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Planta Med 2011; 77(15): 1759-1765
DOI: 10.1055/s-0030-1271038
Biochemistry, Molecular Biology and Biotechnology
Original Papers
© Georg Thieme Verlag KG Stuttgart · New York

Artemisinin Biosynthesis Enhancement in Transgenic Artemisia annua Plants by Downregulation of the β-Caryophyllene Synthase Gene

Jian-Lin Chen1 , 2 , Hua-Ming Fang2 , Yun-Peng Ji1 , 2 , Gao-Bin Pu3 , Yan-Wu Guo2 , Li-Li Huang1 , 2 , Zhi-Gao Du2 , Ben-Ye Liu2 , He-Chun Ye2 , Guo-Feng Li2 , Hong Wang1 , 2
  • 1Graduate University of the Chinese Academy of Sciences, Beijing, China
  • 2Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, China
  • 3Shandong Yingcai University, Jinan, China
Further Information

Publication History

received August 9, 2010 revised March 11, 2011

accepted March 30, 2011

Publication Date:
20 April 2011 (online)

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Abstract

Artemisinin is an effective antimalarial drug isolated from the medicinal plant Artemisia annua L. Due to its increasing market demand and the low yield in A. annua, there is a great interest in increasing its production. In this paper, in an attempt to increase artemisinin content of A. annua by suppressing the expression of β-caryophyllene synthase, a sesquiterpene synthase competing as a precursor of artemisinin, the antisense fragment (750 bp) of β-caryophyllene synthase cDNA was inserted into the plant expression vector pBI121 and introduced into A. annua by Agrobacterium-mediated transformation. PCR and Southern hybridization confirmed the stable integration of multiple copies of the transgene in 5 different transgenic lines of A. annua. Reverse transcription PCR showed that the expression of endogenous CPS in the transgenic lines was significantly lower than that in the wild-type control A. annua plants, and β-caryophyllene content decreased sharply in the transgenic lines in comparison to the control. The artemisinin content of one of the transgenic lines showed an increase of 54.9 % compared with the wild-type control. The present study demonstrated that the inhibition pathway in the precursor competition for artemisinin biosynthesis by anti-sense technology is an effective means of increasing the artemisinin content of A. annua plants.

Key words

Artemisia annua L. - Asteraceae - artemisinin biosynthesis - anti-sense β-caryophyllene synthase gene - genetic transformation

References

  • 1 Cooperative Research Group on Artemisinin Structure . Artemisinin – a new sesquiterpene lactone.  Chin Sci Bull. 1977;  22 142
    PubMedGoogle Scholar
  • 2 Liu J M, Ni M Y, Fan J F, Tu Y Y, Wu Z H, Wu Y L, Zhou W S. Structure and reaction of artemisinin.  Acta Chim Sin. 1979;  37 129-143
    PubMedGoogle Scholar
  • 3 Hua L, Matsuda S P. The molecular cloning of 8-epicedrol synthase from Artemisia annua L.  Arch Biochem Biophys. 1999;  369 208-212
    CrossrefPubMedGoogle Scholar
  • 4 Mercke P, Crock J, Croteau R, Brodelius P E. Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.  Arch Biochem Biophys. 1999;  369 213-222
    CrossrefPubMedGoogle Scholar
  • 5 Mercke P, Bengtsson M, Bouwmeester H J, Posthumus M A, Brodelius P E. Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L.  Arch Biochem Biophys. 2000;  381 173-180
    CrossrefPubMedGoogle Scholar
  • 6 Chang Y J, Song S H, Park S H, Kim S U. Amorpha-4,11-diene synthase of Artemisia annua: cDNA isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis.  Arch Biochem Biophys. 2000;  383 178-184
    CrossrefPubMedGoogle Scholar
  • 7 Wallaart T E, Bouwmeester H J, Hille J, Poppinga L, Maijers N C. Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin.  Planta. 2001;  21 460-465
    PubMedGoogle Scholar
  • 8 Cai Y, Jia J W, Crock J, Lin Z X, Chen X Y, Croteau R. A cDNA clone for beta-caryophyllene synthase from Artemisia annua.  Phytochemistry. 2002;  61 523-529
    CrossrefPubMedGoogle Scholar
  • 9 Picaud S, Brodelius M, Brodelius P E. Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua.  Phytochemistry. 2005;  66 961-967
    CrossrefPubMedGoogle Scholar
  • 10 Bouwmeester H J, Kodde J, Verstappen F W, Altug I G, de Kraker J W, Wallaart T E. Isolation and characterization of two germacrene A synthase cDNA clones from chicory.  Plant Physiol. 2002;  129 134-144
    CrossrefPubMedGoogle Scholar
  • 11 Han J L, Wang H, Ye H C, Liu Y, Li Z Q, Zhang Y, Zhang Y S, Yan F, Li G F. High efficiency of genetic transformation and regeneration of Artemisia annua L. via Agrobacterium tumefaciens-mediated procedure.  Plant Sci. 2005;  168 73-80
    CrossrefPubMedGoogle Scholar
  • 12 Allen G, Flores-Vergara M, Krasynanski S, Kumar S, Thompson W. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide.  Nat Protoc. 2006;  1 2320-2325
    CrossrefPubMedGoogle Scholar
  • 13 Sambrook J, Fritsch E F, Mariatis T. Molecular cloning: A laboratory manual, 2nd edition. New York; Cold Spring Harbor Laboratory Press 1989: 483-490
    Google Scholar
  • 14 Zhao S S, Zeng M Y. Determination of Qinghaosu in Artemisia annua L. by high performance liquid chromatography.  Chin J Pharm Anal. 1986;  6 3-5
    PubMedGoogle Scholar
  • 15 Rasmann S, Köllner T G, Degenhardt J, Hiltpold I, Toepfer S, Kuhlmann U, Gershenzon J, Turlings T C. Recruitment of entomopathogenic nematodes by insect-damaged maize roots.  Nature. 2005;  434 732-737
    CrossrefPubMedGoogle Scholar
  • 16 Wallaart T E, Uden W V, Lubberink H G, Woerdenbag H J, Pras N, Quax W J. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin.  J Nat Prod. 1999;  62 430-433
    CrossrefPubMedGoogle Scholar
  • 17 Rydén A-M, Ruyter-Spira C, Quax W J, Osada H, Muranaka T, Kayser O, Bouwmeester H. The molecular cloning of dihydroartemisinic aldehyde reductase and its implication in artemisinin biosynthesis in Artemisia annua.  Planta Med. 2010;  76 1178-1183
    PubMedGoogle Scholar
  • 18 Covello P S, Teoh K H, Polichuk D R, Reed D W, Nowak G. Functional genomics and the biosynthesis of artemisinin.  Phytochemistry. 2007;  68 1864-1871
    CrossrefPubMedGoogle Scholar
  • 19 Matsushita Y, Kang W, Charlwood B V. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua.  Gene. 1996;  172 207-209
    CrossrefPubMedGoogle Scholar
  • 20 Picaud S, Olofsson L, Brodelius M, Brodelius P E. Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L.  Arch Biochem Biophys. 2005;  436 215-226
    CrossrefPubMedGoogle Scholar
  • 21 Teoh K H, Polichuk D R, Reed D W, Nowak G, Covello P S. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin.  FEBS Lett. 2006;  580 1411-1416
    CrossrefPubMedGoogle Scholar
  • 22 Zhang Y, Teoh K H, Reed D W, Maes L, Goossens A, Olson D J H, Ross A R S, Covello P S. The molecular cloning of artemisinic aldehyde Delta 11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua.  J Biol Chem. 2008;  283 21501-21508
    CrossrefPubMedGoogle Scholar
  • 23 Teoh K H, Polichuk D R, Reed D W, Covello P S. Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua.  Botany. 2009;  87 635-642
    CrossrefPubMedGoogle Scholar
  • 24 Liu Y, Ye H C, Wang H, Li G F. Molecular cloning, Escherichia coli expression and genomic organization of squalene synthase gene from Artemisia annua.  Acta Bot Sin. 2003;  45 608-613
    PubMedGoogle Scholar
  • 25 Yang R Y, Feng L L, Yang X Q, Yin L L, Xu X L, Zeng Q P. Quantitative transcript profiling reveals down-regulation of a sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants.  Planta Med. 2008;  74 1510-1516
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Zhang L, Jing F, Li F, Li M, Wang Y, Wang G, Sun X, Tang K. 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. 2009;  52 199-207
    CrossrefPubMedGoogle Scholar

Dr. Hong Wang

College of Life Sciences
Graduate University of the Chinese Academy of Sciences

19A Yuquanlu

Beijing, 100049

P. R. China

Phone: +86 10 62 83 62 39

Fax: +86 10 88 25 60 80

Email: wangh@ibcas.ac.cn