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Article(id=1220655534286488249, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1220655523473571972, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2020-0278, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1583769600000, receivedDateStr=2020-03-10, revisedDate=1585756800000, revisedDateStr=2020-04-02, acceptedDate=null, acceptedDateStr=null, onlineDate=1768956558056, onlineDateStr=2026-01-21, pubDate=1597161600000, pubDateStr=2020-08-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768956558056, onlineIssueDateStr=2026-01-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768956558056, creator=13701087609, updateTime=1768956558056, updator=13701087609, issue=Issue{id=1220655523473571972, tenantId=1146029695717560320, journalId=1189982191388893191, year='2020', volume='55', issue='8', pageStart='1707', pageEnd='1982', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768956555479, creator=13701087609, updateTime=1768986579152, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1220781451944051235, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1220655523473571972, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1220781451944051236, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1220655523473571972, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1951, endPage=1956, ext={EN=ArticleExt(id=1220655534760444629, articleId=1220655534286488249, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Use of ITS2 and psbA-trnH DNA barcode sequences of wild Phytolacca resources in Guanzhong area of Shaanxi Province, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

In order to explore the use of DNA barcode in the identification of wild Phytolacca resources in the Shaanxi Guanzhong area, 29 DNA samples were amplified and sequenced by using the universal primers ITS2 and psbA-trnH. The sequences were spliced and proof-read by Codon CodeA aligner V3.0, followed by blast comparison and identification analysis; mega 6.0 was used to analyze sequence characteristics, Kimura 2-Parameter (K2P) was used to analyze distance and intraspecific or interspecific variation, and Neighbor-Joining trees were established to evaluate the ability of two pairs of candidate sequences to distinguish Phytolaccae Radix from its adulterants. The results showed that the success rate of PCR amplification and sequencing of ITS2 and psbA-trnH was 100%; the NJ tree showed that both ITS2 and psbA-trnH sequences could separate P. acinosa, P. americana, other species of the same genus like P. japonica, P. exiensis and two adulterant species into a single clade; primer ITS2 had an advantage over psbA-trnH in determining interspecific genetic distances. Therefore, both ITS2 and psbA-trnH sequences can be used for identification of Phytolacca and their adulterants, which provides a theoretical basis for the distribution of wild Phytolacca resources and their rational development and utilization.

, correspAuthors=Gang ZHANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2020 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Rui-hua Lü, Zhao FENG, Tian-yi MA, Rui-hua Lü, Ming-ying ZHANG, Liang PENG, Jing GAO, Gang ZHANG), CN=ArticleExt(id=1220655535850963724, articleId=1220655534286488249, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=陕西关中野生商陆资源的ITS2和psbA-trnH条形码序列研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

为探讨DNA条形码在陕西关中野生商陆资源中的鉴定作用，本研究采用通用引物ITS2和psbA-trnH对29份商陆样品DNA进行PCR扩增和测序，用Codon CodeA-ligner V3.0进行序列拼接和校对，随后进行BLAST比对鉴定分析；同时运用MEGA 6.0分析序列特征、K2P遗传距离及种内或种间变异，并构建系统进化树，评价两对引物对该区域商陆及其混伪品的鉴别能力。研究结果表明，ITS2和psbA-trnH序列的PCR扩增和测序成功率均为100%；NJ系统进化树显示ITS2和psbA-trnH序列均可明显将所有样本分别聚为商陆、垂序商陆并与其同属近源种鄂西商陆、日本商陆及两种混伪品各自聚为不同分支；ITS2序列在种间遗传距离上较psbA-trnH有一定优势。ITS2和psbA-trnH序列均可作为DNA条形码对商陆及其混伪品进行识别和鉴定，为商陆资源品种现状与分布研究及合理开发利用提供理论依据。

, correspAuthors=张岗, authorNote=null, correspAuthorsNote=
*张岗, Tel/Fax:86-29-38185165, E-mail:
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Chin Tradit Herb Drugs (中草药), 2017, 48: 1403-1408., articleTitle=Cluster analysis on psbA-trnH and ITS2 sequences of Microcos paniculata from different geographical populations, refAbstract=null), Reference(id=1220655545250398693, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, doi=null, pmid=null, pmcid=null, year=2018, volume=24, issue=null, pageStart=44, pageEnd=50, url=http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsyfjxzz201804008, language=null, rfNumber=[21], rfOrder=20, authorNames=Zhou KL, Lv WQ, Tang SL, journalName=Chin J Exp Tradit Med Form (中国实验方剂学杂志), refType=null, unstructuredReference= Zhou KL , Lv WQ , Tang SL . Variation patterns of contents of water-soluble extractives and esculentoside A and selection of superior germplasms in Phytolacca acinosa[J]. Chin J Exp Tradit Med Form (中国实验方剂学杂志), 2018, 24: 44-50., articleTitle=Variation patterns of contents of water-soluble extractives and esculentoside A and selection of superior germplasms in Phytolacca acinosa, refAbstract=null)], funds=[Fund(id=1220655542557655353, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, awardId=2017PY32, language=CN, fundingSource=陕西中医药大学科研培育项目(2017PY32), fundOrder=null, country=null), Fund(id=1220655542708650310, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, awardId=2019-QN01, language=CN, fundingSource=陕西中医药大学“秦药”品质评价及资源开发学科创新团队项目(2019-QN01), fundOrder=null, country=null), Fund(id=1220655542805119312, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, awardId=141306200105, language=CN, fundingSource=陕西中医药大学“思邈青年学者”项目(141306200105), fundOrder=null, country=null), Fund(id=1220655542935142749, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, awardId=财社[2018]43号, language=CN, fundingSource=2018年公共卫生服务补助奖金专项“全国中药资源普查项目”(财社[2018]43号), fundOrder=null, country=null), Fund(id=1220655543035806057, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, awardId=null, language=CN, fundingSource=陕西省高校青年杰出人才支持计划项目, fundOrder=null, country=null), Fund(id=1220655543149052271, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, awardId=null, language=CN, fundingSource=咸阳市中青年科技领军人才项目, fundOrder=null, country=null)], companyList=[AuthorCompany(id=1220655536085844764, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, xref=null, ext=[AuthorCompanyExt(id=1220655536094233371, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, companyId=1220655536085844764, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. 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Specimen
No.		 Collection location Longitude Latitude Altitude GenBank accession No.
ITS2 psbA-trnH
1 Yabai Town, Zhouzhi County, Xi'an City 108.0419° E 34.0953° N 460 m MT118138 MT130936
2 Tamiao Village, Zhuyu Town, Zhouzhi County, Xi'an City 108.0003° E 34.1116° N 700 m MT118139 MT130937
7 Xizhai Village, Hengqu Town, Mei County, Baoji City 107.5935° E 34.0712° N 660 m MT118140 MT130938
8 Chaoyang Village, Fengming Town, Qinshan County, Baoji City 107.6043° E 34.4475° N 672 m MT118141 MT130939
9 Chaoyang Village, Fengming Town, Qishan County, Baoji City 107.6055° E 34.4477° N 675 m MT118142 MT130940
10 Yanhe Village, Fengming Town, Qishan County, Baoji City 107.6002° E 34.4500° N 681 m MT118143 MT130941
11 Banpopu Village, Liulin Town, Fengxiang County, Baoji City 107.2421° E 34.5716°N 957 m MT118144 MT130942
13 Shangdengjia Village, Shuigou Town, Qianyang County, Baoji City 107.0460° E 34.7315°N 762 m MT118145 MT130943
14 Zhaojiapo Village, Chengguan Town, Long County, Baoji City 106.8443° E 34.9039° N 917 m MT118146 MT130944
16 Shiqiao Village, Jinhe Town, Jintai District, Baoji City 107.1422° E 34.4237° N 650 m MT118147 MT130945
17 Yimenbu Village, Shennong Town, Weibin District, Baoji City 107.0547° E 34.1937° N 640 m MT118148 MT130946
18 Cuixigou Village, Yangling District, Xianyang City 108.0753° E 34.3027° N 519 m MT118149 MT130947
20 Yangling Vocational & Technical College 108.0649° E 34.2764° N 466 m MT118150 MT130948
22 Tugou Village, Chengguan Town, Bin County, Xianyang City 107.9780° E 35.0674°N 846 m MT118151 MT130949
23 Chengdong Village, Zhaoren Town, Changwu County, Xianyang City 107.8006° E 35.1981° N 1 181 m MT118152 MT130950
24 Yufeng Village, Yongping Town, Yongshou County, Xianyang City 108.0437° E 34.8057° N 1 265 m MT118153 MT130951
25 HanJiabu Village, Qian County, Xianyang City 108.2284° E 34.5565° N 746 m MT118154 MT130952
26 Yongfeng Village, Yongle Town, Jingyang County, Xianyang City 108.9372° E 34.5214° N 391 m MT118155 MT130953
27 Sanglou Village, Xing Town, Pucheng County, Weinan City 109.4167° E 34.9136° N 456 m MT118156 MT130954
28 Leizhuang Village, Chengguan Town, Chengcheng County, Weinan City 109.9353° E 35.1260° N 649 m MT118157 MT130955
29 Nancai Village, Wangcun Town, Heyang County, Weinan City 110.0859° E 35.1956° N 707 m MT118158 MT130956
31 Wengyukou Village, Huashan Town, Huayin County, Weinan City 110.0217° E 34.5240° N 420 m MT118159 MT130957
32 Zhenjiang Village, Chishui Town, Hua County, Weinan City 109.6432° E 34.4925° N 352 m MT118160 MT130958
33 Yaozhu Village, Yushan Town, Lantian County, Xi'an City 109.5162° E 34.2433° N 864 m MT118161 MT130959
34 Yangzhuang Village, Yangzhuang Town, Chang'an District, Xi'an City 109.1449° E 34.0420° N 622 m MT118162 MT130960
35 Taipingzhuang Village, Yuchan Town, Huyi District, Xi'an City 108.5576° E 34.1277° N 410 m MT118163 MT130961
36 Taipingzhuang Village, Yuchan Town, Huyi District, Xi'an City 108.5576° E 34.1277° N 410 m MT118164 MT130962
37 417 hospital, Lintong District, Xi'an City 109.1949° E 34.3645° N 477 m MT118165 MT130963
38 Shaanxi University of Chinese Medicine, Qindu District, Xianyang City 108.7359° E 34.3175° N 381 m MT118166 MT130964
), ArticleFig(id=1220655541265809634, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=CN, label=Table 1, caption=

Source information and GenBank accession No. of wild Phytolacca L. samples

, figureFileSmall=null, figureFileBig=null, tableContent=
Specimen
No.		 Collection location Longitude Latitude Altitude GenBank accession No.
ITS2 psbA-trnH
1 Yabai Town, Zhouzhi County, Xi'an City 108.0419° E 34.0953° N 460 m MT118138 MT130936
2 Tamiao Village, Zhuyu Town, Zhouzhi County, Xi'an City 108.0003° E 34.1116° N 700 m MT118139 MT130937
7 Xizhai Village, Hengqu Town, Mei County, Baoji City 107.5935° E 34.0712° N 660 m MT118140 MT130938
8 Chaoyang Village, Fengming Town, Qinshan County, Baoji City 107.6043° E 34.4475° N 672 m MT118141 MT130939
9 Chaoyang Village, Fengming Town, Qishan County, Baoji City 107.6055° E 34.4477° N 675 m MT118142 MT130940
10 Yanhe Village, Fengming Town, Qishan County, Baoji City 107.6002° E 34.4500° N 681 m MT118143 MT130941
11 Banpopu Village, Liulin Town, Fengxiang County, Baoji City 107.2421° E 34.5716°N 957 m MT118144 MT130942
13 Shangdengjia Village, Shuigou Town, Qianyang County, Baoji City 107.0460° E 34.7315°N 762 m MT118145 MT130943
14 Zhaojiapo Village, Chengguan Town, Long County, Baoji City 106.8443° E 34.9039° N 917 m MT118146 MT130944
16 Shiqiao Village, Jinhe Town, Jintai District, Baoji City 107.1422° E 34.4237° N 650 m MT118147 MT130945
17 Yimenbu Village, Shennong Town, Weibin District, Baoji City 107.0547° E 34.1937° N 640 m MT118148 MT130946
18 Cuixigou Village, Yangling District, Xianyang City 108.0753° E 34.3027° N 519 m MT118149 MT130947
20 Yangling Vocational & Technical College 108.0649° E 34.2764° N 466 m MT118150 MT130948
22 Tugou Village, Chengguan Town, Bin County, Xianyang City 107.9780° E 35.0674°N 846 m MT118151 MT130949
23 Chengdong Village, Zhaoren Town, Changwu County, Xianyang City 107.8006° E 35.1981° N 1 181 m MT118152 MT130950
24 Yufeng Village, Yongping Town, Yongshou County, Xianyang City 108.0437° E 34.8057° N 1 265 m MT118153 MT130951
25 HanJiabu Village, Qian County, Xianyang City 108.2284° E 34.5565° N 746 m MT118154 MT130952
26 Yongfeng Village, Yongle Town, Jingyang County, Xianyang City 108.9372° E 34.5214° N 391 m MT118155 MT130953
27 Sanglou Village, Xing Town, Pucheng County, Weinan City 109.4167° E 34.9136° N 456 m MT118156 MT130954
28 Leizhuang Village, Chengguan Town, Chengcheng County, Weinan City 109.9353° E 35.1260° N 649 m MT118157 MT130955
29 Nancai Village, Wangcun Town, Heyang County, Weinan City 110.0859° E 35.1956° N 707 m MT118158 MT130956
31 Wengyukou Village, Huashan Town, Huayin County, Weinan City 110.0217° E 34.5240° N 420 m MT118159 MT130957
32 Zhenjiang Village, Chishui Town, Hua County, Weinan City 109.6432° E 34.4925° N 352 m MT118160 MT130958
33 Yaozhu Village, Yushan Town, Lantian County, Xi'an City 109.5162° E 34.2433° N 864 m MT118161 MT130959
34 Yangzhuang Village, Yangzhuang Town, Chang'an District, Xi'an City 109.1449° E 34.0420° N 622 m MT118162 MT130960
35 Taipingzhuang Village, Yuchan Town, Huyi District, Xi'an City 108.5576° E 34.1277° N 410 m MT118163 MT130961
36 Taipingzhuang Village, Yuchan Town, Huyi District, Xi'an City 108.5576° E 34.1277° N 410 m MT118164 MT130962
37 417 hospital, Lintong District, Xi'an City 109.1949° E 34.3645° N 477 m MT118165 MT130963
38 Shaanxi University of Chinese Medicine, Qindu District, Xianyang City 108.7359° E 34.3175° N 381 m MT118166 MT130964
), ArticleFig(id=1220655541408415984, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Primer name Sequence (5′-3′)
ITS2 ITS2-F ATGCGATACTTGGTGTGAAT
ITS2-R GACGCTTCTCCAGACTACAAT
psbA-trnH psbA-F GTTATGCATGAACGTAAT GCTC
trnH-R CGCGCATGGTGGATTCACAATCC
), ArticleFig(id=1220655541538439414, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=CN, label=Table 2, caption=

ITS2 and psbA-trnH primers' sequences

, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Primer name Sequence (5′-3′)
ITS2 ITS2-F ATGCGATACTTGGTGTGAAT
ITS2-R GACGCTTCTCCAGACTACAAT
psbA-trnH psbA-F GTTATGCATGAACGTAAT GCTC
trnH-R CGCGCATGGTGGATTCACAATCC
), ArticleFig(id=1220655541681045759, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Conserved
site		 Variation
site		 Parsimony
informative site		 Singleton
site		 K2P genetic distance PCR amplification
success rate		 Sequencing
success rate		 Mutation
rate
Intraspecific Interspecific
ITS2 216 10 10 0 0 0.046 100.00% 100.00% 4.40%
psbA-trnH 508 12 12 0 0 0.027 100.00% 100.00% 2.30%
), ArticleFig(id=1220655541978841351, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=CN, label=Table 3, caption=

Sequence characteristics of ITS2 and psbA-trnH

, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Conserved
site		 Variation
site		 Parsimony
informative site		 Singleton
site		 K2P genetic distance PCR amplification
success rate		 Sequencing
success rate		 Mutation
rate
Intraspecific Interspecific
ITS2 216 10 10 0 0 0.046 100.00% 100.00% 4.40%
psbA-trnH 508 12 12 0 0 0.027 100.00% 100.00% 2.30%
), ArticleFig(id=1220655542092087566, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Specimen No. Length G+C
content		 NCBI TCM-barcode
Identification
result		 Sequence
similarity		 Accession
No.		 Identification
result		 Sequence
similarity		 Accession
No.
ITS2 1, 9, 10, 13, 16, 17, 18, 20, 22, 25, 26, 27, 28, 29, 31, 32, 36, 37, 38 225 bp 60.89% P.americana 100.00% MH813183 P.americana 100.00% YZY029-2
2, 7, 8, 11, 14, 23, 24, 33, 34, 35 226 bp 58.85% P.acinosa 100.00% MH711096 P.acinosa 100.00% SL02
psbA-trnH 1, 9, 10, 13, 16, 17, 18, 20, 22, 25, 26, 27, 28, 29, 31, 32, 36, 37, 38 462 bp 24.68% P.americana 100.00% MH286315 P.americana 100.00% S1841
2, 7, 14, 23, 24, 33, 34, 35 490 bp 23.47% P.acinosa 100.00% MG595711 P.acinosa 99.40% HAP00220
8, 11 477 bp 23.27% P.exiensis 96.65% MG595709 P.acinosa 100.00% SL01
), ArticleFig(id=1220655542226305306, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=CN, label=Table 4, caption=

BLAST analyses of ITS2 and psbA-trnH sequences against NCBI and TCM-barcode database

, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Specimen No. Length G+C
content		 NCBI TCM-barcode
Identification
result		 Sequence
similarity		 Accession
No.		 Identification
result		 Sequence
similarity		 Accession
No.
ITS2 1, 9, 10, 13, 16, 17, 18, 20, 22, 25, 26, 27, 28, 29, 31, 32, 36, 37, 38 225 bp 60.89% P.americana 100.00% MH813183 P.americana 100.00% YZY029-2
2, 7, 8, 11, 14, 23, 24, 33, 34, 35 226 bp 58.85% P.acinosa 100.00% MH711096 P.acinosa 100.00% SL02
psbA-trnH 1, 9, 10, 13, 16, 17, 18, 20, 22, 25, 26, 27, 28, 29, 31, 32, 36, 37, 38 462 bp 24.68% P.americana 100.00% MH286315 P.americana 100.00% S1841
2, 7, 14, 23, 24, 33, 34, 35 490 bp 23.47% P.acinosa 100.00% MG595711 P.acinosa 99.40% HAP00220
8, 11 477 bp 23.27% P.exiensis 96.65% MG595709 P.acinosa 100.00% SL01
), ArticleFig(id=1220655542339551524, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Latin name Genus GenBank accession No.
ITS2 psbA-trnH
Phytolacca exiensis sp.nov. Phytolaccaceae Phytolacca MG595708 MG595709
Phytolacca japonica Makino Phytolaccaceae Phytolacca MG595704 MG595705
Anisodus tanguticus (Maxim.) Pascher Solanaceae Anisodus KM208638 MF786027
Gypsophila oldhamiana Miq. Caryophyllaceae Gypsophila MF063499 MF064598
), ArticleFig(id=1220655542431826223, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1220655534286488249, language=CN, label=Table 5, caption=

Sequence information for neighbor-joining tree

, figureFileSmall=null, figureFileBig=null, tableContent=
Latin name Genus GenBank accession No.
ITS2 psbA-trnH
Phytolacca exiensis sp.nov. Phytolaccaceae Phytolacca MG595708 MG595709
Phytolacca japonica Makino Phytolaccaceae Phytolacca MG595704 MG595705
Anisodus tanguticus (Maxim.) Pascher Solanaceae Anisodus KM208638 MF786027
Gypsophila oldhamiana Miq. Caryophyllaceae Gypsophila MF063499 MF064598
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陕西关中野生商陆资源的ITS2和psbA-trnH条形码序列研究
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吕瑞华 1 , 冯昭 1 , 马添翼 1 , 吕蕊花 1 , 张明英 2 , 彭亮 2 , 高静 2 , 张岗 2, *
药学学报 | 研究论文 2020,55(8): 1951-1956
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药学学报 | 研究论文 2020, 55(8): 1951-1956
陕西关中野生商陆资源的ITS2和psbA-trnH条形码序列研究
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吕瑞华1, 冯昭1, 马添翼1, 吕蕊花1, 张明英2, 彭亮2, 高静2, 张岗2, *
作者信息
  • 1.陕西中医药大学医学技术学院, 陕西 咸阳 712046
  • 2.陕西中医药大学药学院/陕西省秦岭中草药应用开发工程技术研究中心, 陕西 咸阳 712046

通讯作者:

*张岗, Tel/Fax:86-29-38185165, E-mail:
Use of ITS2 and psbA-trnH DNA barcode sequences of wild Phytolacca resources in Guanzhong area of Shaanxi Province
Rui-hua Lü1, Zhao FENG1, Tian-yi MA1, Rui-hua Lü1, Ming-ying ZHANG2, Liang PENG2, Jing GAO2, Gang ZHANG2, *
Affiliations
  • 1. College of Medical Technology, Shaanxi University of Chinese Medicine, Xianyang 712046, China
  • 2. College of Pharmacy and Shaanxi Qinling Application Development and Engineering Center of Chinese Herbal Medicine, Shaanxi University of Chinese Medicine, Xianyang 712046, China
出版时间: 2020-08-12 doi: 10.16438/j.0513-4870.2020-0278
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摘要
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为探讨DNA条形码在陕西关中野生商陆资源中的鉴定作用，本研究采用通用引物ITS2和psbA-trnH对29份商陆样品DNA进行PCR扩增和测序，用Codon CodeA-ligner V3.0进行序列拼接和校对，随后进行BLAST比对鉴定分析；同时运用MEGA 6.0分析序列特征、K2P遗传距离及种内或种间变异，并构建系统进化树，评价两对引物对该区域商陆及其混伪品的鉴别能力。研究结果表明，ITS2和psbA-trnH序列的PCR扩增和测序成功率均为100%；NJ系统进化树显示ITS2和psbA-trnH序列均可明显将所有样本分别聚为商陆、垂序商陆并与其同属近源种鄂西商陆、日本商陆及两种混伪品各自聚为不同分支；ITS2序列在种间遗传距离上较psbA-trnH有一定优势。ITS2和psbA-trnH序列均可作为DNA条形码对商陆及其混伪品进行识别和鉴定，为商陆资源品种现状与分布研究及合理开发利用提供理论依据。

商陆  /  DNA条形码  /  ITS2  /  psbA-trnH  /  分子鉴定

In order to explore the use of DNA barcode in the identification of wild Phytolacca resources in the Shaanxi Guanzhong area, 29 DNA samples were amplified and sequenced by using the universal primers ITS2 and psbA-trnH. The sequences were spliced and proof-read by Codon CodeA aligner V3.0, followed by blast comparison and identification analysis; mega 6.0 was used to analyze sequence characteristics, Kimura 2-Parameter (K2P) was used to analyze distance and intraspecific or interspecific variation, and Neighbor-Joining trees were established to evaluate the ability of two pairs of candidate sequences to distinguish Phytolaccae Radix from its adulterants. The results showed that the success rate of PCR amplification and sequencing of ITS2 and psbA-trnH was 100%; the NJ tree showed that both ITS2 and psbA-trnH sequences could separate P. acinosa, P. americana, other species of the same genus like P. japonica, P. exiensis and two adulterant species into a single clade; primer ITS2 had an advantage over psbA-trnH in determining interspecific genetic distances. Therefore, both ITS2 and psbA-trnH sequences can be used for identification of Phytolacca and their adulterants, which provides a theoretical basis for the distribution of wild Phytolacca resources and their rational development and utilization.

Phytolacca acinosa Roxb.  /  DNA barcode  /  ITS2  /  psbA-trnH  /  molecular identification
吕瑞华, 冯昭, 马添翼, 吕蕊花, 张明英, 彭亮, 高静, 张岗. 陕西关中野生商陆资源的ITS2和psbA-trnH条形码序列研究. 药学学报, 2020 , 55 (8) : 1951 -1956 . DOI: 10.16438/j.0513-4870.2020-0278
Rui-hua Lü, Zhao FENG, Tian-yi MA, Rui-hua Lü, Ming-ying ZHANG, Liang PENG, Jing GAO, Gang ZHANG. Use of ITS2 and psbA-trnH DNA barcode sequences of wild Phytolacca resources in Guanzhong area of Shaanxi Province[J]. Acta Pharmaceutica Sinica, 2020 , 55 (8) : 1951 -1956 . DOI: 10.16438/j.0513-4870.2020-0278
正文
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中药商陆为商陆科商陆属商陆Phytolacca acinosa Roxb.和垂序商陆P. americana L.的干燥根, 逐水消肿、通利二便、解毒散结[1], 药用历史悠久。商陆属共35个种, 我国有包括《中国药典》的两种及日本商陆P. japonica Makino和多雄蕊商陆P. polyandra Batalin等4种, 广泛分布于除东北、内蒙古、青海、新疆及高海拔等地区外的其他地域[2]。垂序商陆, 又名美洲商陆, 原产北美洲, 1935年于杭州地区首次被发现[3], 因其全株有毒且极具入侵竞争性, 已于2016年12月被我国列入中国自然生态系统外来入侵物种名单(第四批)。2017年Xie等[4]报道湖北西部一新种, 命名为鄂西商陆P. exiensissp. nov.。由于植物在长期的自然选择如天然杂交、基因突变或人工选择如物种入侵、良种选育等过程中, 都可能导致遗传变异的累积和遗传多样性的丰富, 这也是物种不断进化的根本原因[5]。而我国商陆野生资源近源物种偏少, 理清野生商陆资源品种现状、分布并阐明遗传多样性成为保护该资源的有效途径。
DNA条形码技术是利用基因组中一段公认标准的、相对较短的DNA片段来进行物种的分子鉴定[6], 被《中国药典》2015年版作为中药材分子鉴定法所收录, 补充了传统鉴别方法的不足。rbcL、matK、psbA-trnH和ITS/ITS2是常用的植物DNA条形码, 2009年国际条形码协会提出将matK和rbcL组合作为植物通用条形码序列[7], 但该组合针对属内物种水平的分辨率较低。2011年我国科学家对来自42个目75个科141个属1757个物种的6286个个体的rbcL、matK、psbA-trnH和ITS序列进行研究, 发现ITS2和psbA-trnH在属和种水平的鉴定效率分别达到99.8%、92.7%和95.4%、67.6%, 高于rbcL和matK[6, 8]。ITS2为核心、psbA-trnH为补充的植物类药材DNA条形码鉴定体系得以建立[9], 并且已在石斛、黄芪、银柴胡等中药材及其混伪品的鉴定中成功应用[10-12]。
目前, 国内外对商陆的研究主要集中在化学成分、药理作用及工农业领域的应用等方面[13, 14]。课题组前期对商陆产地的本草考证揭示陕西关中地区的咸阳、凤翔府自古即为商陆的产地[15, 16]。本研究基于DNA条形码分子鉴定技术, 利用通用引物ITS2和psbA-trnH序列对陕西关中地区野生商陆的29个样本进行鉴定分析, 结合GenBank数据库中的近源物种及混伪品序列构建系统进化树, 探讨两种DNA条形码在商陆中的鉴定作用, 为商陆资源品种现状与分布研究及其合理开发利用提供科学依据。
材料与方法
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样品  本研究样本于2019年8~9月间在陕西关中地区采集, 分别来自西安、宝鸡、咸阳、渭南、杨凌四市一区。所有样品经西北农林科技大学生命科学院植物学教研室刘虎岐副教授鉴定为商陆科商陆属植物Phytolacca L.。取其新鲜幼嫩叶片, 保存于陕西省秦岭中草药应用开发工程技术研究中心-80 ℃冰箱, 并根据其地理位置差异最终筛选出29份样品用于本研究, 见表 1。
仪器与试剂  高速微量冷冻离心机(D3024R, 北京大龙); Mini离心机(LX-800, 海门市其林贝尔仪器制造有限公司); PCR仪(JY-96G, 北京君意); 电泳仪(JY600E, 北京君意); 涡旋混合器(VORTEX KB-3, 海门市其林贝尔仪器制造有限公司); 凝胶成像系统(WSE-6100, ATTO); 超微量紫外可见分光光度计(DS-11, 美国DeNovix); 组织细胞破碎仪(TP-24, 杰灵仪器制造天津有限公司)。
植物基因组提取试剂盒(CW0531S, 北京康为世纪生物科技有限公司); PrimeSTAR® Max DNA聚合酶(TaKaRa); 琼脂糖(Biowest Agarose, 西班牙); 核酸Marker和核酸染料购自生工生物工程(上海)股份有限公司; ITS2和psbA-trnH引物由北京擎科生物科技有限公司合成。
DNA提取和检测  基因组DNA的提取使用冷冻保存的叶片100 mg左右, 加入液氮充分研磨后, 严格按照植物基因组提取试剂盒操作步骤完成。DNA纯度和浓度使用琼脂糖凝胶电泳法及超微量紫外可见分光光度计检测。
PCR扩增及测序  本研究选用DNA条形码国际通用序列ITS2和psbA-trnH。两对引物序列见表 2。PCR反应体系为50 μL, 包含PrimeSTAR Max Premix (2×) 25 μL, DNA模板2 μL, 正反向引物各2 μL, ddH2O 19 μL。ITS2和psbA-trnH序列的PCR扩增程序为95℃预变性5 min; 98 ℃模板变形5 s, 58 ℃退火10 s, 72 ℃延伸30 s, 共计32个循环后72 ℃延伸10 min。PCR产物用1%琼脂糖凝胶电泳检测后, 委托上海生工公司进行双向测序。
数据处理  采用Codon CodeA-ligner V3.0对测序峰图进行校正, 去除低质量序列及引物区。随后使用基于隐马尔可夫模型(HMMER)的注释方法将所有序列及所需的GenBank序列去除两端的5.8S和28S区段, 进而获得ITS2间隔区序列, 根据GenBank数据库中同科属物种psbA-trnH的注释, 去除序列两端的psbA和trnH基因, 获得完整的psbA-trnH基因间隔区序列。将获得序列分别在中药材DNA条形码鉴定系统(http://www.tcmbarcode.cn)和NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi)中进行样品序列的BLAST比对分析。
使用MEGA 6.0对序列特征进行比对分析, 通过不同的碱基组成确定其种内及种间关系, 用Kimura 2-Parameter (K2P)法将所有数据计算遗传距离, 推断样品间的亲缘关系, 并构建Neighbor-joining系统进化树, 各个分支的支持率使用bootstrap重复1 000次进行检验。
结果与分析
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1 DNA扩增及测序
29份样品均来自商陆的新鲜幼嫩叶片, DNA提取浓度为33.85~223.67 ng·μL-1。由图 1可见, 两对DNA条形码候选序列ITS2和psbA-trnH的PCR产物条带清晰、单一, 表明特异性强、纯度高, 可直接用于双向测序, 且其DNA扩增和测序成功率均为100.00% (表 3)。
2 NCBI数据库和中药材DNA条形码鉴定系统比对分析
去除两端的5.8S和28S区段后, 获得的ITS2序列长度在225~226 bp, GC含量为58.85%~60.89%;去除两端的psbA和trnH基因后, 获得的psbA-trnH序列长度在462~490 bp, GC含量为23.27%~24.68% (表 4)。
将序列在NCBI上进行BLAST比对: ITS2序列分析结果中, 所有样品分为商陆P. acinosa和垂序商陆P. americana; 而对psbA-trnH序列的BLAST结果中, 样品分P. acinosa、P. americana和P. exiensis (8号和11号); 同时利用中药材DNA条形码鉴定系统(http://www.tcmbarcode.cn)进行BLAST比对发现, ITS2和psbA-trnH的比对结果一致, 都将样品分为2个种, P. acinosa和P. americana (表 4)。
将鉴定为P.exiensis的样本8、11与P.acinosa进行psbA-trnH序列比对分析, 以样本8(MT130939)与样本2(MT130937, P.acinosa)为例, 图 2结果表明两个样本的psbA-trnH相似度为87.75%, 存在不同位点碱基的转换、颠换、缺失等变异, 说明商陆和鄂西商陆的该序列有一定的保守性。
3 序列特征及遗传距离分析
对ITS2和psbA-trnH序列的保守位点、变异率等特征参数汇总(表 3)。K2P遗传距离计算显示, psbA-trnH序列种间遗传距离为0.027, 小于ITS2序列的种间遗传距离0.046;psbA-trnH序列的变异率为2.30%, 小于ITS2序列的4.40%, 说明psbA-trnH较ITS2序列保守, 两种候选序列种内遗传距离均为0, 小于种间遗传距离, 存在明显的barcoding gap。
4 系统进化树构建分析
采用MEGA 6.0对29个样品的序列与NCBI下载的同属不同种及两种常用混伪品的ITS2和psbA-trnH序列(表 5)分别建立系统进化树。从图 3可以看出, ITS2和psbA-trnH序列均将所有样本明显聚成商陆P.acinosa、垂序商陆P.americana、鄂西商陆(MG595708)与日本商陆(MG595704)及两个混伪品等分支, 鄂西商陆、日本商陆所在分支与商陆亲缘关系较近。
讨论
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传统的中药材鉴定方法主要包括基原、性状、显微、理化鉴定等, 这些方法与生物体的表现型有关, 易受环境等外部因素及鉴定人员的专业性和经验等影响。与RFLP、RAPD等其他分子标记技术相比, DNA条形码鉴定技术不但能够批量鉴定、可重复性强, 又不需要对形态鉴定的相关知识积累, 且不受环境因素、样品形态及材料部位的影响, 因此在中药材基原鉴定方面应用广泛[17]。本研究运用DNA条形码技术对陕西关中地区野生商陆资源品质进行鉴别, 探讨ITS2和psbA-trnH在商陆中的鉴别能力, 为该资源品种鉴定与分类等研究提供依据。
29个野生商陆样本的序列分析结果表明, ITS2和psbA-trnH序列的PCR扩增和测序成功率均为100%。K2P种间明显大于种内且形成明显的间隔区barcoding gap, 这是判断条形码是否理想的标准[9]。Duan等[18]在紫丹参及其混伪品的研究中发现, 根据K2P遗传距离分析, 明显的barcoding gap可以将紫丹参和丹参显著区分开, 因此ITS2序列可作为鉴别紫丹参基原植物及近源种的DNA条形码。Zheng等[19]在对川贝母及其近源种进行分子鉴定时, 发现psbA-trnH序列的种内与种间距离分布重叠度远高于ITS2序列, 因此ITS2适宜贝母物种鉴定及亲缘关系研究。本研究ITS2和psbA-trnH序列的种间K2P遗传距离均明显大于种内, 且形成显著的间隔区barcoding gap, 无重叠区域, 因此ITS2和psbA-trnH序列都符合DNA条形码候选序列的基本要求。
进化树分析表明ITS2和psbA-trnH序列均能明显区分各样本, 利用ITS2和psbA-trnH序列构建的进化树均将在NCBI中BLAST鉴定为鄂西商陆的样本8、11与商陆聚为一支。本研究随后对所有样本特别是样本8、11与鄂西商陆作进一步形态对比, 发现二者更接近商陆, 而非鄂西商陆, 而该物种仅在湖北西部的3个地点发现, 产生原因和时间不详, 基本无扩散可能[4], 推测生境因子可能诱导了类似变异, 这种影响可能导致一些特殊的地理生态型产生[20]。因此, ITS2和psbA-trnH序列均可用于商陆及其混伪品的分子鉴别, ITS2序列较psbA-trnH变异程度大, 种间鉴别更有优势, psbA-trnH能甄别种内变异, 可能有利于从环境或遗传多样性角度研究。
南北朝陶弘景《名医别录》云: “如人形者, 有神。生咸阳[15]。”, 明代《本草品汇精要》记载“生咸阳川谷, 今处处有之, 多生人家园圃中, (道地)并州凤翔府[16]。”, 表明咸阳、凤翔府为古时商陆的道地产区。另有研究对全国23份野生商陆种源的水溶性浸出物、商陆皂苷甲含量进行测定, 发现其中4个高成分含量的优质种源, 有3个出自陕西[21], 说明陕西有着得天独厚的商陆优质资源。本研究表明关中地区商陆属除了入侵物种垂序商陆外, 基本为一个本土物种, 即商陆P. acinosa, 且少数个体发生变异。这种物种高度的保守型或许与关中地区被秦岭和黄土高原包围的地形有关, 这种地形对物种的自然传播起到了天然的屏障作用, 导致了该区域商陆资源较单一, 偶尔发生变异可能与生境因子有关。
致谢 西北农林科技大学陈耀锋教授和刘虎岐副教授在采样过程中给予帮助。
作者贡献:第一作者吕瑞华完成相关实验及论文撰写工作, 通讯作者张岗负责设计、指导实验及论文修改, 冯昭、马添翼参与实验及数据分析, 吕蕊花、高静参与样品采集及修改论文, 张明英、彭亮参与数据分析及对论文的修改。
利益冲突:无任何利益冲突。
基金
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  • 陕西中医药大学科研培育项目(2017PY32)
  • 陕西中医药大学“秦药”品质评价及资源开发学科创新团队项目(2019-QN01)
  • 陕西中医药大学“思邈青年学者”项目(141306200105)
  • 2018年公共卫生服务补助奖金专项“全国中药资源普查项目”(财社[2018]43号)
  • 陕西省高校青年杰出人才支持计划项目
  • 咸阳市中青年科技领军人才项目
文献
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2020年第55卷第8期
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doi: 10.16438/j.0513-4870.2020-0278
  • 接收时间：2020-03-10
  • 首发时间：2026-01-21
  • 出版时间：2020-08-12
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  • 收稿日期：2020-03-10
  • 修回日期：2020-04-02
基金
陕西中医药大学科研培育项目(2017PY32)
陕西中医药大学“秦药”品质评价及资源开发学科创新团队项目(2019-QN01)
陕西中医药大学“思邈青年学者”项目(141306200105)
2018年公共卫生服务补助奖金专项“全国中药资源普查项目”(财社[2018]43号)
陕西省高校青年杰出人才支持计划项目
咸阳市中青年科技领军人才项目
作者信息
    1.陕西中医药大学医学技术学院, 陕西 咸阳 712046
    2.陕西中医药大学药学院/陕西省秦岭中草药应用开发工程技术研究中心, 陕西 咸阳 712046

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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