Plant Biotechnology Reports

, Volume 5, Issue 1, pp 53–60 | Cite as

Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L.

  • Tazyeen Nafis
  • Mohd. Akmal
  • Mauji Ram
  • Pravej Alam
  • Seema Ahlawat
  • Anis Mohd
  • Malik Zainul Abdin
Original Article

Abstract

Artemisinin is effective against both chloroquine-resistant and -sensitive strains of Plasmodium species. However, the low yield of artemisinin from cultivated and wild plants is a serious limitation to the commercialization of this drug. Optimization of artemisinin yield either in vivo or in vitro is therefore highly desirable. To this end, we have overexpressed the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) gene (hmgr) from Catharanthusroseus L. in Artemisia annua L. and analyzed its influence on artemisinin content. PCR and Southern blot analyses revealed that the transgenic plants showed stable integration of the foreign hmgr gene. The reverse transcriptase-PCR results suggested that the hmgr was expressed at the transcriptional level in transgenic lines of Artemisia annua L., while the high-performance liquid chromatography analysis showed that artemisinin content was significantly increased in a number of the transgenic lines. Artemisinin content in one of the A. annua transgenic lines was 38.9% higher than that in non-transgenic plants, and HMGR enzyme activity in transgenic A. annua L. was also higher than that in the non-transgenic lines.

Keywords

Artemisiaannua L. Genetic transformation RT-PCR HPLC Artemisinin 

Abbreviations

6-BA

Benzylaminopurine

HMGR

3-Hydroxy-3-methyl-glutaryl coenzyme A reductase

MVA

Mevalonate

NAA

Naphthaleneacetic acid

NOS

Nopaline opine synthase

NPT

Neomycin phosphotransferase

SISM

Shoot-induction selection medium

SQS

Squalene synthase

References

  1. Abdin MZ, Israr M, Rehman RU, Jain SK (2003) Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production. Planta Med 69:289–293CrossRefPubMedGoogle Scholar
  2. Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisinin in Artemisia annua. Phytochem 16:1927–1930CrossRefGoogle Scholar
  3. Aquil S, Husaini AM, Abdin MZ, Rather GM (2009) Overexpression of the HMG-CoA reductase gene leads to enhanced artemisinin biosynthesis in transgenic Artemisia annua plants. Planta Med 75:1–6CrossRefGoogle Scholar
  4. Argolo ACC, Charlwood BV, Pletsch M (2000) The regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare. Planta Med 66:448–451CrossRefPubMedGoogle Scholar
  5. Ayora-Talavera T, Chappell J, Lozoya-Gloria E, Loyola-Vargas VM (2002) Overexpression in Catharanthus roseus hairy roots of a truncated hamster 3-hydroxy-3-methylglutaryl-CoA reductase gene. Appl Biochem Biotechnol 97:135–145CrossRefPubMedGoogle Scholar
  6. Bach TJ (1986) Hydroxymethylglutaryl-CoA reductase, a key enzyme in phytosterol synthesis. Lipid 21:121–125CrossRefGoogle Scholar
  7. Borrmann S, Szlezak N, Faucher JF, Matsiegui PB, Neubauer R, Biner RK, Lell B, Kremsner PG (2001) Artesunate and praziquantel for the treatment of Schistosoma haematobium infections: a double blind, randomized, placebo-controlled study. J Infect Dis 184:1363–1366CrossRefPubMedGoogle Scholar
  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  9. Chappell J (1995) Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants. Annu Rev Plant Physiol Plant Mol Biol 46:521–547CrossRefGoogle Scholar
  10. Chen D, Ye H, Li G (2000) Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation. Plant Sci 155:179–185CrossRefPubMedGoogle Scholar
  11. Concepcion RM, Gruissem M (1999) Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation. Plant Physiol 119:41–48CrossRefGoogle Scholar
  12. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  13. Efferth T, Dunstan H, Sauerbrey A, Miyachi H, Chitambar CR (2001) The antimalarial artesunate is also active against cancer. Int J Oncol 18:767–773PubMedGoogle Scholar
  14. Gondet L, Weber T, Maillot VP, Benveniste P, Bach TJ (1992) Regulatory role of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in a tobacco mutant that over produces sterols. Biochem Biophys Res Comm 186:888–893CrossRefPubMedGoogle Scholar
  15. Han JL, Wang H, Ye HC, Liu Y, Li ZQ, Zhang Y, Zhang YS, Yan F, Li GF (2005) High efficiency of genetic transformation and regeneration of Artemisia annua L. via Agrobacterium tumefaciens-mediated procedure. Plant Sci 168:73–80CrossRefGoogle Scholar
  16. Han JL, Liu BY, Ye HC, Wang H, Li ZQ, Li GF (2006) Effects of over expression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L. J Integr Plant Biol 48(4):482–487CrossRefGoogle Scholar
  17. Jung M, Schinazi RF (1994) Synthesis and in vitro anti-human immunodeficiency virus acivity of artemisinin (Qinghaousu) related trioxanes. Bioorg Med Chem Lett 4:934–941CrossRefGoogle Scholar
  18. Kudakasseril GJ, Lam L, Staba EJ (1987) Effect of sterol inhibitors on the incorporation of 14C-isopentenyl pyrophosphate into artemisinin by a cell-free system from Artemisia annua tissue cultures and plants. Planta Med 53:280–284CrossRefPubMedGoogle Scholar
  19. Lange BM, Wildung MK, MacCaskill D, Croteau R (1998) A family of transketolases that directs isoprenoid biosynthesis via mevalonate-independent pathway. Proc Nat Acad Sci USA 95:21000–22104CrossRefGoogle Scholar
  20. Laughlin JC (1994) Agricultural production of artemisinin: a review. Trans Royal Soc Trop Med Hyg 88(1):21–22CrossRefGoogle Scholar
  21. Maldonado MIE, Burnett RJ, Nessler CL (1992) Nucleotide sequence of a cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Catharanthus roseus. Plant Physiol 100:1613–1614CrossRefGoogle Scholar
  22. Mauji Ram, Khan MA, Jha P, Khan S, Kiran U, Ahmad MM, Javed S, Abdin MZ (2010) HMG-CoA reductase limits artemisinin biosynthesis and accumulation in Artemisia annua L. Plants. Acta Physiol Plant 32:859–866. doi:10.1007/s11738-010-0470-5
  23. Maurey K, Wolf F, Golbeck J (1986) 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in Ochmmonac malhamensis. Plant Physiol 82:523–527CrossRefPubMedGoogle Scholar
  24. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3):473–497Google Scholar
  25. Newton P, White N (1999) Malaria: new development in treatment and prevention. Annu Rev Med 50:179–192CrossRefPubMedGoogle Scholar
  26. Prols F, Meyer P (1992) The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. Plant J 2:465–475PubMedGoogle Scholar
  27. Romero MR, Efferth T, Serrano MA, Castano B, Macias RI, Briz O, Marin JJ (2005) Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an ‘in vitro’ system. Antiviral Res 68:75–83CrossRefPubMedGoogle Scholar
  28. Russell DW (1985) 3-Hydroxy-3-methylglutaryl-CoA reductases from pea seedlings. Methods Enzymol 110:26–40CrossRefGoogle Scholar
  29. Sa G, Mi M, He-Chun Y, Ben-Ye L, Guo-feng L, Kang C (2001) Effects of ipt gene expression on the physiological and chemical characteristics of Artemisia annua L. Plant Sci 160:691–698CrossRefPubMedGoogle Scholar
  30. Sen R, Bandyopadhyay S, Dutta A, Mandal G, Ganguly S, Saha P, Chatterjee M (2007) Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes. J Med Microbiol 56:1213–1218CrossRefPubMedGoogle Scholar
  31. Singh NP, Lai H (2001) Selective toxicity of dihydroartemisinin and holotransferrin toward human breast cancer cells. Life Sci 70(1):49–56CrossRefPubMedGoogle Scholar
  32. Stermer BA, Bostock MB (1987) Stermer BA, Bostock MB (1987) Involvement of 3-hydroxy-3-methylglutaryl-CoA reductase in the regulation of sesquiterpenois phytoalexin synthesis in potato. Plant Physiol 84:404–408CrossRefPubMedGoogle Scholar
  33. Tang W, Ronald J, Newton D, Weidner A (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58(3):545–554CrossRefPubMedGoogle Scholar
  34. Towler MJ, Weathers PJ (2007) Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways. Plant Cell Rep 26:2129–2136CrossRefPubMedGoogle Scholar
  35. Utzinger J, Xiao S, N’Goran EK, Berquist R, Tanner M (2001) The potential of artemether for the control of schistosomiasis. Int J Parasitol 31:1549–1562CrossRefPubMedGoogle Scholar
  36. Van Agtmael MA, Eggelte TA, Boxtel CJ (1999) Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication. Trends Pharmacol Sci 20:199–205CrossRefPubMedGoogle Scholar
  37. Vergauwe A, Cammaert R, Vandenberghe D, Genetello C, Van Montagu M, Vanden Eeckhout E (1996) Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plant. Plant Cell Rep 15:929–937CrossRefGoogle Scholar
  38. Weathers PJ, Bunk G, McCoy MC (2005) The effect of phytohormones on growth and artemisinin production in Artemisia annua hairy roots. In Vitro Cell Dev Biol Plant 41:47–53CrossRefGoogle Scholar
  39. Zhang L, Fuyuan J, Fupeng L, Li M, Wang Y, Wang G, Sun X, Tang K (2009) Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti-malarial drug, by hairpin-RNA mediated gene silencing. Biotechnol Appl Biochem 52:199–207CrossRefPubMedGoogle Scholar
  40. Zhao SS, Zeng MY (1986) Determination of Qinghaosu in Artemisia annua L. by high performance liquid chromatography. Chin J Pharm Anal 6:3–5Google Scholar

Copyright information

© Korean Society for Plant Biotechnology and Springer 2010

Authors and Affiliations

  • Tazyeen Nafis
    • 1
  • Mohd. Akmal
    • 1
  • Mauji Ram
    • 1
  • Pravej Alam
    • 1
  • Seema Ahlawat
    • 1
  • Anis Mohd
    • 1
  • Malik Zainul Abdin
    • 1
  1. 1.Centre for Transgenic Plant Development, Department of Biotechnology, Faculty of ScienceJamia HamdardNew DelhiIndia

Personalised recommendations