Isolation and Characterization of Squalene Synthase Gene in Three Species of Achillea, a Rich Source of Saponins

Document Type : Research Article


1 Department of Plant Sciences, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

2 Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

3 Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran


Squalene synthase (SQS, EC. is a key enzyme involved in the biosynthesis pathway of triterpenoid and steroidal saponins. The present study aimed to collect molecular information about the SQS gene in Achillea species, the medicinal plants rich in saponins. For this reason, genomic DNA was isolated from leaves of three Achillea species, including A. millefolium, A. wilhelmsii, and A. vermicularis in Iran, then partial SQS gene was amplified through PCR and sequenced (NCBI accession numbers: AmSQS KX589055, AwSQS KX685330, and AvSQS KX685331). AmSQS was 800 bp, containing four exons and three introns; AwSQS and AvSQS were 510 bp and 500 bp, respectively, containing three exons and two introns. Phylogenetic analysis demonstrated that the isolated SQS sequences were significantly similar to each other, and to Artemisia annua, another species of the genus Achillea. Furthermore, in the phylogeny tree, the SQS gene sequences of dicots and monocots were located in separate clades. The deduced amino acid sequences obtained from the isolated SQS gene had also a high similarity to each other and other organisms SQSs (>73% similarity to higher plants and more than 57% and 47% to the yeast and human). The deduced amino acid sequences included two regions overlapping with domains B and C of SQS, comprising an important motif of aspartate-rich (DYLED) for substrate binding via Mg2+-bridge. Data resulting from this study was the first report of SQS gene isolation and characterization in Achillea species, which also showed the ability of this gene in taxonomic classification.


APG III (Angiosperm Phylogeny Group). 2009. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants. Bot J Linn Soc 161(2): 105-121.
Buchanan BB, Gruissem W, Jones RL. 2000. Biochemistry and molecular biology of plants. American Society of Plant Physiology, Rockville, Maryland.
Chandler RF, Hooper SN, Hooper DL, Jamieson WD, Flinn CG, Safe LM. 1982. Herbal remedies the maritime Indians: sterols and triterpenes of Achillea millefolium L. (yarrow). J Pharm Sci 71: 690-693.
Dhar MK, Koul A, Kaul S. 2013. Farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development. N Biotechnol 30(2): 114-123.
Gu P, Ishii Y, Spencer TA, Shechter I. 1998. Function-structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase. J Biol Chem 273: 12515-12525.
Hata S, Sanmiya K, Kouchi H, Matsuoka M, Yamamoto N, Izui K. 1997. cDNA cloning of squalene synthase genes from mono- and dicotyledonous plants, and expression of the gene in rice. Plant Cell Physiol 38(12): 1409-1413.
Hill RA, Connolly JD. 2013. Triterpenoids. Nat Prod Rep 30(7): 1028-1065.
Kalariya KA, Meena RP, Poojara L, Shahi D, Patel S. 2021. Characterization of squalene synthase gene from Gymnema sylvestre R. Br. Beni-Suef Univ J Basic Appl Sci 10(6): 1-11. .
Khanuja SPS, Shasany AK, Darokar MP, Kumar S. 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol Biol Rep 17(1): 74.
Kim TD, Han JY, Huh GH, Choi YE. 2011. Expression and functional characterization of three squalene synthase genes associated with saponin biosynthesis in Panax ginseng. Plant Cell Physiol 52(1): 125-137.
Krak K, A´ lvarez I, Caklova´ P, Costa A, Chrtek J, Fehrer J. 2012. Development of novel low copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes. Am J Bot 99: 74-77.
Krak K, Caklová P, Chrtek J, Fehrer J. 2013. Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae). Heredity 110: 138-151.
Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Choi YE. 2004. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45: 976-984.
Mozaffarian V. 2005. Notes on the tribe Anthemideae (Compositae), new species, new records and new combination for Iran. Iran J Bot 11: 115-127.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15 (3): 473-497.
Nakashima T, Inoue T, Oka A, Nishino T, Osumi T, Hata S. 1995. Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase. Proc Natl Acad Sci USA, 92: 2328-2332.
Nguyen HT, Neelakadan AK, Quach TN, Valliyodan B, Kumar R, Zhang Z, Nguyen HT. 2013. Molecular characterization of Glycine max squalene synthase genes in seed phytosterol biosynthesis. Plant Physiol Biochem 73: 23-32.
Ogbe RJ, Ochalefu DO, Mafulul SG, Olaniru OB. 2015. A review on dietary phytosterols: Their occurrence, metabolism and health benefits. Asian J Plant Sci Res 5(4): 10-21.
Pandit J, Danley DE, Schulte GK, Mazzalupo S, Pauly TA, Hayward CM, Harwood HJ Jr. 2000. Crystal structure of human squalene synthase, A key enzyme in cholesterol biosynthesis. J Biol Chem 275(39): 30610-30617.
Podlech D. 1986. Compositeae VI. Antemideae. In: Flora Iranica (ed: Rechinger KH) Skademische Druck-u., Verlagsans Talt, Graz, Austria.
Poulter CD. 1990. Biosynthesis of non-head-to-tail terpenes. Formation of 1′-1 and 1′-3 linkages. Acc Chem Res 23 (3): 70-77.
Poulter CD, Rilling HC. 1981. Conversion of farnesyl pyrophosphate to squalene. In: Biosynthesis of isoprenoid compounds (eds: Porter JW and Spurgeon SL) Wiley, New York.
Qian J, Liu Y, Ma C, Chao N, Chen Q, Zhang Y, Wu Y. 2019. Positive selection of squalene synthase in Cucurbitaceae plants. Int J Genom 19:1-15.
Rilling HC, Epstein WW. 1969. Studies on the mechanism of squalene biosynthesis. Presqualene, a pyrophosphorylated precursor to squalene. J Am Chem Soc 19: 1041-1042.
Saeidnia S, Gohari AR, Mokhber-Dezfuli N, Kiuchi F. 2011. A review on phytochemistry and medicinal properties of the genus Achillea. Daru J Pharm Sci 19(3): 173-186.
Sang T. 2002. Utility of low-copy nuclear gene sequences in plant phylogenetics. Crit Rev Biochem Mol 37: 121-147.
Sharp PA, Burge CB. 1997. Classification of introns: U2-type or U12-type. Cell 91: 875-879.
Si XT, Zhang ML, Shi QW, Kiyota H. 2006. Chemical constituents of the plants in the genus Achillea. Chem Biodivers 3: 1163-1180.
Thimmappa R, Geisler K, Louveau T, O'Maille P, Osbourn A. 2014. Triterpene biosynthesis in plants. Annu Rev Plant Biol 65: 225-257.
Yan L, He-Chun Y, Hong W, Guo-Feng L. 2003. Molecular cloning, Escherichia coli expression and genomic organization of squalene synthase gene from Artemisia annua. Acta Bot Sin 45:608-613.
Zhang B, Liu Y, Chen M, Feng J, Ma Z, Zhang X, Zhu C. 2018. Cloning, expression analysis and functional characterization of squalene synthase (SQS) from Tripterygium Wilfordii. Molecules 23(2): 269.
Zhao YJ, Chen X, Zhang M, Su P, Liu YJ, Tong YR, Gao W. 2015. Molecular cloning and characterisation of farnesyl pyrophosphate synthase from Tripterygium wilfordii. PLoS One, 10(5): e0125415.