药学学报, 2022, 57(5): 1495-1505
引用本文:
李冉郡, 武立伟, 辛天怡, 廖海, 林余霖, 姚辉, 周嘉裕, 宋经元. 大黄药材基原物种叶绿体基因组分析与特异DNA条形码开发[J]. 药学学报, 2022, 57(5): 1495-1505.
LI Ran-jun, WU Li-wei, XIN Tian-yi, LIAO Hai, LIN Yu-lin, YAO Hui, ZHOU Jia-yu, SONG Jing-yuan. Analysis of chloroplast genomes and development of specific DNA barcodes for identifying the original species of Rhei Radix et Rhizoma[J]. Acta Pharmaceutica Sinica, 2022, 57(5): 1495-1505.

大黄药材基原物种叶绿体基因组分析与特异DNA条形码开发
李冉郡1,2, 武立伟2, 辛天怡2, 廖海1, 林余霖2, 姚辉2, 周嘉裕1*, 宋经元2,3*
1. 西南交通大学生命科学与工程学院, 四川 成都 610031;
2. 中国医学科学院、北京协和医学院药用植物研究所, 国家中医药管理局中药资源保护重点研究室, 北京 100193;
3. 中药资源教育部工程研究中心, 北京 100193
摘要:
大黄是我国常用大宗药材之一,其基原植物为唐古特大黄、药用大黄和掌叶大黄。不同基原的大黄药材活性成分和药效存在差异,为快速准确鉴定大黄药材3个基原物种,本文利用Illumina高通量测序技术对大黄药材基原物种叶绿体基因组进行测序,完成其组装注释与结构特征解析,并基于叶绿体基因组高变区开发精准鉴别大黄药材3个基原物种的特异DNA条形码,最后进行验证。结果如下:唐古特大黄、药用大黄和掌叶大黄叶绿体基因组全长分别为161 039 bp、161 093 bp和161 136 bp,呈典型四分体结构,均编码131个基因,包括86个蛋白质编码基因、37个tRNA基因和8个rRNA基因。基于高变区设计的5对引物均可对42份样品有效扩增,测序结果分析证明rps16-trnQpsaA-ycf3psbE-petLndhF-rpl32trnT-trnL均可作为特异DNA条形码准确鉴定唐古特大黄、药用大黄和掌叶大黄。本文可为大黄药材3个基原物种分类鉴定、保证大黄药材临床用药安全及规范大黄药材市场提供依据。
关键词:    大黄      叶绿体基因组      基原鉴定      特异DNA条形码     
Analysis of chloroplast genomes and development of specific DNA barcodes for identifying the original species of Rhei Radix et Rhizoma
LI Ran-jun1,2, WU Li-wei2, XIN Tian-yi2, LIAO Hai1, LIN Yu-lin2, YAO Hui2, ZHOU Jia-yu1*, SONG Jing-yuan2,3*
1. School of Life and Science, Southwest Jiaotong University, Chengdu 610031, China;
2. Key Laboratory of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China;
3. Engineering Research Center of Chinese Medicine Resource of Ministry of Education, Beijing 100193, China
Abstract:
Rhei Radix et Rhizoma is one of the most used medicinal materials in China. Its original species are Rheum palmatum, Rh. tanguticum, and Rh. officinale. Rhei Radix et Rhizoma derived from different original species are significantly different in their active ingredients and pharmacological effects. To develop an accurate, rapid, and specific identification method, we obtained the chloroplast genomes of the three original species by Illumina Novaseq sequencing. We designed specific DNA barcodes from the hypervariable regions, which can accurately identify the three original species. The experimental results showed that the total length of the chloroplast genomes of Rh. tanguticum, Rh. officinale and Rh. palmatum were 161 039 bp, 161 093 bp, and 161 136 bp, respectively. All the three genomes were represented as typical quadripartite structures. A total of 131 genes, including 86 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes were identified from each chloroplast genome. Five pairs of primers based on the hypervariable regions were designed to efficiently amplify 42 samples. Results confirmed that five hypervariable regions, rps16-trnQ, psaA-ycf3, psbE-petL, ndhF-rpl32, and trnT-trnL, can be used as specific DNA barcodes for the identification of Rh. tanguticum, Rh. officinale, and Rh. palmatum. These results provided genetic information for further species identification of Rhei Radix et Rhizoma, and improve the safety of this clinical medication as well as standardize the market for Rhei Radix et Rhizoma.
Key words:    Rhei Radix et Rhizoma    chloroplast genome    original species identification    specific DNA barcode   
收稿日期: 2021-10-22
DOI: 10.16438/j.0513-4870.2021-1525
基金项目: 国家自然科学基金青年基金项目(82003906);国家重点研发计划(2019YFC1711100).
通讯作者: 周嘉裕,E-mail:spinezhou@home.swjtu.edu.cn;宋经元,E-mail:jysong@implad.ac.cn
Email: spinezhou@home.swjtu.edu.cn;jysong@implad.ac.cn
相关功能
PDF(1202KB) Free
打印本文
0
作者相关文章
李冉郡  在本刊中的所有文章
武立伟  在本刊中的所有文章
辛天怡  在本刊中的所有文章
廖海  在本刊中的所有文章
林余霖  在本刊中的所有文章
姚辉  在本刊中的所有文章
周嘉裕  在本刊中的所有文章
宋经元  在本刊中的所有文章

参考文献:
[1] State Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (中华人民共和国药典)[S]. 2020 Ed. Part I. Beijing:China Medical Science Press, 2020:24.
[2] Chen JQ, Chen YY, Tang YP, et al. Multifunctional regularity of Rhei Radix et Rhizoma in ancient and modern medicine[J]. Chin Tradit Herb Drugs (中草药), 2019, 50:1485-1492.
[3] Xu LH, Huang XJ, Li YR, et al. Effect and potential mechanism of rhubarb on COVID-19 based on the "wenbingzaixiaqiyure" theory[J]. Pharmacol Clin Chin Mater Med (中药药理与临床), 2020, 36:85-90.
[4] Chen YY, Tang YP, Chen JQ, et al. Research progress and utilization strategy on resource chemistry of Rhei Radix et Rhizoma[J]. Chin Tradit Herb Drugs (中草药), 2018, 49:5170-5178.
[5] Wang Y, Yang X, Xia P, et al. Research progress on chemical composition and pharmacological effects of Rhei Radix et Rhizoma and predictive analysis on quality markers[J]. Chin Tradit Herb Drugs (中草药), 2019, 50:4821-4837.
[6] Komatsu K, Nagayama Y, Tanaka K, et al. Comparative study of chemical constituents of rhubarb from different origins[J]. Chem Pharm Bull (Tokyo), 2006, 54:1491-1499.
[7] Wang XM, Hou XQ, Zhang YQ, et al. Morphological variation in leaf dissection of Rheum palmatum complex (Polygonaceae)[J]. PLoS One, 2014, 9:e110760.
[8] Lin YL, Chen SL. Illustrated Handbook of Chinese Medicinal Plants (中国药用植物原色图鉴)[M]. Fuzhou:Fujian Science and Technology Press, 2016.
[9] Ge JH, Liu XH, Xu H, et al. Identification of different varieties of Rhei Radix et Rhizoma based on chemical analysis[J]. China J Chin Mater Med (中国中药杂志), 2015, 40:2309-2313.
[10] Du QT, Wen JL, Yan YS, et al. UPLC fingerprint of Rhei Radix et Rhizoma from different habitats[J]. J Chin Med Mater (中药材), 2013, 36:725-731.
[11] Li MN, Han RL, Han JP, et al. Identification of original plants of Radix et Rhizoma Rhei from its adulterants Rhizoma et Radix Polygoni Cuspidati and Radix Rumicis Obtusifolii by ITS2 sequences[J]. Global Tradit Chin Med (环球中医药), 2012, 5:185-189.
[12] Song JY, Yao H, Li Y, et al. Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique[J]. J Ethnopharmacol, 2009, 124:434-439.
[13] Zhang XQ, Liu CS, Yan XL, et al. Sequence analysis and identification of a chloroplast matK gene in Rhei Rhizoma from different botanical origins[J]. Acta Pharm Sin (药学学报), 2013, 48:1722-1728.
[14] Li RJ, Xin TY, Song LK, et al. Research progress in original species identification in industry chain of Rhei Radix et Rhizoma[J]. China J Chin Mater Med (中国中药杂志), 2021, 46:1060-1066.
[15] Kane NC, Cronk Q. Botany without borders:barcoding in focus[J]. Mol Ecol, 2008, 17:5175-5176.
[16] Jansen RK, Raubeson LA, Boore JL, et al. Methods for obtaining and analyzing whole chloroplast genome sequences[J]. Methods Enzymol, 2005, 395:348-384.
[17] Zhu AD, Guo WH, Gupta S, et al. Evolutionary dynamics of the plastid inverted repeat:the effects of expansion, contraction, and loss on substitution rates[J]. New Phytol, 2016, 209:1747-1756.
[18] Green BR. Chloroplast genomes of photosynthetic eukaryotes[J]. Plant J, 2011, 66:34-44.
[19] Bolger AM, Lohse M, Usadel B. Trimmomatic:a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30:2114-2120.
[20] Dierckxsens N, Mardulyn P, Smits G. Unraveling heteroplasmy patterns with NOVOPlasty[J]. NAR Genom Bioinform, 2019, 2:lqz011.
[21] Liu C, Shi LC, Zhu YJ, et al. CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences[J]. BMC Genomics, 2012, 13:715.
[22] Tillich M, Lehwark P, Pellizzer T, et al. GeSeq-versatile and accurate annotation of organelle genomes[J]. Nucleic Acids Res, 2017, 45:W6-11.
[23] Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs[J]. Nucleic Acids Res, 2005, 33:W686-689.
[24] Liu C, Huang LF. Chloroplast Genome Illustrated Handbook of Chinese Medicinal Plants (中国药用植物叶绿体基因组图谱第一册)[M]. Beijing:Science Press, 2020.
[25] Wu LW, Cui YX, Nie LP, et al. The characteristics of complete chloroplast genome sequence and phylogenetic analysis of Dendrobium moniliforme[J]. Acta Pharm Sin (药学学报), 2020, 55:1056-1066.
[26] Yang JP, Zhu ZL, Fan YJ, et al. Comparative plastomic analysis of three Bulbophyllum medicinal plants and its significance in species identification[J]. Acta Pharm Sin (药学学报), 2020, 55:2736-2745.
[27] Dong BR, Zhao ZL, Ni LH, et al. Molecular markers based upon whole chloroplast genomes and identifying alpine Gentiana waltonii and G. lhassica (Gentianaceae)[J]. Acta Pharm Sin (药学学报), 2021, 56:2584-2591.
[28] Cui YX. Structural Analysis of Complete Chloroplast Genome of Medicinal and Edible Original Plants of Amomum, Chinese Wolfberry, Hawthorn and Ginger (药食两用药材砂仁、枸杞、山楂和姜基原植物叶绿体基因组结构解析)[D]. Beijing:Peking Union Medical College, 2020.
[29] Wang XM, Hou XQ, Zhang YQ, et al. Distribution pattern of genuine species of rhubarb as traditional Chinese medicine[J]. J Med Plants Res, 2010, 4:1865-1876.
[30] Wang XM, Yang R, Feng SF, et al. Genetic variation in Rheum palmatum and Rheum tanguticum (Polygonaceae), two medicinally and endemic species in China using ISSR markers[J]. PLoS One, 2012, 7:e51667.
[31] Wang XM, Hou XQ, Zhang YQ, et al. Genetic diversity of the endemic and medicinally important plant Rheum officinale as revealed by Inter-Simpe Sequence Repeat (ISSR) markers[J]. Int J Mol Sci, 2012, 13:3900-3915.
[32] Ma XC, Xie CX, Guan M, et al. High levels of genetic diversity within one population of Rheum tanguticum on the Qinghai-Tibet Plateau have implications for germplasm conservation[J]. Pharm Crops, 2014, 5:1-8.
[33] Lou Q, Xin TY, Song JY. Application of DNA barcoding technology in the whole industrial chain of traditional Chinese medicine[J]. Acta Pharm Sin (药学学报), 2020, 55:1784-1791.