year 21, Issue 84 (12-2022)                   J. Med. Plants 2022, 21(84): 26-38 | Back to browse issues page

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1- Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Iran ,
2- Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Iran
3- Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Iran
Abstract:   (500 Views)
Background: Cytochrome P450s have essential roles in oxidative reactions during the biosynthesis of secondary metabolites, such as terpenoids. Objective: This research was aimed to identify the gene CYP72A154 as a gene involved in the glycyrrhizin biosynthesis pathway in Iranian licorice. Methods: CYP72A154 gene was isolated from Iranian licorice and cloned it into the PTG19-T vector. After confirmation of fragment length, the recombination plasmid was sent for sequencing. NCBI BLAST was used to analyze the nucleotide/ protein sequence homology between Glycyrrhiza glabra and other plants. The characterization of predicted amino acid sequences such as sequence homology, protein domains and functional sites, was performed using InterProscan. RT-PCR was performed to improve the relative expression this gene in the licorice root. Results: The query length was 314 aa, which after blasting in NCBI had about 78 to 80 % identity to the cytochrome P450 72A154 and 11-oxo-beta-amyrin 30-oxidase in species of G. glabra, G. uralensis and G. pallidiflora, as well as about 64 to 67 % with other species of the Fabaceae family. TMHMM analysis indicated the Exp number of AAs in TMHs was 22.11931, Exp number and first 60 AAs was 20.013. The results of RT-PCR revealed that the expression of this gene was comparable to the β-AS gene and CYP88D6, both being involved in the glycyrrhizin biosynthesis pathway. Conclusion: According to bioinformatics analysis and RT-PCR, it can be stated that the desired fragment belongs to the CYP72A154 gene and is also involved in the biosynthesis of glycyrrhizin.
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Type of Study: Research | Subject: Medicinal Plants
Received: 2022/08/4 | Accepted: 2022/10/17 | Published: 2022/12/1

1. Li Y-L, Yang Y, FU ChH and Yu LJ. Production of Glycyrrhizin in cell suspension of Glycyrrhiza inflate batalin cultured in Bioreactor. Biotech. Biotechn. Equip. 2012; 26(5): 3231-3235. [DOI:10.5504/BBEQ.2012.0083]
2. Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, Sudo H, Akashi T, Aoki T, Saito K and Muranaka T. Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proc. Natl. Acad. Sci. USA Proc. Natl. Acad. Sci. USA 2008; 105(37): 14204-14209. [DOI:10.1073/pnas.0803876105]
3. Matsui S, Matsumoto H, Sonoda Y, Ando K, Aizu-Yokota E, Sato T and Kasahara T. Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line. Int. Immunopharmacol 2004; 4(13): 1633-1644. [DOI:10.1016/j.intimp.2004.07.023]
4. Takahara T, Watanabe A and Shiraki K. Effects of glycyrrhizin on hepatitis B surface antigen: a biochemical and morphological study. J. Hepatol. 1994; 21(4): 601-609. [DOI:10.1016/S0168-8278(94)80108-8]
5. He J-X, Akao T, Nishino T and Tani T. The influence of commonly prescribed synthetic drugs for peptic ulcer on the pharmacokinetic fate of glycyrrhizin from Shaoyao-Gancao-tang. Biol. Pharm. Bull. 2001; 24(12): 1395-1399. [DOI:10.1248/bpb.24.1395]
6. Park H-Y, Park S-H, Yoon H-K, Han M-J and Kim D-H. Anti-allergic activity of 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide. Arch. Pharm. Res. 2004; 27: 57-60. [DOI:10.1007/BF02980047]
7. Ito M, Sato A, Hirabayashi K, Tanabe F, Shigeta Sh, Baba M, Clercq ED, Nakashima H and Yamamoto N. Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV). Antiviral Res. 1988; 10(11): 289-298. [DOI:10.1016/0166-3542(88)90047-2]
8. Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361(9374): 2045-2046. [DOI:10.1016/S0140-6736(03)13615-X]
9. Chrzanowski J, Chrzanowska A and Grabo'n W. Glycyrrhizin: an old weapon against a novel coronavirus. Phytother. Res. 2021; 35(2): 629-636. [DOI:10.1002/ptr.6852]
10. Van de Sand L, Bormann M, Alt M, Schipper L, Heilingloh CS, Steinmann E, Todt D, Dittmer U, Elsner C, Witzke O and Krawczyk A. Glycyrrhizin effectively inhibits SARS-CoV-2 replication by inhibiting the viral main protease. Viruses 2021; 13(4): 1-10. [DOI:10.3390/v13040609]
11. Bao F, Bai H-Y, Wu Z-R and Yang Z-G. Phenolic compounds from cultivated Glycyrrhiza uralensis and their PD-1/PD-L1 inhibitory activities. Natu. Pro. Res. 2021: 35(4): 562-569. [DOI:10.1080/14786419.2019.1586698]
12. Seki H, Sawai S, Ohyama K, Mizutani M, Ohnishi T, Sudo H, Fukushima EO, Akashi T, Aoki T, Saito K and Muranaka T. Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. Plant Cell. 2011; 23(11): 4112-4123. [DOI:10.1105/tpc.110.082685]
13. Hayashi H, Huang P, Kirakosyan A, Inoue K, Hiraoka N, Ikeshiro Y, Kushiro T, Shibuya M and Ebizuka Y. Cloning and characterization of a cDNA encoding. β-amyrin synthase involved in glycyrrhizin and soyasaponin Biosynthesis in Licorice. Bio Pharm Bull 2001; 24(8):912-916. [DOI:10.1248/bpb.24.912]
14. Chen H, Liu Y, Zhang X, Zhan X and Liu C. Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis. Acta Pharmaceutica Sinica B 2013; 3(6):416-424. [DOI:10.1016/j.apsb.2013.09.002]
15. Ali MM, Krishnamurthy P, El-Hadary M.H, Kim J.M, Nawas M.A, Yang S.H and Chung G. Identification and expression profiling of a new β-amyrin synthase gene (GmbAS3) from soybean. Russ. J. Plant Physio. 2016; 63: 383-390. [DOI:10.1134/S1021443716020035]
16. Shirazi Z, Aalami1 A, Tohidfar M and Sohani MM. Isolation, cloning and bioinformatics analysis of β-amyrin 11-oxidase coding sequence from licorice. Plant Omics J. 2016; 9(2): 165-171. [DOI:10.21475/poj.160902.p7778x]
17. Niu Y, Luo H, Sun C, Yang TJ, Dong L, Huang L and Chen S. Expression profiling of the triterpene saponin biosynthesis genes FPS, SS, SE, and DS in the medicinal plant Panax notoginseng. Gene 2014; 533(1): 295-303. [DOI:10.1016/j.gene.2013.09.045]
18. Goyal P, Muzafar Manzoor M, Gupta AP, Pandotra P and Gupta S. Molecular dissection of genes and promoters involved in glycyrrhizin biosynthesis revealed phytohormone induced modulation in Glycyrrhiza glabra L. Gene 2022; 836: 146682. [DOI:10.1016/j.gene.2022.146682]
19. Agbagw I.O, Datta S, Patil PG, Singh P and Nadarajan N. A protocol for high-quality genomic DNA extraction from legumes. Genet Mole Res. 2012; 11(4): 4632-4639. [DOI:10.4238/2012.September.14.1]
20. Ausubel FM, et al. Eds, Current protocols in molecular biology, John Wiley & Sons, Inc., NY, 1997.
21. Chang AY, Chau VWY, Landas JA and Pang Y. Preparation of calcium competent escherichia coli and heat-shock transformation. JEMI methods 2017; 1: 22-25.
22. Li X, Jin H, Wu Z, Rayner S and Wang B. A continuous process to extract plasmid DNA based on alkaline lysis. Nature Protocols. 2008; 3: 176-180. [DOI:10.1038/nprot.2007.526]
23. Kumar S, Strecher G, Li M, Knyaz C and Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms, Mol. Biol. Evol. 2018; 35(6): 1547-1549. [DOI:10.1093/molbev/msy096]
24. Thompson JD, Higgins DG and Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22: 4673-4680. [DOI:10.1093/nar/22.22.4673]
25. Zarei A, Zamani Z, Mousavi A, Fatahi R, Karimi Alavijeh M, Dehsara B and Salami SA. An effective protocol isolation of high-quality RNA from Pomegranate seeds. The Asian and Australasian Journal of Plant Science and Biotechnology 2012; 6(Special Issue 1): 32-37.
26. Chang S, Puryear S and Cairney J. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter 1993; 11(2): 113-116. [DOI:10.1007/BF02670468]
27. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29: 45-49. [DOI:10.1093/nar/29.9.e45]
28. Kyte J and Doolittle RF. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982; 157(1): 105-132. [DOI:10.1016/0022-2836(82)90515-0]
29. Huhman DV and Sumner LW. Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer. Phytochemistry 2002; 59(3): 347-360. [DOI:10.1016/S0031-9422(01)00432-0]
30. Hayashi H and Sudo H. Economic importance of licorice. Plant Biotech. 2009; 26(1): 101-104. [DOI:10.5511/plantbiotechnology.26.101]
31. Shirazi Z, Aalami A, Tohidfar M and Sohani MM. Metabolic engineering of glycyrrhizin pathway by over expression of β amyrin 11 oxidase in transgenic roots of Glycyrrhiza glabra. Mol. Biote. 2018: 60: 412-419. [DOI:10.1007/s12033-018-0082-7]

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