Skip to main content

Advertisement

SpringerLink
Log in

Ri-mediated Transformation of Glycyrrhiza uralensis with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin

  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS) plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight and was about 2.6 times higher than control hairy roots.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

SQS:

squalene synthase

 :

β-amyrin synthase

OSC:

oxido- squalene cyclase

GuSQS:

Glycyrrhiza uralensis squalene synthase

PPT:

phosphinothricin

GgSQS:

Glycyrrhiza glabra squalene synthase

AS:

acetosyringone

Kana:

kanamycin sulphate

cef:

cefotaxim sodium

cb:

carbenicillin

References

  • Abe H, Ohya N, Yamamoto KF, et al. Effects of glycyrrhizin and glycyrrhetinic acid on growth and melanogenesis in cultured B16 melanoma cells. Eur J Cancer Clin Oncol 1987;23:1549–55.

    Article  PubMed  CAS  Google Scholar 

  • Abe I, Rohmer M, Prestwich GD. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem Rev 1993;93:2189–206.

    Article  CAS  Google Scholar 

  • Ayabe S, Takano H, Fujita T, et al. Triterpenois biosynthesis in tissue cultures of Glycyrrhiza glaba var. glanduliera. Plant Cell Rep 1990;9(4):181–4.

    Article  CAS  Google Scholar 

  • Capasso F, Mascolo M, Autore G, et al. Glycyrrhetinic acid, leucocytes and prostaglandins. J Pharm Pharmacol 1983;35:332–5.

    PubMed  CAS  Google Scholar 

  • Chen DH, Liu CJ, Ye HC, et al. Ri-mediated transformation of Artemisia annua with a recombinant farnesyl diphosphate synthase gene for artemisinin production. Plant Cell Tissue Organ Cult 1999;57:157–62.

    Article  CAS  Google Scholar 

  • Dehpour AR, Zolfaghari ME, Samadian T, et al. The protective effect of licorice components and their derivatives against gastric ulcer induced by aspirin in rats. J Pharm Pharmacol 1994;46(2):148–9.

    PubMed  CAS  Google Scholar 

  • Devarenne TP, Shin DH, Back K, et al. Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor. Arch Biochem Biophys 1998;349:205–15.

    Article  PubMed  CAS  Google Scholar 

  • Devarenne TP, Ghosh A, Chappell J, et al. Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco. Plant Physiol 2002;129:1095–106.

    Article  PubMed  CAS  Google Scholar 

  • Doolitile WF. The origin and function of intervening sequences in DNA: a review. Am Nat 1987;130(6):915–28.

    Article  Google Scholar 

  • Duke JA. Handbook of medicinal herbs. Boca Raton, FL: CRC Press; 1985.

    Google Scholar 

  • Gibson, MR. Glycyrrhiza in old and new perspectives. Lloydia 1978;41:348–54.

    PubMed  CAS  Google Scholar 

  • Hamil JD, Prescott A, Martin C, et al. Assessment of the efficiency of cotransformation of the T-DNA of disarmed binary binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes. Plant Mol Biol 1987;9:573–84.

    Article  Google Scholar 

  • Hanrahan C. Gale encyclopedia of alternative medicine, licorice [book on CD-ROM]. Farmington Hills, MI: Thomson Gale; 2001.

    Google Scholar 

  • Haralampidis K, Trojanowska M, Osbourn AE. Biosynthesis of triterpenoid saponins in plants. Adv. Biochem. Eng Biotechnol 2002;75:31–49.

    PubMed  CAS  Google Scholar 

  • Hayashi H, Fukui H, Tabata M, et al. Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra. Plant Cell Rep 1988;7:508–11.

    Article  CAS  Google Scholar 

  • Hayashi H, Sakai T, Fukui H, et al. Formation of soyasaponins in licorice cell suspension cultures. Phytochemistry 1990;29:3127–9.

    Article  CAS  Google Scholar 

  • Hayashi H, Hiraoka N, Ikeshiro Y. Molecular cloning and functional expression of cDNA for Glycyrrhiza glabra squalene synthase. Biol Pharm Bull 1996;19(10):1387–9.

    PubMed  CAS  Google Scholar 

  • Hayashi H, Hirota A, Hiraoka N, Ikeshiro Y. Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase. Biol. Pharm Bull 1999;22(9):947–50.

    PubMed  CAS  Google Scholar 

  • Hayashi H, Pengyu H, Kenichiro I, et al. Up-regulation of soyasaponin biosynthesis by methyl Jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiol 2003;44(4):404–11.

    Article  PubMed  CAS  Google Scholar 

  • Henry M, Chantalat-Dublanche I. Isolation of licorice protoplasts (Glycyrrhiza glabr Var. typical) from cell suspension cultures not producing Glycyrrhetinic acid. C R Acad Sci Ser II 1984;299:899–903.

    CAS  Google Scholar 

  • Hyung KK, Yoonkyung P, Hee NK, et al. Antimicrobial mechanism of b-glycyrrhetinic acid isolated from licorice, Glycyrrhiza glabra. Biotechnol Lett 2002;24:1899–902.

    Article  Google Scholar 

  • Ko, KS. 10th Symposium of Plant Tissue and Cell Culture, Sendai, 33; 1987.

  • Lee MH, Jeong JH, Seo JW, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 2004;45:976–84.

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog FA. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962;15:473–79.

    Article  CAS  Google Scholar 

  • Murray MT. The healing power of herbs. 2nd ed. New York, NY: Three Rivers Press; 1995.

    Google Scholar 

  • Saito K, et al. Plant Cell Rep 1987;8:718.

    Article  Google Scholar 

  • Saito K, Kaneko H, Yamazaki M, et al. Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector. Plant Cell Rep 1990;8:718–21.

    Article  Google Scholar 

  • Saito K, Yamazaki M, Kaneko H, et al. Tissue-specific and stress-enhancing expression of the TR promoter for mannopine synthase in transgenic medicinal plants. Planta 1991;184:40–6.

    Article  CAS  Google Scholar 

  • Seo JW, Jeong JH, Shin CG, et al. Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry 2005;66:869–77.

    Article  PubMed  CAS  Google Scholar 

  • Shahin EA, Sukhapinda K, Simpson RB, et al. Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA, but no Ri-plasmid T-DNA. Theor Appl Genet 1986;72:770–7.

    Article  CAS  Google Scholar 

  • Shibata, S. A drug over the millennia: Pharmacognosy, chemistry, and pharmacology of licorice. Yakugaku Zasshi 2000;120:849–62.

    PubMed  CAS  Google Scholar 

  • Simpson RB, Spielmann A, Margossian L, et al. A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes. Plant Mol Biol 1986;6:403–15.

    Article  CAS  Google Scholar 

  • Stougaard J, Abildsten D, Marcker K. The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Mol Gen Genet 1987;207:251–5.

    Article  CAS  Google Scholar 

  • Suzuki H, Achnine L, Xu R, et al. A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula. Plant J 2002;32:1033–48.

    Article  PubMed  CAS  Google Scholar 

  • Tokuda H, Ohigashi H, Koshimizu K, et al. Inhibitor effects of ursolic and oleanolic acid on skin tumor promotion by 12-Otetradecanoylphorbol-13-acetate. Cancer Lett 1986;3:3:279–85.

    Article  PubMed  CAS  Google Scholar 

  • Van Rossum TG, Vulto AG. Intravenous glycyrrhizin for the treatment of chronic hepatitis C: a double-blind, randomized, placebo-controlled phase I/II trial. J Gastroenterol Hepatol 1999;14:1093–9.

    Article  PubMed  CAS  Google Scholar 

  • Wang ZY, Agarwal R, Zhou ZC, et al. Inhibition if mutagenecity in Salmonella typhimurium and skin tumor initiating and tumor promoting activities in Senecar mice by glycyrrhetinic acid: comparison of 18a- and 18b-stereoisomers. Carcinogenesis 1991;12:187–92.

    Article  PubMed  Google Scholar 

  • Wentzinger LF, Bach TJ, Hartmann MA. Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase. Plant Physiol 2002;130:334–46.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from Nation Crop Molecular Design Center and from Nation ‘863’ Program in Peking University, China. We thank Prof. Xi-Ping Wang and Mrs Xie Yin for their help and advices.

Author information

Authors and Affiliations

  1. Department of Breeding and Genetics, China Pharmaceutical University, 210038, Nanjing, Jiangsu Province, China

    Hong-Yu Lu, Hai-Chao Zhang & Shan-Lin Gao

  2. Chinese Academy of Agricultural Sciences, 100081, Beijing, China

    Jing-Mei Liu

  3. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China, 310029

    Tao Yin

Authors
  1. Hong-Yu Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing-Mei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hai-Chao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tao Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shan-Lin Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shan-Lin Gao.

Additional information

The nucleotide sequences GuSQS1 and GUSQS2 reported in this paper appear in the EMBL nucleotide sequence database with the accession number AM182329 and AM182330, respectively.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, HY., Liu, JM., Zhang, HC. et al. Ri-mediated Transformation of Glycyrrhiza uralensis with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin. Plant Mol Biol Rep 26, 1–11 (2008). https://doi.org/10.1007/s11105-008-0018-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11105-008-0018-7

Keywords

Search

Navigation