药学学报, 2021, 56(7): 2025-2032
引用本文:
杨林, 汪逗逗, 田少凯, 张智新, 侯嘉铭, 肖瑶, 刘颖. 甘草DXS基因过表达及表达沉默对甘草酸生物合成的影响研究[J]. 药学学报, 2021, 56(7): 2025-2032.
YANG Lin, WANG Dou-dou, TIAN Shao-kai, ZHANG Zhi-xin, HOU Jia-ming, XIAO Yao, LIU Ying. The role of licorice DXS knockout and overexpression in glycyrrhizic acid biosynthesis[J]. Acta Pharmaceutica Sinica, 2021, 56(7): 2025-2032.

甘草DXS基因过表达及表达沉默对甘草酸生物合成的影响研究
杨林1, 汪逗逗1, 田少凯1, 张智新1, 侯嘉铭1, 肖瑶2*, 刘颖1*
1. 北京中医药大学生命科学学院, 北京 102488;
2. 北京中医药大学中药学院, 北京 102488
摘要:
1-脱氧-D-木酮糖-5-磷酸合酶(1-deoxy-D-xylulose-5-phosphate synthase,DXS)是萜类化合物前体物质合成途径2-C-甲基-D-赤藻糖醇-4-磷酸(2-C-methyl-D-erythritol 4-phosphate,MEP)途径的第一个限速酶,在甘草次生代谢过程中发挥着重要的调控作用。本课题组在前期转录组研究中发现DXS基因表达水平与甘草的指标性成分甘草酸的含量呈负相关,因此本文拟通过毛状根培养体系,从基因过表达和沉默两方面开展研究,解析DXS基因对甘草酸生物合成的调控作用。本文克隆了甘草DXS基因(GenBank注册号:MN158121);采用基因融合法构建了过表达载体pCA-DXS;根据DXS第一外显子设计sgRNA序列,构建了CRISPR/Cas9基因编辑载体pHSE-DXS;以甘草胚轴为外植体材料,采用发根农杆菌介导法诱导了DXS基因过表达和表达沉默的甘草毛状根体系。同时,诱导了野生型甘草毛状根及含空质粒的阴性对照毛状根。采用UPLC法测定各甘草毛状根样品中甘草酸的含量,结果显示:DXS基因沉默组毛状根样品中甘草酸含量显著高于野生型和阴性对照组,而DXS基因过表达甘草毛状根样品中甘草酸含量则显著低于野生型和阴性对照组。本文通过逆向遗传学策略,从基因过表达和沉默两个方面证实了DXS基因对甘草酸生物合成的负调控作用,为解析DXS基因在萜类化合物代谢中的作用提供了理论依据,也为进一步构建甘草酸生物合成分子调控网络奠定了基础。
关键词:    甘草酸      DXS      基因过表达      CRISPR/Cas9      毛状根     
The role of licorice DXS knockout and overexpression in glycyrrhizic acid biosynthesis
YANG Lin1, WANG Dou-dou1, TIAN Shao-kai1, ZHANG Zhi-xin1, HOU Jia-ming1, XIAO Yao2*, LIU Ying1*
1. School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102488, China;
2. School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 102488, China
Abstract:
1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.
Key words:    glycyrrhizic acid    1-deoxy-D-xylulose-5-phosphate synthase    gene overexpression    CRISPR/Cas9    hairy root   
收稿日期: 2021-02-08
DOI: 10.16438/j.0513-4870.2021-0225
通讯作者: 刘颖,Tel:86-10-53912163,E-mail:liuyliwd@bucm.edu.cn;肖瑶,Tel:86-10-53912163,E-mail:xiaoyao9510@126.com
Email: liuyliwd@bucm.edu.cn;xiaoyao9510@126.com
相关功能
PDF(560KB) Free
打印本文
0
作者相关文章
杨林  在本刊中的所有文章
汪逗逗  在本刊中的所有文章
田少凯  在本刊中的所有文章
张智新  在本刊中的所有文章
侯嘉铭  在本刊中的所有文章
肖瑶  在本刊中的所有文章
刘颖  在本刊中的所有文章

参考文献:
[1] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (中华人民共和国药典)[M]. Part 1. Beijing:China Medical Science Press, 2020:88-89.
[2] Sun ZG, Zhao TT, Lu N, et al. Research progress of glycyrrhizic acid on antiviral activity[J]. Mini Rev Med Chem, 2019, 19:826-832.
[3] Yang R, Yuan BC, Ma YS, et al. The anti-inflammatory activity of licorice, a widely used Chinese herb[J]. Pharm Biol, 2017, 55:5-18.
[4] Sun X, Duan XP, Wang CY, et al. Protective effects of glycyrrhizic acid against non-alcoholic fatty liver disease in mice[J]. Eur J Pharmacol, 2017, 806:75-82.
[5] Gao ZQ, Tian SK, Hou JM, et al. RNA-Seq based transcriptome analysis reveals the molecular mechanism of triterpenoid biosynthesis in Glycyrrhiza glabra[J]. Bioorg Med Chem Lett, 2020, 30:127102.
[6] Rodríguez-Concepción M, Ahumada I, Diez-Juez E, et al. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening[J]. Plant J, 2001, 27:213-222.
[7] Jadaun JS, Sangwan NS, Narnoliya LK, et al. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera:active involvement of plastid isoprenogenic pathway in their biosynthesis[J]. Physiol Plant, 2017, 159:381-400.
[8] Lange BM, Wildung MR, McCaskill D, et al. A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway[J]. Proc Natl Acad Sci U S A, 1998, 95:2100-2104.
[9] Kim BR, Kim SU, Chang YJ. Differential expression of three 1-deoxy-D-xylulose-5-phosphate synthase genes in rice[J]. Biotechnol Lett, 2005, 27:997-1001.
[10] Zhang FY, Liu WH, Xia J, et al. Molecular characterization of the 1-deoxy-D-xylulose 5-phosphate synthase gene family in Artemisia annua[J]. Front Plant Sci, 2018, 9:952.
[11] Cordoba E, Porta H, Arroyo A, et al. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize[J]. J Exp Bot, 2011, 62:2023-2038.
[12] Roy A. Hairy root culture an alternative for bioactive compound production from medicinal plants[J]. Curr Pharm Biotechnol, 2021, 22:136-149.
[13] Ron M, Kajala K, Pauluzzi G, et al. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model[J]. Plant Physiol, 2014, 166:455-469.
[14] Deng CP, Hao XL, Shi M, et al. Tanshinone production could be increased by the expression of SmWRKY2 in Salvia miltiorrhiza hairy roots[J]. Plant Sci, 2019, 284:1-8.
[15] Singh P, Prasad R, Tewari R, et al. Silencing of quinolinic acid phosphoribosyl transferase (QPT) gene for enhanced production of scopolamine in hairy root culture of Duboisia leichhardtii[J]. Sci Rep, 2018, 8:13939.
[16] Nakayasu M, Akiyama R, Lee HJ, et al. Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene[J]. Plant Physiol Biochem, 2018, 131:70-77.
[17] Hou JM, Yin YC, Tian SK, et al. Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots[J]. Acta Pharm Sin (药学学报), 2021, 56:319-327.
[18] Yin YC, Hou JM, Tian SK, et al. Overexpressing chalcone synthase (CHS) gene enhanced flavonoids accumulation in Glycyrrhiza uralensis hairy roots[J]. Bot Lett, 2020, 167:219-231.
[19] Yin YC, Zhang XD, Gao ZQ, et al. Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of Glycyrrhiza uralensis[J]. Chin Herb Med, 2019, 11:192-199.
[20] Hu T, Gao ZQ, Yin YC, et al. Determination of seven flavonoids in Glycyrrhiza uralensis Fisch. and Glycyrrhiza glabra L. by UPLC[J]. Chin J Pharm Anal (药物分析杂志), 2019, 39:763-771.
[21] Bergman ME, Davis B, Phillips MA. Medically useful plant terpenoids:biosynthesis, occurrence, and mechanism of action[J]. Molecules, 2019, 24:3961.
[22] Bach TJ, Boronat A, Campos N, et al. Mevalonate biosynthesis in plants[J]. Crit Rev Biochem Mol Biol, 1999, 34:107-122.
[23] Newman JD, Chappell J. Isoprenoid biosynthesis in plants:carbon partitioning within the cytoplasmic pathway[J]. Crit Rev Biochem Mol Biol, 1999, 34:95-106.
[24] Frank A, Groll M. The methylerythritol phosphate pathway to isoprenoids[J]. Chem Rev, 2017, 117:5675-5703.
[25] Vranová E, Coman D, Gruissem W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis[J]. Annu Rev Plant Biol, 2013, 64:665-700.
[26] Rodríguez-Concepción M, Boronat A. Breaking new ground in the regulation of the early steps of plant isoprenoid biosynthesis[J]. Curr Opin Plant Biol, 2015, 25:17-22.
[27] Xiang S, Usunow G, Lange G, et al. Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis[J]. J Biol Chem, 2007, 282:2676-2682.
相关文献:
1.张智新, 汪逗逗, 杨林, 田少凯, 肖瑶, 刘颖.基于基因过表达及基因沉默解析F5H基因对甘草酸生物合成的调控研究[J]. 药学学报, 2021,56(6): 1719-1726