药学学报, 2018, 53(4): 646-658
引用本文:
张晓冬, 周姗, 尹彦超, 胡婷, 高智强, 韩沂霖, 李文东, 刘颖. 甘草CHS基因多态性与甘草苷含量的相关性分析[J]. 药学学报, 2018, 53(4): 646-658.
ZHANG Xiao-dong, ZHOU Shan, YIN Yan-chao, HU Ting, GAO Zhi-qiang, HAN Yi-lin, LI Wen-dong, LIU Ying. Analysis on the correlation between chalcone synthase gene polymorphism and content of liquiritin in licorice[J]. Acta Pharmaceutica Sinica, 2018, 53(4): 646-658.

甘草CHS基因多态性与甘草苷含量的相关性分析
张晓冬1, 周姗1, 尹彦超1, 胡婷1, 高智强1, 韩沂霖1, 李文东2, 刘颖1
1. 北京中医药大学生命科学学院, 北京 100029;
2. 北京市药品检验所中药成分分析与生物评价北京市重点实验室, 北京 102206
摘要:
2015版《中华人民共和国药典》明确规定,甘草药材中甘草苷含量不得低于0.5%,然而目前市售栽培甘草大量存在甘草苷含量不达标的现象。由于功能基因多态性对于次生代谢产物的积累至关重要,因此本文试图对甘草苷生物合成途径中的关键酶——查尔酮合酶(chalcone synthase,CHS)进行基因多态性分析,筛选出甘草苷高/低含量样品对应的特异基因型,为进一步解析甘草苷生物合成的分子机制提供参考。本文利用HPLC法测得60株甘草样品中4种主要黄酮类化合物(甘草苷、异甘草苷、甘草素、异甘草素)的含量,应用Spearman相关性分析及χ2检验进行统计学分析,发现4种黄酮类化合物含量间具有相关关系且不同基原间含量差异显著,乌拉尔甘草 > 光果甘草 > 胀果甘草。因此筛选出含量最高的5株乌拉尔甘草及含量最低的5株胀果甘草分为黄酮高、低含量组,对其CHS基因进行克隆及序列分析。10株样品共获得了336条长度为1 175 bp的CHS cDNA序列,存在249个变异位点,其中错义突变位点141个,可分为137种单倍型;编码389个氨基酸残基,存在130个变异位点,可分为102种氨基酸序列类型;主流CHS氨基酸序列类型为AA-3,黄酮高含量组甘草样品特有的主流氨基酸序列类型为AA-35,黄酮低含量组甘草样品特有主流氨基酸序列类型为AA-36,AA-35与AA-36在193位与229位处存在I/V、V/T突变。采用Discovery Studio 2.5对蛋白三维结构进行分析,结果表明AA-35的229位的缬氨酸为底物丙二酰辅酶A的结合位点。MEGA 5.0同源性分析表明甘草CHS亲缘关系良好,与其他物种区分度良好。本文筛选获得了黄酮高/低含量组甘草样品的特异CHS基因型,对于指导优质甘草的分子育种具有重要意义。
关键词:    甘草      甘草苷      查尔酮合酶      基因多态性     
Analysis on the correlation between chalcone synthase gene polymorphism and content of liquiritin in licorice
ZHANG Xiao-dong1, ZHOU Shan1, YIN Yan-chao1, HU Ting1, GAO Zhi-qiang1, HAN Yi-lin1, LI Wen-dong2, LIU Ying1
1. School of Life Sciences, Beijing University of Chinese Medicine, Beijing 100029, China;
2. Beijing Institute for Drug Control, Beijing Key Laboratory of Analysis and Evaluation on Chinese Medicine, Beijing 102206, China
Abstract:
Chinese pharmacopoeia stipulates that the content of liquiritin in licorice slices should be no less than 0.5%. However, there are lots of unqualified licorice slices in the herbal medicine markets. Due to the important role of functional gene polymorphism in secondary metabolism, this study attempts to analyze the influence of chalcone synthase (CHS) gene polymorphism on liquiritin biosynthesis and find out the unique haplotypes in licorice samples with high or low content of liquiritin, and to provide a basis for further analysis of molecular mechanism in flavonoid biosynthetic pathway. The contents of the 4 main flavonoids (liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin) in 60 licorice samples were assayed by HPLC and the results were analyzed by Spearman and χ2 tests. The contents of the 4 main flavonoids were related to each other and obviously different in different original plants. They were highest in Glycyrrhiza uralensis samples and lowest in Glycyrriza inflate samples. Five G. uralensis samples with the highest liquiritin contents and five G. inflate samples with the lowest liquiritin contents were selected to clone the CHS cDNA sequences. 336 CHS cDNA sequences with a full length of 1 175 bp were obtained, 249 variable sites (141 missense mutation sites) were found, and 137 haplotypes were determined. 130 variable sites were found in the 336 CHS amino acid sequences and 102 types were determined. AA-3 is the major type of CHS in licorice, AA-35 is the special major type of CHS in the group with high flavonoids contents and AA-36 is the special major type of CHS in the group with low flavonoids contents. The mutation sites between AA-35 and AA-36 are I/V at 193 and V/T at 229. Discovery Studio 2.5 analysis of the three-dimensional structure of the CHS protein shows that the valine at site 229 of AA-35 is combined with malonyl-CoA. Homology analysis indicates that the homology of CHS among different species is low. This study is significant for identification of the unique haplotypes in licorices with high or low content of liquiritin and guiding the further molecular breeding of high-quantity licorice.
Key words:    licorice    liquiritin    chalcone synthase    gene polymorphism   
收稿日期: 2017-12-14
DOI: 10.16438/j.0513-4870.2017-1245
基金项目: 北京中医药大学杰出青年人才项目(2016JYBXJQ002).
通讯作者: 李文东,Tel:86-10-84738646,E-mail:liuyliwd@sina.com;刘颖,Tel:86-10-52779627,E-mail:swlwd76@163.com
Email: liuyliwd@sina.com;swlwd76@163.com
相关功能
PDF(545KB) Free
打印本文
0
作者相关文章
张晓冬  在本刊中的所有文章
周姗  在本刊中的所有文章
尹彦超  在本刊中的所有文章
胡婷  在本刊中的所有文章
高智强  在本刊中的所有文章
韩沂霖  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] Sen L. Shennong's Herbal (神农本草经)[M]. Shanghai:Shanghai Technology and Health Press, 1959:35.
[2] Cui Y, Ao M, Li W, et al. Anti-inflammatory activity of licochalcone A isolated from Glycyhhiza inflate[J]. Z Natur-forsch B, 2008, 63:361-365.
[3] Zhou Y, Ho WS. Combination of liquiritin, isoliquiritin and isoliquirigenin induce apoptotic cell death through upregulating p53 and p21 in the A549 non-small cell lung cancer cells[J]. Oncol Rep, 2014, 31:298-304.
[4] Ai-Turki AI, Ei-Ziney MG, Abdel-Salam AM. Chemical and anti-bacterial characterization of aqueous extracts of oregano, marjoram, sage and licorice and their application in milk and labneh[J]. Int J Food Agric Environ, 2008, 6:39-44.
[5] Zhao Z, Wang W, Guo H, et al. Antidepressant-like effect of liquiritin from Glycyrrhiza uralensis in chronic variable stress induced depression model rats[J]. Behav Brain Res, 2008, 194:108-113.
[6] Abe K, Ikeda T, Wake K, et al. Glycyrrhizin prevents of lipopolysaccharide/D-galactosamine-induced liver injury through down-regulation of matrix metalloproteinase in mice[J]. J Pharm Pharmacol, 2008, 60:91-97.
[7] Huang MJ, Wang WQ, Wei SL, et al. Investigation on medicinal plant resources of Glycyrrhiza uralensis in China and chemical assessment of its underground part[J]. China J Chin Mater Med (中国中药杂志), 2010, 35:947-951.
[8] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. Part 1(中华人民共和国药典一部)[S]. Beijing:China Medical Science Press, 2015:87.
[9] Yang R, Li WD, Ma YS, et al. The molecular identification of licorice species and the quality evaluation of licorice slices[J]. Acta Pharm Sin (药学学报), 2017, 52:318-326.
[10] Yang R, Yuan BC, Ma YS, et al. Simultaneous determination of liquiritin, isoliquiritin, liquiritigenin and liquiritigenin in Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat. by HPLC[J]. Chin J Pharm Anal (药物分析杂志), 2016, 36:1729-1736.
[11] Liu Y, Zhan XJ, Li WD, et al. Copy number variations of functional genes influence contents of glycyrrhizic acid in Glycyrrhiza uralensis[J]. Acta Physiol Plant, 2014, 36:1433-1440.
[12] Liu Y, Liu DJ, Liu CS, et al. Mechanism of genuineness of liquorice Glycyrrhiza uralensis based on CNVs of HMGR, SQS1 and β-AS gene[J]. Acta Pharm Sin (药学学报), 2012, 47:250-255.
[13] Liu Y, Xu QX, Wang XY, et al. Analysis on correlation between 3-hydroxy-3-methylglutary-coenzyme A reductase gene polymorphism of Glycyrrhiza uralensis ans content of glycyr-rhizic acid[J]. China J Chin Mater Med (中国中药杂志), 2012, 37:3789-3792.
[14] Liu Y, Zhang N, Wang XY, et al. Researches on influence of squalene synthase gene polymorphism on catalytic efficiency of its encode enzyme in Glycyrrhiza uralensis[J]. China J Chin Mater Med (中国中药杂志), 2012, 37:3777-3783.
[15] Liu Y, Zhu XQ, Li WD, et al. Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis[J]. Acta Pharm Sin B, 2014, 4:161-166.
[16] Koes RE, Francesca Q, Joseph NM. The flavonoid biosynthetic pathway in plants:function and evolution[J]. Bioessays, 1994, 16:123-132.
[17] Reimold U, Kröger M, Kreuzaler F, et al. Coding and 3' non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme[J]. Embo J, 1983, 2:1801-1805.
[18] Xu HF. Cell Culture And Cloning of Chalcone Synthase Gene from Glycyrrhiza inflata Bat. (胀果甘草细胞培养及查尔酮合成酶基因的cDNA克隆)[D]. Baoding:Hebei University, 2011.
[19] Zhou S, Ma YS, Hu T, et al. Cloning and sequence analysis of chalcone synthase gene from Glycyrrhiza uralensis Fisch.[J]. Lett in Biotech (生物技术通讯), 2017, 28:812-817.
[20] Wang X, Zhang H, Chen L, et al. Liquorice, a unique "guide drug" of traditional Chinese medicine:a review of its role in drug interactions[J]. J Ethnopharmacol, 2013, 150:781-790.
[21] Durbin ML, Mccaig B, Clegg MT. Molecular evolution of the chalcone synthase multigene family in the morning glory genome[J]. Plant Mol Biol, 2000, 42:79-92.
[22] Kamiishi Y, Otani M, Takagi H, et al. Flower color alteration in the liliaceous ornamental Tricyrtis sp. by RNA interference-mediated suppression of the chalcone synthase gene[J]. Mol Breeding, 2012, 30:671-680.
[23] Blokland RV, Geest NVD, Mol JNM, et al. Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida, results from an increase in RNA turnover[J]. Plant J, 2010, 6:861-877.
[24] Taylor LP, Jorgensen R. Conditional male fertility in chalcone synthase-deficient Petunia[J]. J Hered, 1991, 83:11-17.
[25] Dao TTH, Linthorst HJM, Verpoorte R. Chalcone synthase and its functions in plant resistance[J]. Phytochem Rev, 2011, 10:397-412.
[26] Cushnie TP, Lamb AJ. Antimicrobial activity of flavonoids[J]. Int J Antimicrob Agents, 2005, 26:243-356.
[27] Courtney-Gutterson N, Napoli C, Lemieux C, et al. Modifi-cation of flower color in florist's chrysanthemum:production of a white-flowering variety through molecular genetics[J]. Biotechnology (NY), 1994, 12:268-271.
[28] Pollak PE, Vogt T, Mo Y, et al. Chalcone synthase and flavonol accumulation in stigmas and anthers of Petunia hybrida[J]. Plant Physiol, 1993, 102:925-932.
[29] Qi FJ, Gao XW, Wang JS, et al. Accumulation of pal and chs gene transcripts of rice carrying different bacterial blight resistance genes interacted with Xanthomonas oryzae pv. Oryzae[J]. J Agric Biotechnol (农业生物技术学报), 2000, 8:337-340.
[30] Rao C, Lei W, Li P, et al. Cloning of Scutellaria viscidula CHS gene and consteuction of its sense and antisense plant expression vectors[J]. J Chin Med Mater (中药材), 2009, 32:1661-1664.
[31] Shang J, Chen AL, Pan WZ. Cloning and bioinformation analysis of chalcone synthase gene from Lycium barbarum in Ningxia[J]. Chin Tradit Herb Drugs (中草药), 2013, 44:2898-2903.
[32] Fu M, Wei L, Yu J, et al. cDNA cloning and protein sequence analysis of chalcone synthase gene in leaves of Ampelopsis grossdentata[J]. Chin Tradit Herb Drugs (中草药), 2013, 44:85-89.
[33] Liu XM, Lu WH, Li J, et al. Over-expression of chalcone synthase of safflower increases flavonoid content in Arabidopsis thaliana[J]. Chin J Cell Biol (中国细胞生物学学报), 2017, 39:182-190.
[34] Liu GM, Lei W, Shui XR. Molecular clone, sequence analysis and expression vector construction with high efficiency of CHS gene from Scutellaria baicalensis Georgi[J]. Genomics Appl Biol (基因组学与应用生物学), 2016, 35:2814-2818.
[35] Meng HL, Zhang W, Liu BY, et al. Cloning and expression of chalcone synthase gene from Dendrobium officiale[J]. J South Agric (南方农业学报), 2016, 47:2015-2019.
[36] Mochida K, Sakurai K, Seki H, et al. Draft genome assembly and annotation of Glycyrrhiza uralensis, a medical legume[J]. Plant J, 2017, 89:181-195.
[37] Cheel J, Antwerpen PV, Tumoval L, et al. Free radical-scavenging, antioxidant and immunostimulating effects of a licorice infusion (Glycyrrhiza glabra L.)[J]. Food Chem, 2010, 122:508-517.
[38] Yang Y, Bian GX, Lv QJ. Neuroprotection and neurontrophism effects of liquiritin on primary cultured hippocampal cells[J]. China J Chin Mater Med (中国中药杂志), 2008, 33:931-935.
相关文献:
1.刘颖, 陈宏昊, 文浩, 高雅, 王礼强, 刘春生.甘草鲨烯合酶1基因多态性及其与β-香树脂醇合成酶共表达对β-香树脂醇积累的影响研究[J]. 药学学报, 2014,49(5): 734-741
2.吴青青, 陈 彦, 辛 然, 王晋艳, 周 蕾, 袁 菱, 贾晓斌.甘草主成分及其提取物肠吸收差异的多元化分析[J]. 药学学报, 2012,47(5): 657-663
3.邢盼盼 吴文华 杜 鹏 韩凤梅 陈 勇.马钱子碱与甘草次酸、甘草苷配伍后对大鼠肝脏CYP450的影响(英文)[J]. 药学学报, 2011,46(5): 573-580
4.王俊俊 廖晓欢 叶 敏 陈 勇.Caco-2单层细胞模型上士的宁的体外吸收机制及其与甘草苷的转运相互作用[J]. 药学学报, 2010,45(9): 1160-1164
5.韩博;李晴暖;吴胜伟;李俊纲;陈文;李文新.多壁碳纳米管与甘草苷和异甘草苷的选择性吸附作用[J]. 药学学报, 2007,42(11): 1222-1226