org/10.1007/s00299-011-1099-6

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  • DOI: https://doi.org/10.1007/s00299-011-1099-6

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