Molecular cloning and characterization of GuHMGR, an HMG-CoA reductase gene from liquorice (Glycyrrhiza uralensis)

Abstract

A full length cDNA encoding HMGR (designated as GuHMGR) was isolated from liquorice (Glycyrrhiza uralensis) based on degenerated PCR and genome walking. The full length cDNA of GuHMGR was 2330 bp with a 1518-bp open reading frame (ORF) encoding a 505-aa polypeptide. Bioinformatics analysis indicated that there were two trans-membrane domains in GuHMGR. A molecular model of tertiary structure showed that GuHMGR is a novel HMGR with a similar spatial structure to other plant HMGRs. The deduced polypeptide of GuHMGR has an isoelectric point (pI) of 6.41 and a calculated molecular weight of about 54.7 kDa. Sequence comparison and phylogenetic tree analysis showed that GuHMGR had the highest homology with HMGRs from Pisum sativum and Medicago truncatula, indicating that GuHMGR belongs to the plant HMGR group. Expression analysis showed the similar amount of transcript level of GuHMGR in roots and leaves, suggesting that this gene was expressed constitutively in plants. Therefore, this novel HMGR gene would possibly provide a new strategy for studying the glycyrrhizin metabolism at the molecular level in the future.

Keywords

cloning / liquorice / glycyrrhiza uralensis / genome walker / HMG-CoA reductase

References

[1] Bach T J (1995). Some new aspects of isoprenoid biosynthesis in plants—a review. Lipids, 30(3): 191–202

[2] Caelles C, Ferrer A, Balcells L, Hegardt F G, Boronat A (1989). Isolation and structural characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Mol Biol, 13(6): 627–638

[3] Chang S J, Puryear J, Cairney J (1993). A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep, 11(2): 113–116

[4] Chappell J (1995). Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants. Ann Rev Plant Physio Plant Mol Biol, 46(1): 521–547

[5] Choi D, Ward B L, Bostock R M (1992). Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid. Plant Cell, 4(10): 1333–1344

[6] Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr H W (2003). Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet, 361(9374): 2045–2046

[7] Enjuto M, Balcells L, Campos N, Caelles C, Arró M, Boronat A (1994). Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme. Proc Natl Acad Sci USA, 91(3): 927–931

[8] Goldstein J L, Brown M S (1990). Regulation of the mevalonate pathway. Nature, 343 (6257): 425–430

[9] Hayashi H, Hirota A, Hiraoka N, Ikeshiro Y (1999). Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase. Biol Pharm Bull, 22(9): 947–950

[10] Hayashi H, Huang P, Kirakosyan A, Inoue K, Hiraoka N, Ikeshiro Y, Kushiro T, Shibuya M, Ebizuka Y (2001). Cloning and characterization of a cDNA encoding β-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice. Biol Pharm Bull, 24(8): 912–916

[11] Ito M, Nakashima H, Baba M, Pauwels R, De Clercq E, Shigeta S, Yamamoto N (1987). Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)]. Antiviral Res, 7(3): 127–137

[12] Jelesko J G, Jenkins S M, Rodríguez-Concepció M, Gruissem W (1999). Regulation of tomato HMG1 during cell proliferation and growth. Planta, 208(3): 310–318

[13] Jiang J H, Kai G Y, Cao X Y, Chen F M, He D N, Liu Q (2006). Molecular cloning of a HMG-CoA reductase gene from Eucommia ulmoides Oliver. Biosci Rep, 26(2): 171–181

[14] Learned R M, Fink G R (1989). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes. Proc Natl Acad Sci USA, 86(8): 2779–2783

[15] Liao Z H, Tan Q M, Chai Y R, Zuo K J, Chen M, Gong Y F, Wang P, Pi Y, Tan F, Sun X F, Tang K X (2004). Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast. Funct Plant Biol, 31: 73–81

[16] Maldonado-Mendoza I E, Burnett R J, Nessler C L (1992). Nucleotide sequence of a cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Catharanthus roseus. Plant Physiol, 100(3): 1613–1614

[17] Maldonado-Mendoza I E, Vincent R M, Nessler C L (1997). Molecular characterization of three differentially expressed members of the Camptotheca acuminata 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) gene family. Plant Mol Biol, 34(5): 781–790

[18] Matsui S, Matsumoto H, Sonoda Y, Ando K, Aizu-Yokota E, Sato T, Kasahara T (2004). Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line. Int Immunopharmacol, 4(13): 1633–1644

[19] Park H, Denbow C J, Cramer C L (1992). Structure and nucleotide sequence of tomato HMG2 encoding 3-hydroxy-3-methyl-glutaryl coenzyme A reductase. Plant Mol Biol, 20(2): 327–331

[20] Schwede T, Kopp J, Guex N, Peitsch M C (2003). SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res, 31(13): 3381–3385

[21] Shen G A, Pang Y Z, Wu W S, Liao Z H, Zhao L X, Sun X F, Tang K X (2006). Cloning and characterization of a root-specific expressing gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Ginkgo biloba. Mol Biol Rep, 33(2): 117–127

[22] Shibata S (2000). A drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice. Yakugaku Zasshi, 120(10): 849–862

[23] Van Rossum T G J, Vulto, De Man R A, Brouwer J T, Schalm S W (1998). glycyrrhizin as a potential treatment for chronic hepatitis C. Aliment Pharmacol Ther, 12(3): 199–205

Acknowledgements

This research was supported by the State Natural Sciences Foundation Projects (No. 30572328 and No. 30672615), and the Ministry of Science and Technology of the People’s Republic of China. It was also supported by the Province Natural Sciences Foundation Projects of Hebei (No. C2009000583).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg