药学学报, 2021, 56(1): 319-327
引用本文:
侯嘉铭, 尹彦超, 田少凯, 张智新, 杨林, 李文东, 刘颖. 过表达CHI基因提高甘草毛状根中黄酮类化合物含量的研究[J]. 药学学报, 2021, 56(1): 319-327.
HOU Jia-ming, YIN Yan-chao, TIAN Shao-kai, ZHANG Zhi-xin, YANG Lin, LI Wen-dong, LIU Ying. Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots[J]. Acta Pharmaceutica Sinica, 2021, 56(1): 319-327.

过表达CHI基因提高甘草毛状根中黄酮类化合物含量的研究
侯嘉铭1, 尹彦超2, 田少凯1, 张智新1, 杨林1, 李文东3, 刘颖1
1. 北京中医药大学生命科学学院, 北京 102488;
2. 华中科技大学同济医学院附属同济医院药学部, 湖北 武汉 430000;
3. 北京市药品检验所, 北京 102206
摘要:
查尔酮异构酶(chalcone isomerase,CHI,EC5.5.1.6)是甘草黄酮类有效成分生物合成途径中的第二个限速酶,发挥重要的调控作用。本课题组在前期研究基础上,筛选出黄酮高含量甘草特异对应的CHI基因型,通过基因融合法构建了过表达CHI基因的植物双元表达载体,并通过电转法将其转化到发根农杆菌ACCC10060中,用于侵染甘草子叶和胚轴,获得过表达CHI基因的甘草毛状根系,利用qRT-PCR法测定各甘草毛状根系中CHI基因的拷贝数,并利用UPLC法测定各甘草毛状根系中4种黄酮类化合物的含量。结果显示获得了拷贝数分别为1和5的过表达CHI基因甘草毛状根系,且其总黄酮、甘草苷、甘草素和异甘草素的含量均显著高于野生型毛状根,表明过表达CHI基因能显著提高甘草毛状根中黄酮类化合物的含量。本文为解析CHI基因的功能提供了理论依据,筛选出3个过表达CHI基因甘草毛状根系用于后续扩大培养,可为离体积累甘草黄酮类化合物奠定基础。
关键词:    甘草      查尔酮异构酶      过表达      毛状根      UPLC      qRT-PCR     
Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots
HOU Jia-ming1, YIN Yan-chao2, TIAN Shao-kai1, ZHANG Zhi-xin1, YANG Lin1, LI Wen-dong3, LIU Ying1
1. School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102488, China;
2. Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China;
3. Beijing Institute for Drug Control, Beijing 102206, China
Abstract:
Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.
Key words:    Glycyrrhiza uralensis    chalcone isomerase    overexpression    hairy root    UPLC    qRT-PCR   
收稿日期: 2020-06-11
DOI: 10.16438/j.0513-4870.2020-0951
通讯作者: 李文东,Tel:86-10-53912163,E-mail:liuyliwd@bucm.edu.cn;刘颖,Tel:86-10-52779628,E-mail:sxlwd76@163.com
Email: liuyliwd@bucm.edu.cn;sxlwd76@163.com
相关功能
PDF(984KB) Free
打印本文
0
作者相关文章
侯嘉铭  在本刊中的所有文章
尹彦超  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
杨林  在本刊中的所有文章
李文东  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, 2015:86-87.
[2] Kim YW, Zhao RJ, Park SJ, et al. Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-κB-dependent iNOS and proinflammatory cytokines production[J]. Br J Pharmacol, 2008, 154:165-173.
[3] Grienke U, Braun H, Seidel N, et al. Computer-guided approach to access the anti-influenza activity of licorice constituents[J]. J Nat Prod, 2014, 77:563-570.
[4] Zhou Y, Ho WS. Combination of liquiritin, isoliquiritin and isoliquirigenin induce apoptotic cell death through upregulating p53 and p21 in the A549 nonsmall cell lung cancer cells[J]. Oncol Rep, 2014, 31:298-304.
[5] Boland MJ, Wong E. Purification and kinetic properties of chalcone-flavanone isomerase from soya bean[J]. Eur J Biochem, 1975, 50:383-389.
[6] Shimada N. A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume specific 5-deoxy (iso) flavonoids in Lotus japonicas[J]. Plant Physiol, 2003, 131:941-951.
[7] Jez JM, Bowman ME, Dixon RA, et al. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase[J]. Nat Struct Biol, 2000, 7:786-791.
[8] Mehdy MC, Lamb CJ. Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection[J]. EMBO J, 1988, 86:1527-1533.
[9] Van TAJ, Koes RE, Spelt CE, et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida:coordinate, light-regulated and differential expression of flavonoid genes[J]. EMBO J, 1988, 7:1257-1263.
[10] Blyden ER, Doerner PW, Lamb CJ, et al. Sequence analysis of a chalcone isomerase cDNA of Phaseolus vulgaris L.[J]. Plant Mol Biol, 1991, 16:167-169.
[11] Grotwold E, Peterson T. Isolation and characterization of a maize gene encoding chalcone flavonone isomerase[J]. Mol Gen Genet, 1994, 242:1-8.
[12] Mckhann HI, Hirsch AM. Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.):highest transcript levels occur in young roots and root tips[J]. Plant Mol Biol, 1994, 24:767-777.
[13] Li F, Jin Z, Qu W, et al. Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco[J]. Plant Physiol Biochem, 2006, 44:455-461.
[14] Muir SR, Collins GJ, Robinson S, et al. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols[J]. Nat Biotechnol, 2001, 19:470-474.
[15] Kim S, Jones R, YooK S, et al. Gold color in onions (Allium cepa):a natural mutation of the chalcone isomerase gene resulting in a premature stop codon[J]. Mol Genet Genomics, 2004, 272:411-419.
[16] Li JH. Study on Flavonoid Prenyltransferases from Glycyrrhiza uralensis (乌拉尔甘草黄酮异戊烯基转移酶的研究)[D]. Beijing:Peking Union Medical College, 2014
[17] Elkin YN, Kulesh NI, Stepanova AY, et al. Methylated flavones of the hairy root culture Scutellaria baicalensis[J]. J Plant Physiol, 2018, 231:277-280.
[18] Wang XY, Cui HG, Huang LQ, et al. A full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase cloning and analysis of introduced gene expression in Salvia miltiorrhiza[J]. Acta Pharm Sin (药学学报), 2008, 43:1251-1257.
[19] Kochan E, Nowak A, Zakłos-Szyda M, et al. Panax quinquefolium L. ginsenosides from hairy root cultures and their clones exert cytotoxic, genotoxic and pro-apoptotic activity towards human colon adenocarcinoma cell line Caco-2[J]. Molecules, 2020, 25:E2262.
[20] Zhang J, Zhou L, Zheng X, et al. Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots[J]. Plant Cell Rep, 2017, 36:1297-1309.
[21] Kim YK, Kim JK, Kim YB, et al. Enhanced accumulation of phytosterol and triterpene in hairy root cultures of Platycodon grandiflorum by overexpression of Panax ginseng 3-hydroxy-3-methylglutaryl-coenzyme A reductase[J]. J Agric Food Chem, 2013, 61:1928-1934.
[22] Li B, Cui G, Shen G, et al. Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza[J]. Sci Rep, 2017, 7:43320.
[23] Zhang XD. Molecular Mechanism of Licorice Flavonoid Biosynthesis Based on CHI Gene Polymorphism (基于CHI基因多态性的甘草黄酮类化合物生物合成分子机制研究)[D]. Beijing:Beijing University of Chinese Medicine, 2019.
[24] 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.
[25] 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.
[26] Liu Y, Xu QX, Xi PY, et al. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis[J]. Acta Pharm Sin (药学学报), 2013, 48:773-779.
[27] Liu Y, Zhang N, Wang XY, et al. Study on the effects of Glycyrrhiza uralensis Fisch squalene synthase gene polymorphism on its enzyme catalytic efficiency[J]. China J Chin Mater Med (中国中药杂志), 2012, 37:3777-3783.
[28] Yin YC, Zhang XD, Gao ZQ, et al. Enhancing glycyrrhizic acid accumulation by over-expressing β-amyrin synthase gene (GuBAS) root-specifically in hairy roots of Glycyrrhiza uralensis[J]. Chin Herb Med, 2019, 11:192-199.
[29] Zhou L, Wang Y, Ren L, et al. Overexpression of Ps-CHI1, a homologue of the chalcone isomerase gene from tree peony (Paeonia suffruticosa), reduces the intensity of flower pigmentation in transgenic tobacco[J]. Plant Cell Tissue Organ Culture, 2013, 116:285-295.
[30] Park NI, Xu H, Li X, et al. Enhancement of flavone levels through overexpression of chalcone isomerase in hairy root cultures of Scutellaria baicalensis[J]. Funct Integr Genomics, 2011, 11:491-496.
[31] Gao Y, Wu CH, Piao XC, et al. Optimization of culture medium components and culture period for production of adventitious roots of Echinacea pallida (Nutt.)[J]. Plant Cell Tissue Organ Culture, 2018, 135:299-307.
[32] Jisha S, Gouri PR, Anith KN, et al. Piriformospora indica cell wall extract as the best elicitor for asiaticoside production in Centella asiatica (L.) Urban, evidenced by morphological, physiological and molecular analyses[J]. Plant Physiol Biochem, 2018, 125:106-115.
[33] Mercier L, Lafitte C, Borderies G, et al. The algal polysaccharide carrageenans can act as an elicitor of plant defence[J]. New Phytol, 2010, 149:43-51.
[34] Zhou X, Yang Y. Differential expression of rice Nramp genes in response to pathogen infection, defense signal molecules and metal ions[J]. Physiol Mol Plant Pathol, 2004, 65:235-243.
[35] Logemann E, Wu SC, Schröder J, et al. Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes[J]. Plant J Cell Mol Biol, 2010, 8:865-876.