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Article(id=1218290943799772111, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218290941232861879, articleNumber=1001-2494(2024)15-1393-07, orderNo=null, doi=10.11669/cpj.2024.15.005, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1687622400000, receivedDateStr=2023-06-25, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768392795753, onlineDateStr=2026-01-14, pubDate=1723046400000, pubDateStr=2024-08-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768392795753, onlineIssueDateStr=2026-01-14, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768392795753, creator=13701087609, updateTime=1768392795753, updator=13701087609, issue=Issue{id=1218290941232861879, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='15', pageStart='1361', pageEnd='1452', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768392795141, creator=13701087609, updateTime=1768394622953, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1218298607682376061, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218290941232861879, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1218298607682376062, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218290941232861879, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1393, endPage=1399, ext={EN=ArticleExt(id=1218290944030458834, articleId=1218290943799772111, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Molecular Identification of Fritillaria taipaiensis and Its Relatives Based on Indel Marker, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To construct molecularidentification of Fritillaria taipaiensis and its relatives based on Indel marker. METHODS One hundred and three chloroplast genomes of 13 Fritillaria species were downloaded from GenBank and were subsequently used to screen Indel marker based on sequence alignment. Then, in accordance with the conserved upstream and downstream of Indel marker, a pair of specific primers was designed accordingly, by which a method including routine PCR and electrophoresis was constructed. Furthermore, the specificity, sensitivity, and optimal reaction temperature of the method were assessed. RESULTS One deletion of 137 bp was found in the accD-psaI spacer region of F. taipaiensis chloroplast genome according to the multiple sequence alignments, which was defined as the Indel marker for F. taipaiensis. The constructed method exhibited high specificity due to the fact that only F. taipaiensis exhibited a band of 302 bp after routine PCR and electrophoresis. The limit of detection for F. taipaiensis DNA template was 0.239 ng·μL-1, and the optimal melting temperature (Tm) value was 58 ℃. CONCLUSION This accurate, quick and cost-affordable method provides not only identification of F. taipaiensis and its related species, but also technical reference for identification of other traditional medicines.

, correspAuthors=Jiayu ZHOU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, 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=Hai LIAO, Xinyi YU, Xue HUANG, Tian ZHANG, Huige QUAN, Yuchen LI, Jiayu ZHOU), CN=ArticleExt(id=1218290945246807028, articleId=1218290943799772111, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=利用Indel标记鉴定太白贝母及其近缘种, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 建立基于插入缺失(Indel)标记的常规聚合酶链反应(PCR)方法对太白贝母及其近缘种开展分子鉴定研究。方法 从GenBank下载103条来自不同贝母来源植物的叶绿体基因组序列,序列比对筛选太白贝母的特异性Indel标记。在该Indel标记的上下游保守区中设计引物,通过常规PCR与凝胶电泳方法对不同贝母来源植物开展鉴定,并考察方法的特异性、灵敏性与最适反应温度。结果 太白贝母在accD-psaI基因间隔区具有一段长度为137 bp的缺失,可以作为太白贝母的特异性Indel标记。建立的常规PCR与凝胶电泳方法对太白贝母有良好的特异性,仅有太白贝母产生302 bp的条带,从而与暗紫贝母等其余12 种不同贝母来源植物明显区分。该方法对贝母DNA模板的检测下限为0.239 ng·μL-1,最适熔解温度(Tm)值为58 ℃。结论 该检测方法具有特异性高、快捷与成本低等特点,不仅能够实现对太白贝母及其近缘种的准确鉴定,还能够为其他重要来源植物的分子鉴定提供开发思路。

, correspAuthors=周嘉裕, authorNote=null, correspAuthorsNote=
* 周嘉裕,女,博士,副教授 研究方向:中药分子鉴定研究 Tel:(028)87103202
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廖海,男,博士,副教授 研究方向:中药分子鉴定研究

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廖海,男,博士,副教授 研究方向:中药分子鉴定研究

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廖海,男,博士,副教授 研究方向:中药分子鉴定研究

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The code after each Latin name was the accession number of its chloroplast genome; The species name in red font represented F. taipaiensis; The species names in orange font represented the other five original plants of bulbus Fritillariae cirrhosae.

, figureFileSmall=tu7GSjKctk36udDtqoEVHw==, figureFileBig=Lbl+hpfjrsWO9MLJnezDVw==, tableContent=null), ArticleFig(id=1218484896054559669, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=CN, label=图1, caption=基于叶绿体基因组的川贝母及其常见混伪品的系统进化树

植物拉丁名后的代号为叶绿体基因组登录号;红色字体为太白贝母;橙色字体为川贝母的其余5种来源植物。

, figureFileSmall=tu7GSjKctk36udDtqoEVHw==, figureFileBig=Lbl+hpfjrsWO9MLJnezDVw==, tableContent=null), ArticleFig(id=1218484896167805884, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=EN, label=Fig.2, caption=Alignment based on accD-psaI regions of 13 Fritillaria specie

→-the forward primer;←-the reverse primer; “55924” and “56238” represented the base position in chloroplast genome of F. taipaiensis, respectively.

, figureFileSmall=xmmjSbKMHRb+aFaaa35A7A==, figureFileBig=Omb2HLy6yJdfa5xmddi8Og==, tableContent=null), ArticleFig(id=1218484896255886273, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=CN, label=图2, caption=13种贝母来源植物的accD-psaI区域多重序列比对

→-正向引物;←-反向引物;“55924”与“56238”对应太白贝母叶绿体基因组的碱基位置。

, figureFileSmall=xmmjSbKMHRb+aFaaa35A7A==, figureFileBig=Omb2HLy6yJdfa5xmddi8Og==, tableContent=null), ArticleFig(id=1218484896339772357, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=EN, label=Fig.3, caption=Agarose electrophoresis of PCR products of 14 Fritillaria species

A-agarose electrophoresis of PCR products of the original plants of Beimu herbs(except that F. hupehensis is powder herb, other Fritillaria samples are made of leaves): 1-14 indicated F. unibracteata, F. delavayi, F. cirrhosa, F. taipaiensis, F. przewalskii, F. unibracteata Hsiaoet K.C.Hsia var.wabuensis, F. sinica, F. cirrhosa var.ecirrhosa, F. pallidiflora, F. mellea, F. thunbergii, F. walujewii and F. hupehensis, respectively: B-Agarose electrophoresis of PCR products of commercial herbs; 1-4 indicated Shongbei, Qingbei, Lubei herbs and bulbs of F. taipaiensis, respectively; M-DNA Marker; Arrow indicated the band of 302 bp.

, figureFileSmall=C/nnOdOp2FIqf3xmMwJbXw==, figureFileBig=+nXAFq/PI75rnT9Uhzz/lA==, tableContent=null), ArticleFig(id=1218484896440435659, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=CN, label=图3, caption=特异性引物对14种贝母样本聚合酶链反应(PCR)扩增的电泳结果

A-贝母来源植物的常规PCR结果(除湖北贝母为粉末状药材,其余材料选用叶片):1~14分别代表暗紫贝母、梭砂贝母、卷叶贝母、太白贝母、甘肃贝母、瓦布贝母、中华贝母、康定贝母、伊犁贝母、浓蜜贝母、浙贝母、平贝母、新疆贝母与湖北贝母;B-川贝母药材的常规PCR结果:1~4分别代表市售松贝、市售青贝、市售炉贝与太白贝母鳞茎;M-DNA相对分子质量标准物;箭头指示为302 bp条带。

, figureFileSmall=C/nnOdOp2FIqf3xmMwJbXw==, figureFileBig=+nXAFq/PI75rnT9Uhzz/lA==, tableContent=null), ArticleFig(id=1218484896511738828, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=EN, label=Fig.4, caption=Limit of detection base on PCR

M-DNA marker; 1-8 indicated the F. taipaiensis samples with DNA concentration of 238.54, 23.584, 2.39, 0.239, 0.023 9, 0.002 39, 0.000 239 and 0.000 023 9 ng·μL-1, respectively; Arrow indicated the band of 302 bp.

, figureFileSmall=/PrFXTa9famV7YDSXJpMvw==, figureFileBig=iDs7kdnAI3KEyUMVKl2lzA==, tableContent=null), ArticleFig(id=1218484896599819215, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=CN, label=图4, caption=PCR反应的灵敏度检测

M-DNA相对分子质量标准物;1~8分别代表DNA质量浓度为238.54、23.584、2.39、0.239、0.023 9、0.002 39、0.000 239与0.000 023 9 ng·μL-1的太白贝母样品;箭头指示为302 bp条带。

, figureFileSmall=/PrFXTa9famV7YDSXJpMvw==, figureFileBig=iDs7kdnAI3KEyUMVKl2lzA==, tableContent=null), ArticleFig(id=1218484896683705299, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=EN, label=Fig.5, caption=PCR results at various Tm values

A-F indicated the PCR results at Tm value of 55, 56, 57, 58, 59 and 60 ℃, respectively; M-DNA marker; 1-14 contained the same samples as those in Fig.3A; Arrow indicated the band of 302 bp.

, figureFileSmall=BE0LMFwxZn5mEkKZIpJvgQ==, figureFileBig=eV8mjBRhBiilePyre5dhIw==, tableContent=null), ArticleFig(id=1218484896788562905, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=CN, label=图5, caption=不同熔解温度(Tm)值的PCR结果

A~F分别为Tm=55、56、57、58、59与60 ℃时的PCR结果图;M-DNA相对分子质量标准物;1~14顺序与图3A相同;箭头指示为302 bp条带。

, figureFileSmall=BE0LMFwxZn5mEkKZIpJvgQ==, figureFileBig=eV8mjBRhBiilePyre5dhIw==, tableContent=null), ArticleFig(id=1218484896880837598, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=EN, label=Tab.1, caption=

Accession numbers of chloroplast genome from Chuan-Beimu and its adulterants.

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Species name Accession number of chloroplasts Number of samples
1 Fritillaria unibracteata NC_044629.1,MH244909.1,MH593352.1,MH593351.1,OM807063.1,MW849272.1,MN126570.1,MN148410.1,
MN148409.1,MN148410.1		 3
2 F. cirrhosa MH244906.1,NC_024728.1,KF769143.1,MT806755.1,MH593346.1,MH593345.1,MH593344.1,MH593343.1,
MH593342.1,KY646167.1		 3
3 F. taipaiensis MH244910.1,MT806754.1,MT806753.1,MT806752.1,MT806751.1,MT806750.1,MT806749.1,MH593359.1,MH593358.1,
MH593357.1,MH593356.1,OQ433931.1,OQ433930.1,OQ411258.1,NC_023247.1,KC543997.1,MK642356.1,
KF769144.1,KC713823.1,KC713822.1		 3
4 F. przewalskii NC_044636.1,MH244908.1,MT806747.1,MH593349.1,MH593348.1,MH593347.1,MW849274.1 3
5 F. delavayi MN480806.1,MH593355.1,MH593354.1,MH593353.1,MW849275.1,MN148401.1 3
6 F. unibracteata Hsiaoet
K. C. Hsia var. wabuensis		 KF769142.1 3
7 F. sinica NC_044631.1,MN810971.1,MH244912.1,MW849273.1 3
8 F. pallidiflora NC_037216.1,MG211822.1,MH593366.1,MH593364.1,MN148405.1,MN148404.1,MN148403.1 3
9 F. thunbergii MH244914.1,MH593362.1,MH593361.1,MH593360.1,NC_034368.1,KY646165.1,MN148408.1,MN148407.1,MN148406.1 3
10 F. hupehensis NC_024736.1,MN811003.1,MN811002.1,MN811001.1,KF712486.1,OK631821.1,MN148402.1 1
11 F. ussuriensis NC_034369.1,MH593369.1,MH593368.1,MH593367.1,KY646166.1,MN148414.1,MN148413.1,MN148412.1 3
12 F. walujewii NC_037215.1,MN810991.1,MN810990.1,MN810989.1,MN810988.1,MG211820.1 3
13 F. anhuiensis NC_045861.1,MN810981.1,MN810980.1,MN810979.1,MN810978.1,MN810977.1,MH593363.1,MK258148.1 NA
14 F. cirrhosa var. ecirrhosa NA 3
15 Amana edulis KY401425.1 NA
16 Bolbostemma paniculatum NC_072567.1 NA
), ArticleFig(id=1218484896985695202, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218290943799772111, language=CN, label=表1, caption=

川贝母及其常见混伪品的叶绿体基因组序列

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Species name Accession number of chloroplasts Number of samples
1 Fritillaria unibracteata NC_044629.1,MH244909.1,MH593352.1,MH593351.1,OM807063.1,MW849272.1,MN126570.1,MN148410.1,
MN148409.1,MN148410.1		 3
2 F. cirrhosa MH244906.1,NC_024728.1,KF769143.1,MT806755.1,MH593346.1,MH593345.1,MH593344.1,MH593343.1,
MH593342.1,KY646167.1		 3
3 F. taipaiensis MH244910.1,MT806754.1,MT806753.1,MT806752.1,MT806751.1,MT806750.1,MT806749.1,MH593359.1,MH593358.1,
MH593357.1,MH593356.1,OQ433931.1,OQ433930.1,OQ411258.1,NC_023247.1,KC543997.1,MK642356.1,
KF769144.1,KC713823.1,KC713822.1		 3
4 F. przewalskii NC_044636.1,MH244908.1,MT806747.1,MH593349.1,MH593348.1,MH593347.1,MW849274.1 3
5 F. delavayi MN480806.1,MH593355.1,MH593354.1,MH593353.1,MW849275.1,MN148401.1 3
6 F. unibracteata Hsiaoet
K. C. Hsia var. wabuensis		 KF769142.1 3
7 F. sinica NC_044631.1,MN810971.1,MH244912.1,MW849273.1 3
8 F. pallidiflora NC_037216.1,MG211822.1,MH593366.1,MH593364.1,MN148405.1,MN148404.1,MN148403.1 3
9 F. thunbergii MH244914.1,MH593362.1,MH593361.1,MH593360.1,NC_034368.1,KY646165.1,MN148408.1,MN148407.1,MN148406.1 3
10 F. hupehensis NC_024736.1,MN811003.1,MN811002.1,MN811001.1,KF712486.1,OK631821.1,MN148402.1 1
11 F. ussuriensis NC_034369.1,MH593369.1,MH593368.1,MH593367.1,KY646166.1,MN148414.1,MN148413.1,MN148412.1 3
12 F. walujewii NC_037215.1,MN810991.1,MN810990.1,MN810989.1,MN810988.1,MG211820.1 3
13 F. anhuiensis NC_045861.1,MN810981.1,MN810980.1,MN810979.1,MN810978.1,MN810977.1,MH593363.1,MK258148.1 NA
14 F. cirrhosa var. ecirrhosa NA 3
15 Amana edulis KY401425.1 NA
16 Bolbostemma paniculatum NC_072567.1 NA
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利用Indel标记鉴定太白贝母及其近缘种
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廖海 , 喻心怡 , 黄雪 , 张田 , 全慧鸽 , 李宇辰 , 周嘉裕 *
中国药学杂志 | 论著 2024,59(15): 1393-1399
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中国药学杂志 | 论著 2024, 59(15): 1393-1399
利用Indel标记鉴定太白贝母及其近缘种
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廖海, 喻心怡, 黄雪, 张田, 全慧鸽, 李宇辰, 周嘉裕*
作者信息
  • 西南交通大学生命科学与工程学院, 成都 610031

    • 廖海,男,博士,副教授 研究方向:中药分子鉴定研究

通讯作者:

* 周嘉裕,女,博士,副教授 研究方向:中药分子鉴定研究 Tel:(028)87103202
Molecular Identification of Fritillaria taipaiensis and Its Relatives Based on Indel Marker
Hai LIAO, Xinyi YU, Xue HUANG, Tian ZHANG, Huige QUAN, Yuchen LI, Jiayu ZHOU*
Affiliations
  • School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
出版时间: 2024-08-08 doi: 10.11669/cpj.2024.15.005
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摘要
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目的 建立基于插入缺失(Indel)标记的常规聚合酶链反应(PCR)方法对太白贝母及其近缘种开展分子鉴定研究。方法 从GenBank下载103条来自不同贝母来源植物的叶绿体基因组序列,序列比对筛选太白贝母的特异性Indel标记。在该Indel标记的上下游保守区中设计引物,通过常规PCR与凝胶电泳方法对不同贝母来源植物开展鉴定,并考察方法的特异性、灵敏性与最适反应温度。结果 太白贝母在accD-psaI基因间隔区具有一段长度为137 bp的缺失,可以作为太白贝母的特异性Indel标记。建立的常规PCR与凝胶电泳方法对太白贝母有良好的特异性,仅有太白贝母产生302 bp的条带,从而与暗紫贝母等其余12 种不同贝母来源植物明显区分。该方法对贝母DNA模板的检测下限为0.239 ng·μL-1,最适熔解温度(Tm)值为58 ℃。结论 该检测方法具有特异性高、快捷与成本低等特点,不仅能够实现对太白贝母及其近缘种的准确鉴定,还能够为其他重要来源植物的分子鉴定提供开发思路。

叶绿体基因组  /  太白贝母  /  accD-psaI  /  插入缺失标记  /  分子鉴定

OBJECTIVE To construct molecularidentification of Fritillaria taipaiensis and its relatives based on Indel marker. METHODS One hundred and three chloroplast genomes of 13 Fritillaria species were downloaded from GenBank and were subsequently used to screen Indel marker based on sequence alignment. Then, in accordance with the conserved upstream and downstream of Indel marker, a pair of specific primers was designed accordingly, by which a method including routine PCR and electrophoresis was constructed. Furthermore, the specificity, sensitivity, and optimal reaction temperature of the method were assessed. RESULTS One deletion of 137 bp was found in the accD-psaI spacer region of F. taipaiensis chloroplast genome according to the multiple sequence alignments, which was defined as the Indel marker for F. taipaiensis. The constructed method exhibited high specificity due to the fact that only F. taipaiensis exhibited a band of 302 bp after routine PCR and electrophoresis. The limit of detection for F. taipaiensis DNA template was 0.239 ng·μL-1, and the optimal melting temperature (Tm) value was 58 ℃. CONCLUSION This accurate, quick and cost-affordable method provides not only identification of F. taipaiensis and its related species, but also technical reference for identification of other traditional medicines.

chloroplast genome  /  Fritillaria taipaiensis  /  accD-psaI  /  Indel marker  /  molecular identification
廖海, 喻心怡, 黄雪, 张田, 全慧鸽, 李宇辰, 周嘉裕. 利用Indel标记鉴定太白贝母及其近缘种. 中国药学杂志, 2024 , 59 (15) : 1393 -1399 . DOI: 10.11669/cpj.2024.15.005
Hai LIAO, Xinyi YU, Xue HUANG, Tian ZHANG, Huige QUAN, Yuchen LI, Jiayu ZHOU. Molecular Identification of Fritillaria taipaiensis and Its Relatives Based on Indel Marker[J]. Chinese Pharmaceutical Journal, 2024 , 59 (15) : 1393 -1399 . DOI: 10.11669/cpj.2024.15.005
正文
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川贝母为百合科贝母属植物的鳞茎,属名贵道地中药材,其药用价值始载于《神农本草经》,归肺、心经,具有润肺止咳、化痰平喘与散结消痈之功效[1]。目前,以川贝母为原料生产的中成药已超过100种[2],年需求量缺口达1 900吨[3-4],市场上也出现众多混伪品。为了缓解供应与需求矛盾,2020年版《中国药典》将卷叶贝母(Fritillaria cirrhosa D. Don)、暗紫贝母(F. unibracteata Hsiao &K. C. Hsia)、太白贝母(F. taipaiensis P. Y. Li)、甘肃贝母 (F. przewalskii Maxim.)、梭砂贝母(F. delavayi Franch.)和瓦布贝母[F. unibracteata Hsiao et K. C. Hsiavar. wabuensis (S. Y. Tang et S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen]均纳入川贝母项下。尽管扩大植物来源部分缓解了川贝母野生资源的匮乏,但不同来源川贝母在生物碱类型与含量上仍有较大差别。已有研究报道,太白贝母主要富集贝母素乙与番茄碱[2],卷叶贝母主要富含西贝母碱[5],本课题组发现贝母辛较多富集于暗紫贝母[6]。因此,建立适合于不同来源的、准确、快速鉴定方法,是确保药用效能的前提。
目前用于区分不同来源川贝母及其混伪品的传统方法有形态法与化学法。前者需要较丰富的鉴定经验,且受主观判断力影响,推广难度较大[7]。借助不同品种植物间的化学成分差异,利用高效液相色谱法(HPLC)及指纹图谱也可用于鉴定不同来源川贝母及其混伪品[7-8]。然而,化学成分的形成和积累与生长阶段、生长环境条件有着密切关系,极可能出现品种相同但成分不同的现象,且化学成分检测需要昂贵的硬件设施,难以普及[9]。DNA作为遗传物质,其序列变异受环境影响相对较小,种间特异性更高,为中药材鉴定工作提供了便利[10]。例如,Yun等[11]将Taqman技术成功用于冬虫夏草的分子鉴定。以ITS2为代表的DNA 条形码是近年来建立的分子鉴定方法[12-13],已用于毛莨科、虎耳草科等植物鉴别。
本课题组在前期研究中,曾利用限制性片段长度多态性(PCR-RFLP)[14]、DNA条形码[14]、叶绿体基因组[15]与TagMan-小沟结合(TaqMan-MGB)实时荧光聚合酶链反应(PCR)[16]等方法对川贝母开展了分子鉴定研究。相较于其他DNA序列,叶绿体基因组拥有较高的多态性与变异位点,可能开发出较丰富的候选分子标记。插入缺失(insertion-deletion,Indel)是指在基因组同一位点发生数量不等的DNA片段的插入或缺失,具有变异稳定、检测便捷等优点,已成功用于川芎[17]、人参与西洋参[9]等中药的分子鉴别。本研究分析13种不同贝母来源植物的103条叶绿体基因组序列,发现太白贝母在accD-psaI基因区间中有一段长度为137 bp的DNA片段缺失,这段缺失即作为太白贝母专属性的Indel标记。本研究基于该Indel标记,建立一种成本较低、准确与快速鉴定太白贝母的方法,为太白贝母资源的合理开发、川贝母产业链的管理和监管提供技术支撑。
1 材料和方法
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1.1 植物材料
6种川贝母来源植物[卷叶贝母(F. cirrhosa)、暗紫贝母(F. unibracteata)、太白贝母(F. taipaiensis)、甘肃贝母 (F. przewalskii)、梭砂贝母(F. delavayi)和瓦布贝母(F. unibracteata Hsiaoet K. C. Hsia var. wabuensis)]、中华贝母(F. sinica)、浓蜜贝母(F. mellea)、伊犁贝母(F. pallidiflora Schrenk)与浙贝母(F. thunbergii Miq.)采自青海省西宁市互助土族自治县;康定贝母(F. cirrhosa var. ecirrhosa Franch)采自四川省康定市;平贝母(F. ussuriensis)采自陕西省汉中市;新疆贝母(F. walujewii Regel)由中国科学院苏志豪研究员与天津理工大学刘明玉教授馈赠。湖北贝母(F. hupehensis Hsiao et K. C. Hsia)药材(形态:粉末)购自中国食品药品检定研究院,批号:120962-201005。所有来源植物及药材由西南交通大学生命科学与工程学院周嘉裕副教授鉴定,并保存于生命科学与工程学院。
1.2 实验仪器
多功能酶标仪(美国Bio-Tek公司);DYY-6C 型电泳仪(北京六一生物科技有限公司);96-Well Thermal Cycler PCR仪(美国Thermo Fisher公司)。植物DNA 提取试剂盒 (DP305,北京天根生化有限公司);DNA相对分子质量标准物、PCR试剂、电泳试剂与核酸染料(北京擎科生物科技有限公司)。
1.3 序列比对与Indel引物设计
分别从GenBank下载13种贝母来源植物的103条叶绿体基因组信息,见表1。
筛选103条叶绿体基因组中accD-psaI间隔序列,利用DNAMAN 6.0进行序列比对,确定太白贝母的Indel专属性标记。利用Primer Premier 6.0设计引物,并由北京擎科生物科技有限公司合成。
1.4 系统进化树构建
分别选择MH244909.1、MH593355、KF769143、KC543997、KF769142.1、MH244908、MN810981、MH244912.1、MH244914、KF712486、MH593364.1、MH593369.1、MN810991.1、KY401425.1与NC_072567.1作为13种贝母及2种混伪品的叶绿体基因组代表。序列选择原则为该序列曾用于构建系统进化树[15]。利用MEGA7.0软件,构建Maximum Likelihood(ML)系统进化树,并以2种非贝母类混伪品的叶绿体基因组为外源序列,自检次数为1 000次,记录Bootstrap值。
1.5 样品基因组DNA提取
参考Zhang等[16]的方法,取不同贝母样品50~60 mg,植物基因组 DNA 提取试剂盒(DP305)提取基因组DNA。以灭菌水作空白对照,酶标仪检测DNA的浓度。
1.6 常规聚合酶链式反应(PCR)体系
PCR体系的总体积为25 μL,含有2×Master Mix试剂12.5 μL,正反向引物(浓度均为10 μmol·L-1)各1.0 μL,DNA模板2.0 μL,加水补足。PCR反应条件为:95 ℃预变性10 min(95 ℃变性,30 s;58 ℃退火,30 s;72 ℃延伸,50 s;共35次循环),72 ℃延伸,7 min。3%琼脂糖电泳检测PCR扩增产物,加样量为8 μL,恒定电压100 V。
1.7 常规PCR 灵敏度与最优熔解温度(Tm)值检测
选取太白贝母基因组DNA样本,用灭菌水稀释成8个梯度,每个浓度梯度各取2 μL作为DNA模板,按“1.6”项下方法进行灵敏度检测。
设置6个不同的Tm值(55~60 ℃),按“1.6”项下方法进行最优Tm值检测。
2 结果与分析
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2.1 川贝母及其混伪品的系统进化树与序列比对结果
基于叶绿体基因组构建ML进化树(图1),自展值超过90的节点占92.3%,表明该进化树的可信度较高,能够准确反映物种间的系统进化关系。进一步分析发现,老鸦瓣(Amana edulis)与土贝母(Bolbostemma paniculatum)与13种贝母来源植物的亲缘关系较远,形成了不同的聚类。其中,来自百合科贝母属的13种贝母来源植物形成单系群,推测它们可能起源于某个共同原始物种。6种川贝母来源植物与中华贝母(F. sinica)聚类为单系群(自展值为100),组成川贝母复合群,亲缘关系较近。分布于华东地区的湖北贝母(F. hupehensis)、浙贝母(F. thunbergii)与安徽贝母(F. anhuiensis)聚类为单系群(自展值为100),形成华东贝母群。川贝母复合群与华东贝母群互为姐妹群,共同组成了南方贝母群。然而,分布于北方的3种贝母属植物[平贝母(F. ussuriensis)、伊犁贝母(F. pallidiflora)与新疆贝母(F. walujewii)]未形成单系群,其中,平贝母(F. ussuriensis)与其余12种贝母属植物互为姐妹群,推测其与其他贝母属植物的分化时间较早。有趣的是,中药伊贝母的2种来源植物[新疆贝母(F. walujewii)与伊犁贝母(F. pallidiflora)]形成单系群(自展值为100)。由于叶绿体基因组能够区分不同贝母及其混伪品,且可信度高,提示有可能基于叶绿体基因组,筛选出物种鉴定用分子标记。
2.2 太白贝母鉴定用Indel标记发现
比对13种贝母来源植物的103条叶绿体基因组序列,发现20条太白贝母基因组在accD-psaI间隔区均含有一段长度为137 bp的DNA片段缺失(碱基TTCTT与AGGAT之间)。由于这段缺失,使得有可能利用常规PCR方法,根据PCR产物长度差异快速鉴定太白贝母。随后,以该137 bp的缺失序列为核心,分别设计正向与反向PCR 引物,以用于太白贝母的特异性鉴定。正向引物(TBF:5'-GCGAACGAGTATTTAGTTCATC-3')位于accD基因内部,反向引物(TBR:5'-AGGGTTCTTTCACTCCTTTCT-3')位于psaI基因内部,引物位置见图2。使用该对引物对太白贝母样品进行PCR扩增,经Sanger测序进一步确认该137 bp的片段缺失。
2.3 PCR鉴定太白贝母
当以植物叶片DNA为模板时,仅有太白贝母的PCR扩增产物在302 bp位置显示为阳性结果,其余贝母品种在302 bp位置均显示为阴性结果(图3A)。使用该方法鉴定购买的药材(松贝、青贝、炉贝),并以太白贝母鳞茎为标准品,也仅有太白贝母标准品产生302 bp的特异条带(图3B)。由此,本方法不仅可用于来源植物的鉴定,也可为太白贝母药材的鉴定提供了较为理想的候选方法。
2.4 PCR的检测灵敏度
采用不同浓度的太白贝母DNA,作为PCR扩增的模板,结果显示,当DNA模板质量浓度为238.54~0.239 ng·μL-1时,均能产生良好的PCR扩增效果,在302 bp位置显示阳性结果(图4),表明该鉴定方法有较高的灵敏度。
2.5 最适Tm值的选择
为了摸索更好的检测条件,我们设置不同的Tm值(55~60 ℃)。当Tm值范围为55~59 ℃时,太白贝母样品的PCR扩增效率较高,均在302 bp处显示清晰的电泳条带(图5),并且,当Tm值为59 ℃时,仅有太白贝母样品出现扩增条带。当Tm值范围为55~60 ℃时,平贝母与湖北贝母样品未能有效扩增。中华贝母与浓蜜贝母样品对Tm值要求较高,仅在58 ℃时,出现440 bp附近的扩增条带。其余贝母样品在Tm值范围为55~58 ℃时,取得较高的PCR扩增效率,在440 bp附近显示清晰的电泳条带。综合考虑,55~59 ℃可作为该PCR方法的合适Tm区间,58 ℃为最适Tm值。
3 讨论
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植物来源鉴定对于确保川贝母等贝母类产品的质量和安全是非常必要的。基于DNA的分子鉴定方法具有重现性好,不易受到植物生长状况、加工方式与仪器参数的优势,而备受关注。PCR-RFLP、DNA条形码、超级条形码(细胞器基因组)与Taqman探针等分子鉴定方法,已用于川贝母及其近缘物种的鉴定。然而,基于ITS1区域的PCR-RFLP方法,因为ITS1的多态性较低,鉴定效果有限,无法实现特定物种的鉴定;以ITS2为代表的DNA条形码,也由于相似度太高,难以实现特定来源物种的特异性鉴定[14];叶绿体与线粒体等细胞器基因组具有较高的多态性与分辨率,能够有效实现特异性鉴定,然而,该方法需要新鲜样品,耗时,且测序成本过高,难以广泛应用[15];Taqman探针法的灵敏度较高,能够实现低丰度样品的特异性鉴定,但是该方法需要合成荧光探针,且需要使用荧光定量PCR仪,导致实验成本过高[16]。
通过文献比较,基于叶绿体基因组的系统进化树的分辨率与可信度要明显优于ITS1与ITS2等通用DNA条形码[14,16]。例如,Zheng等[14]报道,14种不同贝母属植物ITS2的相似度超过95%,构建的系统进化树中仅有29%的分支拥有超过90的自展值;Zhang等[16]发现基于13种贝母属植物ITS1的系统进化树,也仅有30%的分支拥有超过90的自展值。本研究构建基于叶绿体基因组的系统进化树中,有92.3%的分支处的自展值超过90,证实其可靠性要明显优于DNA条形码。随后,为了保证序列比对结果具有代表性,我们下载了GenBank中所选植物的全部叶绿体基因组序列(103条),发现太白贝母在accD-psaI基因区间含有一段长度为137 bp的缺失。由于该缺失仅存在于太白贝母的叶绿体基因组中,推测缺失发生的时间可能晚于太白贝母的物种形成时间,且可能作为太白贝母鉴定用的Indel标记。为此,在该Indel标记的两侧,选择高度保守的区域分别设计上下游引物,实现了对太白贝母的特异性鉴定,而且能有效地检测到DNA模板量低至0.239 ng·μL-1的样品。尽管部分太白贝母样本在302 bp主带的下方有少量非特异性扩增,但不影响对样本的整体鉴定,对此我们将进行进一步的实验条件优化。在对市售川贝母药材开展常规PCR检测时,3类川贝母药材中均未含有太白贝母。其可能原因与本次实验所用的川贝母药材产自四川阿坝州与甘孜州有关。由于上述区域并非太白贝母的主产区,因此我们将重点收集重庆、陕西等主产区的川贝母药材,开展进一步的验证。
相较于已有方法,常规PCR方法具有特异性强、简便快捷与成本更低等优势。第一,由于该137 bp的Indel缺失仅存在于太白贝母的叶绿体基因组中,特异性较强。第二,本鉴定方法较为简便,仅需检测是否含有302 bp的电泳条带,即可判定样品是否为太白贝母;且在较宽泛的Tm区间(55~59 ℃)与较低DNA样本质量浓度(0.239 ng·μL-1)时,均能实现太白贝母样品的有效扩增,因此可以作为太白贝母鉴定的理想分子标记体系。尽管本方法的灵敏度弱于本实验室前期建立的Taqman-MGB探针方法[16],但是前者不需要昂贵的MGB探针;且仅需低成本的常规PCR仪,而非荧光定量PCR仪,较易在一般企业中推广。第三,本方法同时适用于太白贝母的叶片与鳞茎,表明本方法不仅可用于来源植物的鉴定,也可为药材的鉴定提供候选方法。第四,本方法的操作步骤包含:DNA提取、PCR扩增、凝胶电泳、凝肽成像与结果分析,操作时间仅需2~3 h。相较于PCR-RFLP、DNA条形码及超级条形码等方法[14,18-19],本方法无须酶切与核酸测序等步骤,大大缩短检测周期。
在贝母属植物叶绿体基因组陆续公布的背景下,可以更好地依托叶绿体基因组开发出更多的Indel标记,进一步用于其他川贝母及其他贝母类样品的来源鉴定。并且,该Indel分子标记的开发方法也可以为其他中药材的来源鉴定提供方法借鉴。
基金
收起
  • 四川省科技项目(2018SZ0061)
  • 四川省科技项目(2021ZHFP0170)
  • 四川省中医药管理局面上项目(2021MS116)
  • 成都市科技局项目(2022-YF05-01357-SN)
  • 西南交通大学医工结合项目(2682021ZTPY017)
文献
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参考文献 引证文献
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2024年第59卷第15期
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文章信息
doi: 10.11669/cpj.2024.15.005
  • 接收时间：2023-06-25
  • 首发时间：2026-01-14
  • 出版时间：2024-08-08
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  • 收稿日期：2023-06-25
基金
四川省科技项目(2018SZ0061)
四川省科技项目(2021ZHFP0170)
四川省中医药管理局面上项目(2021MS116)
成都市科技局项目(2022-YF05-01357-SN)
西南交通大学医工结合项目(2682021ZTPY017)
作者信息
    西南交通大学生命科学与工程学院, 成都 610031

通讯作者:

* 周嘉裕,女,博士,副教授 研究方向:中药分子鉴定研究 Tel:(028)87103202
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https://castjournals.cast.org.cn/joweb/zgyxzz/CN/10.11669/cpj.2024.15.005
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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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