Article(id=1322631243469013616, tenantId=1146029695717560320, journalId=1302319053441957962, issueId=1322631228000424183, articleNumber=null, orderNo=null, doi=10.7501/j.issn.0253-2670.2026.18.025, pmid=null, cstr=null, oa=null, hot=0, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1770048000000, receivedDateStr=2026-02-03, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1793269461742, onlineDateStr=2026-10-29, pubDate=null, pubDateStr=null, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793269461742, onlineIssueDateStr=2026-10-29, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793269461742, creator=13701087609, updateTime=1793269461742, updator=13701087609, issue=Issue{id=1322631228000424183, tenantId=1146029695717560320, journalId=1302319053441957962, year='2026', volume='57', issue='18', pageStart='7073', pageEnd='7500', issueExtLink='null', onlineDate='null', pubDate='1790524800000', pubDateStr='2026-09-28', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1793269458054, creator='13701087609', updateTime=1793269548245, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1322631606339228314, tenantId=1146029695717560320, journalId=1302319053441957962, issueId=1322631228000424183, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1322631606339228315, tenantId=1146029695717560320, journalId=1302319053441957962, issueId=1322631228000424183, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=7388, endPage=7396, ext={EN=ArticleExt(id=1322631245570359923, articleId=1322631243469013616, tenantId=1146029695717560320, journalId=1302319053441957962, language=EN, title=Systematic screening of reference genes and expression analysis of genes involved in gastrodin biosynthesis pathway in Gastrodia elata , columnId=null, journalTitle=Chinese Traditional and Herbal Drugs, columnName=null, runingTitle=null, highlight=null, articleAbstract=Objective To screen stable reference genes in Gastrodia elata and analyze the expression patterns of key enzyme genes in the gastrodin biosynthesis pathway, providing reliable normalization tools for molecular studies of G. elata . Methods Two transcriptome datasets, encompassing various tissues (tubers, stems, and flowers) at the jian-ma stage and four developmental stages post-inoculation, were integrated. Expression matrices were constructed using Transcripts Per Million (TPM) and normalized via the TMM method. Candidate reference genes were screened based on a coefficient of variation (CV) threshold of < 0.05, and specific primers were designed using the PrimerServer2 package. The expression levels of these candidates across different tissues and developmental stages were validated by qRT-PCR. Stability was comprehensively evaluated using delta-Ct, geNorm, NormFinder, BestKeeper, and RefFinder. Furthermore, the identified optimal reference gene was used to normalize the expression of six key enzyme genes in the gastrodin biosynthesis pathway. Results Gelref1 exhibited the highest stability across all samples, serving as a universal reference gene for various tissues and developmental stages of G. elata . Expression analysis revealed distinct patterns for the six key enzyme genes, with most showing high expression levels during the mid-to-late stages of tuber development. Conclusion This study establishes a systematic workflow for reference gene screening in G. elata based on RNA-Seq data, providing a reliable foundation for further research into the molecular mechanisms and functional validation of genes in gastrodin biosynthesis., authors=SU Huijun1 , SHI Yasen1 , TIAN Fang2 , MAO Jiajing1 , LUO Ming3 , MIAO Yuhuan1,3 , LI Xiaohua1,3 , LIU Dahui1,3 , SU Wei1,3 , authorsList=SU Huijun, SHI Yasen, TIAN Fang, MAO Jiajing, LUO Ming, MIAO Yuhuan, LI Xiaohua, LIU Dahui, SU Wei, authorCompany=1 School of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China; 2 Peoples Hospital of Luotian County, Huanggang 438600, China; 3 Hubei Shizhen Laboratory, Wuhan 430065, China, correspAuthors=null, 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, fund=null), CN=ArticleExt(id=1322631245494862450, articleId=1322631243469013616, tenantId=1146029695717560320, journalId=1302319053441957962, language=CN, title=天麻内参基因的系统筛选及天麻素合成途径基因的表达分析, columnId=1304140203967410414, journalTitle=中草药, columnName=药材与资源, runingTitle=null, highlight=null, articleAbstract=目的 利用RNA-Seq数据筛选天麻Gastrodia elata 稳定表达的通用内参基因,并分析天麻素合成途径相关基因的表达模式,为天麻分子生物学研究提供技术支撑。方法 收集了天麻剑麻阶段块茎、茎、花的转录组数据集,以及接菌后4个不同发育时期的转录组数据集共2个RNA-Seq数据集。通过分别计算2个数据集的Transcripts Per Million(TPM)表达矩阵,并采用M值结尾均值(trimmed mean of M-values,TMM)方法进行标准化,获得最终的表达量文件genes.TMM.TPM.matrix。以变异系数(coefficient of variation,CV)均小于0.05为阈值,从2个表达量文件中共同筛选出稳定表达基因作为候选内参基因,并基于本地PrimerServer2软件包设计特异性引物。利用实时荧光定量PCR分别检测候选基因在天麻块茎不同发育时期及组织部位的表达水平,使用delta-CT、geNorm、NormFinder、BestKeeper及RefFinder软件进行稳定性综合评价。同时,以筛选获得的最优内参基因分析天麻素合成途径上的6个关键酶基因的表达模式。结果 Gelref1 基因在不同组织部位及发育时期样品中表达最稳定,可作为本研究条件下天麻qRT-PCR分析的适宜内参基因。以该基因为内参,6个天麻素合成途径关键酶基因在不同发育时期组织部位的表达模式存在明显差异,其中多数基因在块茎发育中后期均高表达。结论 提出了一套基于RNA-Seq数据的天麻内参基因系统筛选流程,为后续天麻素生物合成途径的分子机制研究及功能基因验证奠定了基础。, authors=苏慧君1 , 时娅森1 , 田芳2 , 毛家婧1 , 罗鸣3 , 苗玉焕1,3 , 李晓华1,3 , 刘大会1,3 , 苏威1,3 , authorsList=苏慧君, 时娅森, 田芳, 毛家婧, 罗鸣, 苗玉焕, 李晓华, 刘大会, 苏威, authorCompany=1 湖北中医药大学药学院, 湖北 武汉 430065; 2 罗田县人民医院, 湖北 黄冈 438600; 3 湖北时珍实验室, 湖北 武汉 430065, correspAuthors=刘大会, authorNote=苏慧君: 苏慧君,硕士研究生,研究方向为中药资源。E-mail:shj13545708117@163.com, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=CrpHs/jK7WEaQMAQU95jbQ==, pdfFileSize=6742812, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=湖北省中医药创新发展联合基金培育项目 (2026AFC0923); 国家自然科学基金项目 (32500333); 湖北省中医药管理局中医药创新团队项目 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copyrightInformation=null, country=null, option=, provinceCode=null, provinceName=null, collectFlag=false, interPubPlatform=, interPubPlatformUrl=null), detailUrlCn=https://castjournals.cast.org.cn/joweb/zcy/CN/10.7501/j.issn.0253-2670.2026.18.025, detailUrlEn=https://castjournals.cast.org.cn/joweb/zcy/EN/10.7501/j.issn.0253-2670.2026.18.025, pdfUrlCn=https://castjournals.cast.org.cn/joweb/zcy/CN/PDF/10.7501/j.issn.0253-2670.2026.18.025, pdfUrlEn=https://castjournals.cast.org.cn/joweb/zcy/EN/PDF/10.7501/j.issn.0253-2670.2026.18.025, aliStartDate=null, aliEndDate=null, collectionFlag=false, citedCount=null, citedUrl=null, previewStatus=0, delFlag=0, hasFullText=0, orderTime=1790524800000, fullTextJson=null, articleText=null, reference=朋冬琴,罗蜜蜜,郭欣慰,等.当归实时荧光定量PCR内参基因筛选[J]. 中草药, 2024, 55(1):269-278. 李德华,程波,叶力夏提·达那别克,等.基于特异性PCR鉴别维药蜘蛛香及其混淆品[J]. 中草药, 2025,56(24):9165-9171. Bustin S A, Benes V, Garson J A, et al. The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments[J]. Clin Chem,2009, 55(4):611-622. Gutierrez L, Mauriat M, Guénin S, et al. The lack of a systematic validation of reference genes:A serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR)analysis in plants[J]. Plant Biotechnol J, 2008, 6(6):609-618. Guénin S, Mauriat M, Pelloux J, et al. Normalization of qRT-PCR data:The necessity of adopting a systematic,experimental conditions-specific, validation of references[J]. J Exp Bot, 2009, 60(2):487-493. Ahmed U, Xie Q, Shi X P, et al. Development of reference genes for horticultural plants[J]. Crit Rev Plant Sci, 2022,41(3):190-208. Xie Q, Ahmed U, Qi C, et al. A protocol for identifying universal reference genes within a genus based on RNASeq data:A case study of poplar stem gene expression[J]. For Res, 2024, 4:e021. 蒲钧逸,顾宇星,黄微,等.基于网络药理学、分子对接及分子动力学模拟技术探讨天麻治疗脑梗死的作用机制[J]. 现代药物与临床, 2026, 41(1):58-69. 中国药典[S]. 一部. 2025:123-124. 雷小湖,李志平,刘书磊,等.天麻及其上市药品的组方规律与临床应用研究进展[J]. 中草药, 2026, 57(7):2787-2798. 李波,熊仕英,王丽,等.天麻素延长秀丽隐杆线虫寿命及改善衰老相关神经退行性疾病的影响[J]. 世界中医药, 2026, 21(1):47-52. 陈春光,米华玲.天麻素生物合成途径相关基因的分析[J]. 植物生理学报, 2021, 57(9):1819-1828. 徐德宏,崔培梧,罗怀浩,等.天麻素生物合成的研究进展[J]. 中草药, 2020, 51(22):5877-5883. Liu B C, Chen J Y, Zhang W J, et al. The gastrodin biosynthetic pathway in Pholidota chinensis Lindl.revealed by transcriptome and metabolome profiling[J]. Front Plant Sci, 2022, 13:1024239. Pfaffl M W, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper:Excelbased tool using pair-wise correlations[J]. Biotechnol Lett,2004, 26(6):509-515. Andersen C L, Jensen J L,Ørntoft T F. Normalization of realtime quantitative reverse transcription-PCR data:A modelbased variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64(15):5245-5250. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3(7):RESEARCH0034. Shan T Y, Yin M Z, Wu J X, et al. Comparative transcriptome analysis of tubers, stems, and flowers of Gastrodia elata Blume reveals potential genes involved in the biosynthesis of phenolics[J]. Fitoterapia, 2021,153:104988. Liu Q, Wu Z Q, Gao Y G, et al. Ge ARF5/GeIAA33-Ge SWEET14 module balances the secondary metabolic biosynthesis to increase the yield and quality in Gastrodia elata[J]. J Integr Plant Biol, 2026, 68(4):1155-1175.)
中草药
|药材与资源
2026
, 57
(18) :
7388
-7396
天麻内参基因的系统筛选及天麻素合成途径基因的表达分析
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苏慧君1 , 时娅森1 , 田芳2 , 毛家婧1 , 罗鸣3 , 苗玉焕1,3 , 李晓华1,3 , 刘大会1,3 , 苏威1,3
作者信息
1 湖北中医药大学药学院, 湖北 武汉 430065; 2 罗田县人民医院, 湖北 黄冈 438600; 3 湖北时珍实验室, 湖北 武汉 430065
通讯作者:
刘大会
作者简介:
苏慧君: 苏慧君,硕士研究生,研究方向为中药资源。E-mail:shj13545708117@163.com
Systematic screening of reference genes and expression analysis of genes involved in gastrodin biosynthesis pathway in Gastrodia elata
SU Huijun1 , SHI Yasen1 , TIAN Fang2 , MAO Jiajing1 , LUO Ming3 , MIAO Yuhuan1,3 , LI Xiaohua1,3 , LIU Dahui1,3 , SU Wei1,3
Affiliations
1 School of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China; 2 Peoples Hospital of Luotian County, Huanggang 438600, China; 3 Hubei Shizhen Laboratory, Wuhan 430065, China
doi: 10.7501/j.issn.0253-2670.2026.18.025
文章导航
目的 利用RNA-Seq数据筛选天麻Gastrodia elata 稳定表达的通用内参基因,并分析天麻素合成途径相关基因的表达模式,为天麻分子生物学研究提供技术支撑。方法 收集了天麻剑麻阶段块茎、茎、花的转录组数据集,以及接菌后4个不同发育时期的转录组数据集共2个RNA-Seq数据集。通过分别计算2个数据集的Transcripts Per Million(TPM)表达矩阵,并采用M值结尾均值(trimmed mean of M-values,TMM)方法进行标准化,获得最终的表达量文件genes.TMM.TPM.matrix。以变异系数(coefficient of variation,CV)均小于0.05为阈值,从2个表达量文件中共同筛选出稳定表达基因作为候选内参基因,并基于本地PrimerServer2软件包设计特异性引物。利用实时荧光定量PCR分别检测候选基因在天麻块茎不同发育时期及组织部位的表达水平,使用delta-CT、geNorm、NormFinder、BestKeeper及RefFinder软件进行稳定性综合评价。同时,以筛选获得的最优内参基因分析天麻素合成途径上的6个关键酶基因的表达模式。结果 Gelref1 基因在不同组织部位及发育时期样品中表达最稳定,可作为本研究条件下天麻qRT-PCR分析的适宜内参基因。以该基因为内参,6个天麻素合成途径关键酶基因在不同发育时期组织部位的表达模式存在明显差异,其中多数基因在块茎发育中后期均高表达。结论 提出了一套基于RNA-Seq数据的天麻内参基因系统筛选流程,为后续天麻素生物合成途径的分子机制研究及功能基因验证奠定了基础。
天麻
/
RNA-Seq
/
内参基因
/
天麻素
/
基因表达
Objective To screen stable reference genes in Gastrodia elata and analyze the expression patterns of key enzyme genes in the gastrodin biosynthesis pathway, providing reliable normalization tools for molecular studies of G. elata . Methods Two transcriptome datasets, encompassing various tissues (tubers, stems, and flowers) at the jian-ma stage and four developmental stages post-inoculation, were integrated. Expression matrices were constructed using Transcripts Per Million (TPM) and normalized via the TMM method. Candidate reference genes were screened based on a coefficient of variation (CV) threshold of < 0.05, and specific primers were designed using the PrimerServer2 package. The expression levels of these candidates across different tissues and developmental stages were validated by qRT-PCR. Stability was comprehensively evaluated using delta-Ct, geNorm, NormFinder, BestKeeper, and RefFinder. Furthermore, the identified optimal reference gene was used to normalize the expression of six key enzyme genes in the gastrodin biosynthesis pathway. Results Gelref1 exhibited the highest stability across all samples, serving as a universal reference gene for various tissues and developmental stages of G. elata . Expression analysis revealed distinct patterns for the six key enzyme genes, with most showing high expression levels during the mid-to-late stages of tuber development. Conclusion This study establishes a systematic workflow for reference gene screening in G. elata based on RNA-Seq data, providing a reliable foundation for further research into the molecular mechanisms and functional validation of genes in gastrodin biosynthesis.
Gastrodia elata Blume
/
RNA-Seq
/
reference genes
/
gastrodin
/
gene expression
苏慧君, 时娅森, 田芳, 毛家婧, 罗鸣, 苗玉焕, 李晓华, 刘大会, 苏威.
天麻内参基因的系统筛选及天麻素合成途径基因的表达分析.
中草药,
2026
, 57
(18)
: 7388
-7396
.
DOI: 10.7501/j.issn.0253-2670.2026.18.025
SU Huijun, SHI Yasen, TIAN Fang, MAO Jiajing, LUO Ming, MIAO Yuhuan, LI Xiaohua, LIU Dahui, SU Wei.
Systematic screening of reference genes and expression analysis of genes involved in gastrodin biosynthesis pathway in Gastrodia elata [J].
Chinese Traditional and Herbal Drugs ,
2026
, 57
(18)
: 7388
-7396
.
DOI: 10.7501/j.issn.0253-2670.2026.18.025
湖北省中医药创新发展联合基金培育项目 (2026AFC0923); 国家自然科学基金项目 (32500333); 湖北省中医药管理局中医药创新团队项目 (ZY2025J002); 中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目” (2060302)
参考文献
引证文献
朋冬琴,罗蜜蜜,郭欣慰,等.当归实时荧光定量PCR内参基因筛选[J]. 中草药, 2024, 55(1):269-278. 李德华,程波,叶力夏提·达那别克,等.基于特异性PCR鉴别维药蜘蛛香及其混淆品[J]. 中草药, 2025,56(24):9165-9171. Bustin S A, Benes V, Garson J A, et al. The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments[J]. Clin Chem,2009, 55(4):611-622. Gutierrez L, Mauriat M, Guénin S, et al. The lack of a systematic validation of reference genes:A serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR)analysis in plants[J]. Plant Biotechnol J, 2008, 6(6):609-618. Guénin S, Mauriat M, Pelloux J, et al. Normalization of qRT-PCR data:The necessity of adopting a systematic,experimental conditions-specific, validation of references[J]. J Exp Bot, 2009, 60(2):487-493. Ahmed U, Xie Q, Shi X P, et al. Development of reference genes for horticultural plants[J]. Crit Rev Plant Sci, 2022,41(3):190-208. Xie Q, Ahmed U, Qi C, et al. A protocol for identifying universal reference genes within a genus based on RNASeq data:A case study of poplar stem gene expression[J]. For Res, 2024, 4:e021. 蒲钧逸,顾宇星,黄微,等.基于网络药理学、分子对接及分子动力学模拟技术探讨天麻治疗脑梗死的作用机制[J]. 现代药物与临床, 2026, 41(1):58-69. 中国药典[S]. 一部. 2025:123-124. 雷小湖,李志平,刘书磊,等.天麻及其上市药品的组方规律与临床应用研究进展[J]. 中草药, 2026, 57(7):2787-2798. 李波,熊仕英,王丽,等.天麻素延长秀丽隐杆线虫寿命及改善衰老相关神经退行性疾病的影响[J]. 世界中医药, 2026, 21(1):47-52. 陈春光,米华玲.天麻素生物合成途径相关基因的分析[J]. 植物生理学报, 2021, 57(9):1819-1828. 徐德宏,崔培梧,罗怀浩,等.天麻素生物合成的研究进展[J]. 中草药, 2020, 51(22):5877-5883. Liu B C, Chen J Y, Zhang W J, et al. The gastrodin biosynthetic pathway in Pholidota chinensis Lindl.revealed by transcriptome and metabolome profiling[J]. Front Plant Sci, 2022, 13:1024239. Pfaffl M W, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper:Excelbased tool using pair-wise correlations[J]. Biotechnol Lett,2004, 26(6):509-515. Andersen C L, Jensen J L,Ørntoft T F. Normalization of realtime quantitative reverse transcription-PCR data:A modelbased variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64(15):5245-5250. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3(7):RESEARCH0034. Shan T Y, Yin M Z, Wu J X, et al. Comparative transcriptome analysis of tubers, stems, and flowers of Gastrodia elata Blume reveals potential genes involved in the biosynthesis of phenolics[J]. Fitoterapia, 2021,153:104988. Liu Q, Wu Z Q, Gao Y G, et al. Ge ARF5/GeIAA33-Ge SWEET14 module balances the secondary metabolic biosynthesis to increase the yield and quality in Gastrodia elata[J]. J Integr Plant Biol, 2026, 68(4):1155-1175.
2026年第57卷第18期
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doi: 10.7501/j.issn.0253-2670.2026.18.025
接收时间:2026-02-03
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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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