Article(id=1201124483122815547, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201124478286786612, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0666, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1684944000000, receivedDateStr=2023-05-25, revisedDate=1692028800000, revisedDateStr=2023-08-15, acceptedDate=null, acceptedDateStr=null, onlineDate=1764299992590, onlineDateStr=2025-11-28, pubDate=1710172800000, pubDateStr=2024-03-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764299992590, onlineIssueDateStr=2025-11-28, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764299992590, creator=13701087609, updateTime=1764299992590, updator=13701087609, issue=Issue{id=1201124478286786612, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='3', pageStart='493', pageEnd='788', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764299991434, creator=13701087609, updateTime=1764300490467, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1201126571420639892, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201124478286786612, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1201126571420639893, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201124478286786612, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=751, endPage=763, ext={EN=ArticleExt(id=1201124483944899166, articleId=1201124483122815547, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Identification, expression and protein interaction analysis of Aux/IAA and ARF gene family in Senna tora L., columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

The early response of plant auxin gene family Aux/IAA (auxin/indole-3-acetic acid) and its interaction with auxin response factor (ARF) are important pattern to regulate plant growth and development. This work identified 28 StoIAA and 24 StoARF members based on the whole genome data of the medicinal plant Senna tora L., which were classified into 10 and 8 subfamilies, respectively. Phylogenetic tree and collinearity analysis showed that S. tora has close evolutionary relationship with the IAA and ARF homologous genes of Glycine max, Medicago truncatula, and the segment duplication events dominate the expansion of StoIAA and StoARF. Gene structure analysis showed that the vast majority of StoIAA and StoARF contain characteristic conserved domain. Transcriptome data showed that StoIAAs and StoARFs were expressed in leaves, roots and seeds, some members had tissue specific expression. The StoIAA and StoARF promoter region most contain functional elements related to stress response, growth and development, hormone induction and secondary metabolism. In addition, gene expression analysis showed that many StoIAAs and StoARFs can quickly respond to drought and salt stress and exhibited same expression patterns under both stress condition. The yeast two-hybrid experiment confirmed that StoARF8 and StoARF10 exhibit varying degrees of interaction with multiple StoIAA proteins, respectively. The above results provide a basis for further biological functional analysis of the Aux/IAA and ARF gene family of S. tora.

, correspAuthors=Ren-jun MAO, Gang ZHANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 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=Zhao FENG, Shi-peng LIU, Rui-hua LÜ, Rui-hua LÜ, Xiao-chen HU, Ming-ying ZHANG, Ren-jun MAO, Gang ZHANG), CN=ArticleExt(id=1201124487895933729, articleId=1201124483122815547, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=决明Aux/IAAARF基因家族鉴定、表达及蛋白互作分析, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

植物生长素早期响应基因家族(auxin/indole-3-acetic acid, Aux/IAA) 及其与生长素响应转录因子(auxin response factor, ARF) 的互作是调控植物生长发育的重要方式。本文基于药用植物决明(Senna tora L.) 全基因组数据鉴定到28个StoIAA和24个StoARF成员, 分别归为10个和8个亚家族。系统发育树和共线性分析发现, 决明与大豆(Glycine max)、蒺藜苜蓿(Medicago truncatula) 的IAAARF同源基因均存在密切的进化关系, 且基因片段重复事件主导了StoIAAStoARF的扩张。基因结构分析显示, 绝大多数StoIAAsStoARFs含有特征保守结构域。转录组数据分析显示, StoIAAsStoARFs在叶、根和种子中均有表达, 部分成员具有组织表达特异性。StoIAAStoARF启动子区域多含有与胁迫响应、生长发育、激素诱导、次生代谢等相关的作用元件。基因表达分析显示许多StoIAAsStoARFs能够迅速响应干旱和盐胁迫, 且在两种胁迫条件下表现出相似的表达模式。酵母双杂交实验证实StoARF8和StoARF10分别与多个StoIAA蛋白之间同时存在不同程度的互作。以上研究结果为进一步开展决明Aux/IAAARF基因家族的生物学功能分析提供基础。

, correspAuthors=毛仁俊, 张岗, authorNote=null, correspAuthorsNote=
*毛仁俊, Tel: 86-911-2332030, E-mail: ;
张岗, Tel: 86-29-38185165, E-mail:
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College of Life Science, Yan'an University, Yan'an 716000, China), AuthorCompanyExt(id=1201166977214673003, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, companyId=1201166977197895786, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.延安大学生命科学学院, 陕西 延安 716000)])], figs=[ArticleFig(id=1201166980402344131, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=FBbjIOnRzOIB0zUx29TOkA==, figureFileBig=+9M83Tow+Ddb3M+zYPQjGQ==, tableContent=null), ArticleFig(id=1201166980507201733, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 1, caption= Phylogenetic analyses of IAA (A) and ARF (B) proteins among <i>S. tora</i> (Sto), <i>A. thaliana</i> (At), <i>M. truncatula</i> (Mt) and <i>G. max</i> (Gm) , figureFileSmall=FBbjIOnRzOIB0zUx29TOkA==, figureFileBig=+9M83Tow+Ddb3M+zYPQjGQ==, tableContent=null), ArticleFig(id=1201166980624642247, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=iNEHlTY9VvX6ofxJNzaUOQ==, figureFileBig=RepWMHda8F/fZB+NyCVNbA==, tableContent=null), ArticleFig(id=1201166980725305546, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 2, caption= Segmental duplication analysis of <i>Aux/IAA</i> and <i>ARF</i> genes in Sto. Pink curves represent the <i>StoIAA</i> gene pairs; green curves represent <i>StoARF</i> gene pairs , figureFileSmall=iNEHlTY9VvX6ofxJNzaUOQ==, figureFileBig=RepWMHda8F/fZB+NyCVNbA==, tableContent=null), ArticleFig(id=1201166980817580235, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=lQsc1mAKtDWhOAmISjP7xA==, figureFileBig=VdThFeb9rLtWWl4b2I6Xrg==, tableContent=null), ArticleFig(id=1201166980897272012, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 3, caption= Synteny analysis of <i>IAA</i> (A) and <i>ARF</i> (B) gene families of Sto and At, Mt and Gm. The green, yellow, red and blue rectangles represent the chromosomes of <i>A. thaliana</i>, <i>M. truncatula</i>, <i>G. max</i> and <i>S. tora</i>, respectively; the green, yellow and red curves represent the collinear gene pairs between <i>A. thaliana</i>, <i>M. truncatula</i> and <i>G. max</i> with <i>S. tora</i> respectively , figureFileSmall=lQsc1mAKtDWhOAmISjP7xA==, figureFileBig=VdThFeb9rLtWWl4b2I6Xrg==, tableContent=null), ArticleFig(id=1201166980985352398, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=IxUIsWjEFiiZ+rLZ2Nk27g==, figureFileBig=oeptylRefdMjsb9pP8JfVQ==, tableContent=null), ArticleFig(id=1201166981094404303, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 4, caption= Number of putative <i>cis</i>-regulatory elements in promoter region of <i>IAA</i> and <i>ARF</i> genes in <i>S. tora</i> , figureFileSmall=IxUIsWjEFiiZ+rLZ2Nk27g==, figureFileBig=oeptylRefdMjsb9pP8JfVQ==, tableContent=null), ArticleFig(id=1201166981178290384, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=/GMwKvHQO/toZyKo6XD+Dw==, figureFileBig=VLna6wN4NEBMmCjX0zw2yg==, tableContent=null), ArticleFig(id=1201166981232816338, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 5, caption= Heatmap of <i>StoIAAs</i> (A) and <i>StoARFs</i> (B) expression in different tissues and development stages , figureFileSmall=/GMwKvHQO/toZyKo6XD+Dw==, figureFileBig=VLna6wN4NEBMmCjX0zw2yg==, tableContent=null), ArticleFig(id=1201166981308313812, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=RXRdD3qIKhhTu4Eu1DL5pw==, figureFileBig=puJCjcteizBCxbHuK8u8ZQ==, tableContent=null), ArticleFig(id=1201166981362839766, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 6, caption= Expression analysis of <i>StoIAAs</i> (A, C) and <i>StoARFs</i> (B, D) in response to salinity (A, B) and drought (C, D) treatment. <i>n</i> = 40, <i><span class="mag-xml-overline" style="border-top:1px solid black">x</span></i> ± <i>s</i>. <i>P</i> < 0.05 indicates significant differences, values with borders represent no significant differences , figureFileSmall=RXRdD3qIKhhTu4Eu1DL5pw==, figureFileBig=puJCjcteizBCxbHuK8u8ZQ==, tableContent=null), ArticleFig(id=1201166981446725848, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=Yol+Lwe+sPoakmfZDJ7o9A==, figureFileBig=/l1NHG2GPYvyRnrxNrW7WA==, tableContent=null), ArticleFig(id=1201166981530611929, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Figure 7, caption= Y2H-based interactions between StoIAAs and StoARFs. AD: Active domain; BD: Binding domain; DDO: SD-Leu/-Trp medium; TDO: SD-Leu/-Trp/-His medium; QDO+X-<i>α</i>-gal: SD-Leu/-Trp/-His/-Ade+X-<i>α</i>-gal medium , figureFileSmall=Yol+Lwe+sPoakmfZDJ7o9A==, figureFileBig=/l1NHG2GPYvyRnrxNrW7WA==, tableContent=null), ArticleFig(id=1201166981618692315, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
GeneGene IDForward primer sequenceReverse primer sequenceSize/bp
StoIAA1Sto01g002110ACTGGATGCTTGCTGGGGTCCAAGCAGCACTCGCAA150
StoIAA2Sto01g002120ATTGTTGATGCCGGCGGTGGCCACCCCACAACTTGA95
StoIAA3Sto01g003090TGGGATGGCCTCCAATTCGGCAACAACAACACCACGCT133
StoIAA4Sto02g036010GTTAGGCCGACGGAAGGGCGCCGCATACCCAGAGAG124
StoIAA5Sto03g064360GCTAAGCCACCTGCAGCTTCCTCCTCCTGTCTCGCC126
StoIAA6Sto03g064380GCAGCATGGGAGGCATCAAGGGTGCTTGTTGCCACT89
StoIAA7Sto05g123870GTCCCCCAGGCCTACTCATCTCGAGTGCGGCCTTTG119
StoIAA8Sto06g204060GGAGCTCAGCCAACCTCGAGCTGGTGCACTGCCATT120
StoIAA9Sto08g232830GGTTGGCCACCAGTTCGATTCACAAACGTTGCGCCG89
StoIAA10Sto08g235250CAGCAGCAACCACCCACTAGGCTGCCCCCTACTTGA142
StoIAA11Sto08g238370GCTGTGCCTCTTCCTCCCGATCTGCCCCCACGACAG134
StoIAA12Sto08g238390CCAACCCTCCTGCCAAGGTGGAGCTCCATCCATGCT149
StoIAA13Sto08g239310TGAACGGACTGCAGCCAGAGCATCCAGTCTCCCTCCT144
StoIAA14Sto08g252470ACCTCAAAGCCACCGAGCGAAACCCCTCTTGGCCCC107
StoIAA15Sto09g281850GTCCTGTGGTGGGATGGCTCCCAGCAAGCTTGTCCAC126
StoIAA16Sto09g282170TTGGGCTTCCTGGCTGTGAGCCTCTCTTAGATCCAGCCA117
StoIAA17Sto09g282820ATGGTGGTGACGACGGTGTCCCCTCCTCCTCCTCCT84
StoIAA18Sto09g282850CCAAAGCGGGCACGAAACTCCGATCCCTTTGGTGCG118
StoIAA19Sto09g310410TCCTCTGTGTCCCTCGAAGATTCCCCTCGGAGAAGCCA81
StoIAA20Sto10g348320GCTACGGCTCGGGTTACCAACCAGCGTCAATGGCGT140
StoIAA21Sto11g355830ACTGTGCAACCTGAAGCCTCCAATGCACTGAGTTTGGCA91
StoIAA22Sto12g391490CGATTCGGCCCCTCCTTCATCCCACCACTCTCGCCT102
StoIAA23Sto12g391500GATGCTCCTTCCGCCTCCTCTGCCCCTCCTGAAGCA106
StoIAA24Sto13g437070GTGACTCCATGCTCACCGAAGCTGTTACGTTGGCCCC113
StoIAA25Sto13g437970TTGGATGCTTGTCGGCGATGCATGGCTCTTGGTGCA108
StoIAA26Sto13g438000GGGATGGCCACCTGTGAGGCACCAGCCATGCTCACT96
StoIAA27Sto13g440900ACCATTCTCTTGGGGACGGGGAACATCCCCAACCACGA104
StoIAA28Sto13g443170GGATGGCCACCTGTTCGAAGGAGCTCCATCCATGCT102
StoARF1Sto02g025950GGGGTCAACCAAGGAGGCGCAGCCCTACGGATTCCC136
StoARF2Sto02g051230GGAGCTGCGTGTTGGAGTGCAGTGGCCAGAACACCA102
StoARF3Sto02g052010CCACCCTTTCCTGGCAGGCTGGTGCTGCTAACCCCC146
StoARF4Sto03g060360TGGCATGCATGTGCTGGAGCTGCAACTTGCTCGCTG92
StoARF5Sto03g087170CCTGTGCTGGTCCCCTTGACGCCTCCACCTGCTCTA87
StoARF6Sto04g089020GCGGACCAGCATATGCCTGCTCCGGAACCAACGTCA124
StoARF7Sto04g116880ACAATCCAAGGGCCAGCCCTGCGCACTCCAGACTCC130
StoARF8Sto05g119260TCAGTACCTCGCAGGGCTCTCTTCGGCTGACCACGG140
StoARF9Sto05g122210CTGCCGCGTATCCTTCGTAGGTGCATCGGCGGATTC139
StoARF10Sto05g157220ACCGGCTCAAGAAGGGGAGTTGAAGCGGCAACCTGC141
StoARF11Sto05g157230GGGTATCCGGCGAGCAAAGCTATTTGTGGCAGCGGC115
StoARF12Sto06g173650AGGCAGAGCAAGCAGGTGCTGCTCGACCAACAGCCA82
StoARF13Sto06g192810ACGACGTTGAATCGCGGTGTCGACAAGAGGTCCCGC119
StoARF14Sto07g215360AATGGTGGCGGCTTCTCCGACGTCCTTGGCCGAGAC105
StoARF15Sto07g215710GGGCGGCTGAAGACTGTTAAGCAGATGCCGCCTTGG137
StoARF16Sto08g237050CGCCGGCCCTTCTGTTAAAAGGCAGCGACCTGTTCC84
StoARF17Sto08g240730ACAGAGGAACGCCAAGGCTGAATCCCCCGCAACGAG83
StoARF18Sto08g255920GGCTGTAACTTCCGCGGTGCTCTCACAGACGAGGCC99
StoARF19Sto08g260200CTGGGCATCTGGGGCTTCATTGCCCTGCTCCAGCTG93
StoARF20Sto09g302520CTGCTGGGACTCGTGTGGTCAACTGGGGTGGCAAGC117
StoARF21Sto12g377610GACGACTGGGTGGAGCACGGCGAATCCCCACACACA104
StoARF22Sto13g414350GCCCCGATGATCCTCTGCACATGTCCAATGGCGGCA144
StoARF23Sto13g425100GCTTCGGGTTTGGCTCCTGCTGCTCAAAGTGGCCCT145
StoARF24Sto13g430430CGTGTTGCGGTGGATCCTTGTGGCTGCCCTAACTGC80
), ArticleFig(id=1201166981715161308, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Table 1, caption=

Primers used for quantitative real-time PCR (qRT-PCR) in this study. StoIAAs: Auxin/indole-3-acetic acid (Aux/IAA) gene from S. tora (Sto); StoARFs: Auxin response factor gene (ARF) from Sto

, figureFileSmall=null, figureFileBig=null, tableContent=
GeneGene IDForward primer sequenceReverse primer sequenceSize/bp
StoIAA1Sto01g002110ACTGGATGCTTGCTGGGGTCCAAGCAGCACTCGCAA150
StoIAA2Sto01g002120ATTGTTGATGCCGGCGGTGGCCACCCCACAACTTGA95
StoIAA3Sto01g003090TGGGATGGCCTCCAATTCGGCAACAACAACACCACGCT133
StoIAA4Sto02g036010GTTAGGCCGACGGAAGGGCGCCGCATACCCAGAGAG124
StoIAA5Sto03g064360GCTAAGCCACCTGCAGCTTCCTCCTCCTGTCTCGCC126
StoIAA6Sto03g064380GCAGCATGGGAGGCATCAAGGGTGCTTGTTGCCACT89
StoIAA7Sto05g123870GTCCCCCAGGCCTACTCATCTCGAGTGCGGCCTTTG119
StoIAA8Sto06g204060GGAGCTCAGCCAACCTCGAGCTGGTGCACTGCCATT120
StoIAA9Sto08g232830GGTTGGCCACCAGTTCGATTCACAAACGTTGCGCCG89
StoIAA10Sto08g235250CAGCAGCAACCACCCACTAGGCTGCCCCCTACTTGA142
StoIAA11Sto08g238370GCTGTGCCTCTTCCTCCCGATCTGCCCCCACGACAG134
StoIAA12Sto08g238390CCAACCCTCCTGCCAAGGTGGAGCTCCATCCATGCT149
StoIAA13Sto08g239310TGAACGGACTGCAGCCAGAGCATCCAGTCTCCCTCCT144
StoIAA14Sto08g252470ACCTCAAAGCCACCGAGCGAAACCCCTCTTGGCCCC107
StoIAA15Sto09g281850GTCCTGTGGTGGGATGGCTCCCAGCAAGCTTGTCCAC126
StoIAA16Sto09g282170TTGGGCTTCCTGGCTGTGAGCCTCTCTTAGATCCAGCCA117
StoIAA17Sto09g282820ATGGTGGTGACGACGGTGTCCCCTCCTCCTCCTCCT84
StoIAA18Sto09g282850CCAAAGCGGGCACGAAACTCCGATCCCTTTGGTGCG118
StoIAA19Sto09g310410TCCTCTGTGTCCCTCGAAGATTCCCCTCGGAGAAGCCA81
StoIAA20Sto10g348320GCTACGGCTCGGGTTACCAACCAGCGTCAATGGCGT140
StoIAA21Sto11g355830ACTGTGCAACCTGAAGCCTCCAATGCACTGAGTTTGGCA91
StoIAA22Sto12g391490CGATTCGGCCCCTCCTTCATCCCACCACTCTCGCCT102
StoIAA23Sto12g391500GATGCTCCTTCCGCCTCCTCTGCCCCTCCTGAAGCA106
StoIAA24Sto13g437070GTGACTCCATGCTCACCGAAGCTGTTACGTTGGCCCC113
StoIAA25Sto13g437970TTGGATGCTTGTCGGCGATGCATGGCTCTTGGTGCA108
StoIAA26Sto13g438000GGGATGGCCACCTGTGAGGCACCAGCCATGCTCACT96
StoIAA27Sto13g440900ACCATTCTCTTGGGGACGGGGAACATCCCCAACCACGA104
StoIAA28Sto13g443170GGATGGCCACCTGTTCGAAGGAGCTCCATCCATGCT102
StoARF1Sto02g025950GGGGTCAACCAAGGAGGCGCAGCCCTACGGATTCCC136
StoARF2Sto02g051230GGAGCTGCGTGTTGGAGTGCAGTGGCCAGAACACCA102
StoARF3Sto02g052010CCACCCTTTCCTGGCAGGCTGGTGCTGCTAACCCCC146
StoARF4Sto03g060360TGGCATGCATGTGCTGGAGCTGCAACTTGCTCGCTG92
StoARF5Sto03g087170CCTGTGCTGGTCCCCTTGACGCCTCCACCTGCTCTA87
StoARF6Sto04g089020GCGGACCAGCATATGCCTGCTCCGGAACCAACGTCA124
StoARF7Sto04g116880ACAATCCAAGGGCCAGCCCTGCGCACTCCAGACTCC130
StoARF8Sto05g119260TCAGTACCTCGCAGGGCTCTCTTCGGCTGACCACGG140
StoARF9Sto05g122210CTGCCGCGTATCCTTCGTAGGTGCATCGGCGGATTC139
StoARF10Sto05g157220ACCGGCTCAAGAAGGGGAGTTGAAGCGGCAACCTGC141
StoARF11Sto05g157230GGGTATCCGGCGAGCAAAGCTATTTGTGGCAGCGGC115
StoARF12Sto06g173650AGGCAGAGCAAGCAGGTGCTGCTCGACCAACAGCCA82
StoARF13Sto06g192810ACGACGTTGAATCGCGGTGTCGACAAGAGGTCCCGC119
StoARF14Sto07g215360AATGGTGGCGGCTTCTCCGACGTCCTTGGCCGAGAC105
StoARF15Sto07g215710GGGCGGCTGAAGACTGTTAAGCAGATGCCGCCTTGG137
StoARF16Sto08g237050CGCCGGCCCTTCTGTTAAAAGGCAGCGACCTGTTCC84
StoARF17Sto08g240730ACAGAGGAACGCCAAGGCTGAATCCCCCGCAACGAG83
StoARF18Sto08g255920GGCTGTAACTTCCGCGGTGCTCTCACAGACGAGGCC99
StoARF19Sto08g260200CTGGGCATCTGGGGCTTCATTGCCCTGCTCCAGCTG93
StoARF20Sto09g302520CTGCTGGGACTCGTGTGGTCAACTGGGGTGGCAAGC117
StoARF21Sto12g377610GACGACTGGGTGGAGCACGGCGAATCCCCACACACA104
StoARF22Sto13g414350GCCCCGATGATCCTCTGCACATGTCCAATGGCGGCA144
StoARF23Sto13g425100GCTTCGGGTTTGGCTCCTGCTGCTCAAAGTGGCCCT145
StoARF24Sto13g430430CGTGTTGCGGTGGATCCTTGTGGCTGCCCTAACTGC80
), ArticleFig(id=1201166981794853086, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
GeneRestriction enzymePrimer sequence
StoARF8EcoR IFor: TGGCCATGGAGGCCGAATTCATGGCTTTCGTGGAAGACAAGATT
BamH IRev: CGCTGCAGGTCGACGGATCCCTATATGAAGCCACCCGACATGGA
StoARF10EcoR IFor: TGGCCATGGAGGCCGAATTCATGAGGCTCTCTTCATCTGGACT
BamH IRev: CGCTGCAGGTCGACGGATCCTCAACTACCAGATTCATGCAATTCA
StoIAA2EcoR IFor: GCCATGGAGGCCAGTGAATTCATGACTCTTGGACTTGAGATC
BamH IRev: CAGCTCGAGCTCGATGGATCTCACTTCGTTTTGCCCTTCAAAGATCC
StoIAA5EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGCCGCTTTGGGATTGA
BamH IRev: CAGCTCGAGCTCGATGGATCTTAGCTTCTGCTTTTGCATTTTTC
StoIAA8EcoR IFor: GCCATGGAGGCCAGTGAATTCATGTCTACGCCAGTGTTAGTT
BamH IRev: CAGCTCGAGCTCGATGGATCCTAGCTCCTGCTCTTGGATT
StoIAA10EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGGCAAAGGGTCAAGCTC
BamH IRev: CAGCTCGAGCTCGATGGATCTTAGCACCCAAATGCATCTACCC
StoIAA13EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGATGGGTTACCAATTGG
BamH IRev: CAGCTCGAGCTCGATGGATCCTACCCCCAAGGTACATCT
StoIAA17EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGGGAAGGAAGAATTGGGAG
BamH IRev: CAGCTCGAGCTCGATGGATCTTACTCCCAAGGGACATC
StoIAA20EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGAGAAAAGCTCCGATAAGTTTTC
BamH IRev: CAGCTCGAGCTCGATGGATCTTAATTACGACTTTTGCACTT
StoIAA21EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGATTCTAACACTTCCAGCTTTG
BamH IRev: CAGCTCGAGCTCGATGGATCTTAGGTGTAGCTAGAAGAACAA
StoIAA27EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGGCAAACCCTCTGCTTC
BamH IRev: CAGCTCGAGCTCGATGGATCCTATACCCTGGTGATCCTTAG
StoIAA28EcoR IFor: GCCATGGAGGCCAGTGAATTCATGAGTGGGAAAGATATGAAGG
BamH IRev: CAGCTCGAGCTCGATGGATCTCATATGGCGCAGCCCAATCCTT
), ArticleFig(id=1201166981958430944, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Table 2, caption=

Primer sequences used for vector construction for yeast 2 hybrid (Y2H) analysis

, figureFileSmall=null, figureFileBig=null, tableContent=
GeneRestriction enzymePrimer sequence
StoARF8EcoR IFor: TGGCCATGGAGGCCGAATTCATGGCTTTCGTGGAAGACAAGATT
BamH IRev: CGCTGCAGGTCGACGGATCCCTATATGAAGCCACCCGACATGGA
StoARF10EcoR IFor: TGGCCATGGAGGCCGAATTCATGAGGCTCTCTTCATCTGGACT
BamH IRev: CGCTGCAGGTCGACGGATCCTCAACTACCAGATTCATGCAATTCA
StoIAA2EcoR IFor: GCCATGGAGGCCAGTGAATTCATGACTCTTGGACTTGAGATC
BamH IRev: CAGCTCGAGCTCGATGGATCTCACTTCGTTTTGCCCTTCAAAGATCC
StoIAA5EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGCCGCTTTGGGATTGA
BamH IRev: CAGCTCGAGCTCGATGGATCTTAGCTTCTGCTTTTGCATTTTTC
StoIAA8EcoR IFor: GCCATGGAGGCCAGTGAATTCATGTCTACGCCAGTGTTAGTT
BamH IRev: CAGCTCGAGCTCGATGGATCCTAGCTCCTGCTCTTGGATT
StoIAA10EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGGCAAAGGGTCAAGCTC
BamH IRev: CAGCTCGAGCTCGATGGATCTTAGCACCCAAATGCATCTACCC
StoIAA13EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGATGGGTTACCAATTGG
BamH IRev: CAGCTCGAGCTCGATGGATCCTACCCCCAAGGTACATCT
StoIAA17EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGGGAAGGAAGAATTGGGAG
BamH IRev: CAGCTCGAGCTCGATGGATCTTACTCCCAAGGGACATC
StoIAA20EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGAGAAAAGCTCCGATAAGTTTTC
BamH IRev: CAGCTCGAGCTCGATGGATCTTAATTACGACTTTTGCACTT
StoIAA21EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGATTCTAACACTTCCAGCTTTG
BamH IRev: CAGCTCGAGCTCGATGGATCTTAGGTGTAGCTAGAAGAACAA
StoIAA27EcoR IFor: GCCATGGAGGCCAGTGAATTCATGGGCAAACCCTCTGCTTC
BamH IRev: CAGCTCGAGCTCGATGGATCCTATACCCTGGTGATCCTTAG
StoIAA28EcoR IFor: GCCATGGAGGCCAGTGAATTCATGAGTGGGAAAGATATGAAGG
BamH IRev: CAGCTCGAGCTCGATGGATCTCATATGGCGCAGCCCAATCCTT
), ArticleFig(id=1201166982046511330, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
ProteinGene IDCDS length/bpProtein length/aaMolecular mass/kDapISubcellular localization
StoIAA1Sto01g00211064221323.688.44Cytoplasmic
StoIAA2Sto01g00212056718820.887.62Nuclear
StoIAA3Sto01g00309074724827.106.97Nuclear
StoIAA4Sto02g03601055818519.7810.00Cytoplasmic
StoIAA5Sto03g06436062420722.888.82Cytoplasmic
StoIAA6Sto03g06438071123626.506.54Cytoplasmic
StoIAA7Sto05g1238701 07135638.617.15Cytoplasmic
StoIAA8Sto06g2040601 06835538.247.61Nuclear
StoIAA9Sto08g23283071723826.178.63Cytoplasmic
StoIAA10Sto08g23525052817519.205.69Chloroplast
StoIAA11Sto08g23837071123626.356.51Chloroplast
StoIAA12Sto08g23839073224326.978.52Nuclear
StoIAA13Sto08g239310258859.394.88Nuclear
StoIAA14Sto08g25247072924226.368.62Nuclear
StoIAA15Sto09g28185091230333.256.02Nuclear
StoIAA16Sto09g28217083127630.705.91Nuclear
StoIAA17Sto09g28282049216318.564.81Nuclear
StoIAA18Sto09g28285058819521.568.70Cytoplasmic
StoIAA19Sto09g3104101 03234338.096.47Nuclear
StoIAA20Sto10g34832082827529.888.22Chloroplast
StoIAA21Sto11g35583065421724.835.74Nuclear
StoIAA22Sto12g39149060320022.085.36Nuclear
StoIAA23Sto12g39150094531434.914.96Nuclear
StoIAA24Sto13g43707077125627.896.01Nuclear
StoIAA25Sto13g43797039913214.747.65Chloroplast
StoIAA26Sto13g43800057018921.265.42Chloroplast
StoIAA27Sto13g44090042914215.726.21Chloroplast
StoIAA28Sto13g44317058819522.005.24Chloroplast
), ArticleFig(id=1201166982159757539, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Table 3, caption=

Analysis of physical and chemical properties of StoIAA gene family. CDS: Coding sequece; pI: Isoelectric point

, figureFileSmall=null, figureFileBig=null, tableContent=
ProteinGene IDCDS length/bpProtein length/aaMolecular mass/kDapISubcellular localization
StoIAA1Sto01g00211064221323.688.44Cytoplasmic
StoIAA2Sto01g00212056718820.887.62Nuclear
StoIAA3Sto01g00309074724827.106.97Nuclear
StoIAA4Sto02g03601055818519.7810.00Cytoplasmic
StoIAA5Sto03g06436062420722.888.82Cytoplasmic
StoIAA6Sto03g06438071123626.506.54Cytoplasmic
StoIAA7Sto05g1238701 07135638.617.15Cytoplasmic
StoIAA8Sto06g2040601 06835538.247.61Nuclear
StoIAA9Sto08g23283071723826.178.63Cytoplasmic
StoIAA10Sto08g23525052817519.205.69Chloroplast
StoIAA11Sto08g23837071123626.356.51Chloroplast
StoIAA12Sto08g23839073224326.978.52Nuclear
StoIAA13Sto08g239310258859.394.88Nuclear
StoIAA14Sto08g25247072924226.368.62Nuclear
StoIAA15Sto09g28185091230333.256.02Nuclear
StoIAA16Sto09g28217083127630.705.91Nuclear
StoIAA17Sto09g28282049216318.564.81Nuclear
StoIAA18Sto09g28285058819521.568.70Cytoplasmic
StoIAA19Sto09g3104101 03234338.096.47Nuclear
StoIAA20Sto10g34832082827529.888.22Chloroplast
StoIAA21Sto11g35583065421724.835.74Nuclear
StoIAA22Sto12g39149060320022.085.36Nuclear
StoIAA23Sto12g39150094531434.914.96Nuclear
StoIAA24Sto13g43707077125627.896.01Nuclear
StoIAA25Sto13g43797039913214.747.65Chloroplast
StoIAA26Sto13g43800057018921.265.42Chloroplast
StoIAA27Sto13g44090042914215.726.21Chloroplast
StoIAA28Sto13g44317058819522.005.24Chloroplast
), ArticleFig(id=1201166982268809445, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
ProteinGene IDCDS length/bpProtein length/aaMolecular mass/kDapISubcellular localization
StARF1Sto02g0259501 99266372.486.33Nuclear
StARF2Sto02g0512302 991996112.028.1Plasma membrane
StARF3Sto02g0520103 0721 023114.517.96Nuclear
StARF4Sto03g0603603 2971 098122.466.02Nuclear
StARF5Sto03g0871702 59886596.386.22Nuclear
StARF6Sto04g0890202 69489799.466.32Nuclear
StARF7Sto04g1168804 1041 367152.806.2Nuclear
StARF8Sto05g1192602 805934103.225.23Nuclear
StARF9Sto05g1222101 92063971.138.69Chloroplast
StARF10Sto05g15722075024927.696.19Chloroplast
StARF11Sto05g1572302 16672179.476.32Nuclear
StARF12Sto06g1736502 11870578.735.78Nuclear
StARF13Sto06g1928102 49383092.895.77Nuclear
StARF14Sto07g2153602 02567475.728.58Nuclear
StARF15Sto07g2157102 40980288.526.59Nuclear
StARF16Sto08g2370502 22674182.316.2Nuclear
StARF17Sto08g2407302 02867574.046.85Nuclear
StARF18Sto08g2559201 82160666.746.74Nuclear
StARF19Sto08g2602003 2071 068117.496.07Nuclear
StARF20Sto09g3025202 54784894.616.06Nuclear
StARF21Sto12g3776102 01967274.226.94Nuclear
StARF22Sto13g4143501 85761868.915.87Nuclear
StARF23Sto13g4251002 01667173.847.29Nuclear
StARF24Sto13g4304302 67088998.825.87Nuclear
), ArticleFig(id=1201166982419804389, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124483122815547, language=CN, label=Table 4, caption=

Analysis of physical and chemical properties of StoARF gene family

, figureFileSmall=null, figureFileBig=null, tableContent=
ProteinGene IDCDS length/bpProtein length/aaMolecular mass/kDapISubcellular localization
StARF1Sto02g0259501 99266372.486.33Nuclear
StARF2Sto02g0512302 991996112.028.1Plasma membrane
StARF3Sto02g0520103 0721 023114.517.96Nuclear
StARF4Sto03g0603603 2971 098122.466.02Nuclear
StARF5Sto03g0871702 59886596.386.22Nuclear
StARF6Sto04g0890202 69489799.466.32Nuclear
StARF7Sto04g1168804 1041 367152.806.2Nuclear
StARF8Sto05g1192602 805934103.225.23Nuclear
StARF9Sto05g1222101 92063971.138.69Chloroplast
StARF10Sto05g15722075024927.696.19Chloroplast
StARF11Sto05g1572302 16672179.476.32Nuclear
StARF12Sto06g1736502 11870578.735.78Nuclear
StARF13Sto06g1928102 49383092.895.77Nuclear
StARF14Sto07g2153602 02567475.728.58Nuclear
StARF15Sto07g2157102 40980288.526.59Nuclear
StARF16Sto08g2370502 22674182.316.2Nuclear
StARF17Sto08g2407302 02867574.046.85Nuclear
StARF18Sto08g2559201 82160666.746.74Nuclear
StARF19Sto08g2602003 2071 068117.496.07Nuclear
StARF20Sto09g3025202 54784894.616.06Nuclear
StARF21Sto12g3776102 01967274.226.94Nuclear
StARF22Sto13g4143501 85761868.915.87Nuclear
StARF23Sto13g4251002 01667173.847.29Nuclear
StARF24Sto13g4304302 67088998.825.87Nuclear
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决明Aux/IAAARF基因家族鉴定、表达及蛋白互作分析
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冯昭 1 , 刘世鹏 1 , 吕蕊花 1 , 吕瑞华 1 , 胡晓晨 2 , 张明英 2 , 毛仁俊 3, * , 张岗 2, *
药学学报 | 研究论文 2024,59(3): 751-763
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药学学报 | 研究论文 2024, 59(3): 751-763
决明Aux/IAAARF基因家族鉴定、表达及蛋白互作分析
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冯昭1, 刘世鹏1, 吕蕊花1, 吕瑞华1, 胡晓晨2, 张明英2, 毛仁俊3, * , 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, 陕西省中医药管理局"秦药"研发重点实验室, 陕西 咸阳 712046
  • 3.延安大学生命科学学院, 陕西 延安 716000

通讯作者:

*毛仁俊, Tel: 86-911-2332030, E-mail: ;
张岗, Tel: 86-29-38185165, E-mail:
Identification, expression and protein interaction analysis of Aux/IAA and ARF gene family in Senna tora L.
Zhao FENG1, Shi-peng LIU1, Rui-hua LÜ1, Rui-hua LÜ1, Xiao-chen HU2, Ming-ying ZHANG2, Ren-jun MAO3, * , Gang ZHANG2, *
Affiliations
  • 1. College of Medical Technology, Shaanxi University of Chinese Medicine, Xianyang 712046, China
  • 2. Key Laboratory for Research of "Qin Medicine" of Shaanxi Administration of Chinese Medicine, College of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang 712046, China
  • 3. College of Life Science, Yan'an University, Yan'an 716000, China
出版时间: 2024-03-12 doi: 10.16438/j.0513-4870.2023-0666
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植物生长素早期响应基因家族(auxin/indole-3-acetic acid, Aux/IAA) 及其与生长素响应转录因子(auxin response factor, ARF) 的互作是调控植物生长发育的重要方式。本文基于药用植物决明(Senna tora L.) 全基因组数据鉴定到28个StoIAA和24个StoARF成员, 分别归为10个和8个亚家族。系统发育树和共线性分析发现, 决明与大豆(Glycine max)、蒺藜苜蓿(Medicago truncatula) 的IAAARF同源基因均存在密切的进化关系, 且基因片段重复事件主导了StoIAAStoARF的扩张。基因结构分析显示, 绝大多数StoIAAsStoARFs含有特征保守结构域。转录组数据分析显示, StoIAAsStoARFs在叶、根和种子中均有表达, 部分成员具有组织表达特异性。StoIAAStoARF启动子区域多含有与胁迫响应、生长发育、激素诱导、次生代谢等相关的作用元件。基因表达分析显示许多StoIAAsStoARFs能够迅速响应干旱和盐胁迫, 且在两种胁迫条件下表现出相似的表达模式。酵母双杂交实验证实StoARF8和StoARF10分别与多个StoIAA蛋白之间同时存在不同程度的互作。以上研究结果为进一步开展决明Aux/IAAARF基因家族的生物学功能分析提供基础。

决明  /  Aux/IAA  /  生长素响应转录因子  /  激素  /  蛋白互作

The early response of plant auxin gene family Aux/IAA (auxin/indole-3-acetic acid) and its interaction with auxin response factor (ARF) are important pattern to regulate plant growth and development. This work identified 28 StoIAA and 24 StoARF members based on the whole genome data of the medicinal plant Senna tora L., which were classified into 10 and 8 subfamilies, respectively. Phylogenetic tree and collinearity analysis showed that S. tora has close evolutionary relationship with the IAA and ARF homologous genes of Glycine max, Medicago truncatula, and the segment duplication events dominate the expansion of StoIAA and StoARF. Gene structure analysis showed that the vast majority of StoIAA and StoARF contain characteristic conserved domain. Transcriptome data showed that StoIAAs and StoARFs were expressed in leaves, roots and seeds, some members had tissue specific expression. The StoIAA and StoARF promoter region most contain functional elements related to stress response, growth and development, hormone induction and secondary metabolism. In addition, gene expression analysis showed that many StoIAAs and StoARFs can quickly respond to drought and salt stress and exhibited same expression patterns under both stress condition. The yeast two-hybrid experiment confirmed that StoARF8 and StoARF10 exhibit varying degrees of interaction with multiple StoIAA proteins, respectively. The above results provide a basis for further biological functional analysis of the Aux/IAA and ARF gene family of S. tora.

Senna tora  /  Aux/IAA  /  auxin response factor  /  hormone  /  protein interaction
冯昭, 刘世鹏, 吕蕊花, 吕瑞华, 胡晓晨, 张明英, 毛仁俊, 张岗. 决明Aux/IAAARF基因家族鉴定、表达及蛋白互作分析. 药学学报, 2024 , 59 (3) : 751 -763 . DOI: 10.16438/j.0513-4870.2023-0666
Zhao FENG, Shi-peng LIU, Rui-hua LÜ, Rui-hua LÜ, Xiao-chen HU, Ming-ying ZHANG, Ren-jun MAO, Gang ZHANG. Identification, expression and protein interaction analysis of Aux/IAA and ARF gene family in Senna tora L.[J]. Acta Pharmaceutica Sinica, 2024 , 59 (3) : 751 -763 . DOI: 10.16438/j.0513-4870.2023-0666
植物生长素(auxin, Aux) 在植物生长发育和环境胁迫生理适应方面发挥重要作用, 其种类繁多, 吲哚-3-乙酸(3-indoleacetic acid, IAA) 就是一种典型的植物生长激素[1]。生长素通过调节早期响应基因, 如Aux/IAA基因、生长素响应因子(ARF) 和生长素上调小RNA (small auxin upregulated RNA, SAUR) 等[2]发挥作用。其中, Aux/IAAs和ARFs对于生长素介导转录调控至关重要, 在生长素缺乏的情况下, Aux/IAA能与ARFs蛋白互作调控基因表达[3]。研究证实, Aux/IAA参与调控植物体生长发育进程, 例如, 敲除拟南芥IAA3/SHY2影响生长素平衡和侧根形成[4]。TIR1/AFB2与AtIAA2、AtIAA6、AtIAA9或AtIAA17能够形成特殊的传感复合物, 经生长素处理能够调节植物茉莉酸平衡和不定根的起始发育[5]Aux/IAA基因还参与多种逆境胁迫响应, 如水稻OsIAA9OsIAA20受干旱和盐胁迫诱导表达[6], 干旱条件下, 大豆GmIAA47GmIAA49在生殖阶段叶片的转录水平显著增加[7]
Aux/IAA蛋白主要含有四个保守结构域。结构域Ⅰ含有一个保守的亮氨酸重复基序(LxLxLx motif), 与一种联合抑制蛋白TOPLESS (TPL) 共同介导依赖生长素的转录抑制过程[8]。结构域Ⅱ作为生长素降解区, 通过泛素降解降低Aux/IAA蛋白的稳定性[9]。Ⅲ/Ⅳ区域内包含一个羟基末端的PB1 (Phox和Bem1) 基序, Aux/IAA不直接与目标基因生长素反应元件(AuxRE) 结合, 但可通过该结构域与ARF蛋白互作参与调控下游基因表达。ARF蛋白通常包含一个N端高度保守的DNA结合结构域(DNA-binding domain, DBD)、中间区域(middle region, MR) 和C端二聚体结构域(C-terminal domain, CTD)[2]。DBD赋予ARFs特异性结合许多生长素响应基因的TGTCTC元件, MR为非保守区域, 有转录激活或抑制作用[10], CTD参与Aux/IAAs和ARFs的二聚化[11]ARF基因功能多样, 拟南芥AtARF7和AtARF19通过调节单个或部分重叠的目标基因, 对生长素调节的植物发育过程至关重要[12]。另有证据表明, 在生长素调控次级代谢产物的生物合成过程中, Aux/IAA-ARF信号通路介导并发挥关键作用[13, 14]
决明(Senna tora L.) 为豆科决明属草本植物, 其成熟干燥种子作中药材决明子药用历史悠久, 主治目赤涩痛, 羞明多泪, 头痛眩晕, 大便秘结[15]。决明中含有甾体化合物、大黄酚、大黄素等活性成分, 还有人体必需的微量元素, 具有抗菌、降血压、降血脂、抗血小板凝集、保肝等功效[16, 17]。决明原产于我国, 喜光照, 稍耐旱, 不耐寒, 主要分布在安徽、四川、广东及陕西等省适生区[18]。然而, 过度开发和环境污染已严重影响药用植物生态农业的发展, 抗逆新品种的培育和提高药材品质是解决该问题的关键。本草基因组学从基因组水平研究中药, 为次生代谢产物的生物合成和基因组辅助分子育种提供重要基础[19]。虽然Aux/IAAARF在许多物种中得以鉴定, 但其在药用植物中的研究仍较匮乏。本研究利用决明基因组数据[20]开展Aux/IAAARF基因家族鉴定、表达和蛋白互作等研究, 旨在为进一步明确其在决明次生代谢调控及药材品质形成中的生物学功能奠定基础。
决明IAAARF成员鉴定  决明全基因组注释文件和文件下载自NABIC (http://nabic.rda.go.kr/Species/Senna_tora2)。已报道的拟南芥IAAARF基因序列下载自TAIR数据库(http://www.arabidopsis.org/), 用作参考序列查询决明。蒺藜苜蓿和大豆IAA与ARF蛋白序列分别下载自https://www.mdpi.com/article/10/3390/ijms221910494/s1.和SoyBase (https://www.soybase.org/)。根据Aux/IAA和ARF家族结构域的主要特性对经双向BLAST比对后获得的成员进一步筛选, 并利用SMART (http://smart.embl-heidelberg.de/) 和InterProScan (http://www.ebi.ac.uk/interpro) 检测所有生成的非冗余蛋白序列中是否存在特征结构, 最终确定决明IAA和ARF成员及蛋白序列。
决明IAA和ARF蛋白序列分析  使用ExPASy的ProParam工具(http://web.expasy.org/protparam/), 分析StoIAA和StoARF蛋白的理化性质, 包括蛋白分子质量(MW)、理论等电点(pI)、亲水性总平均值(GRAVY)。利用ClustalX分别对StoIAA和StoARF氨基酸序列进行多序列比对。使用MEGA-X的邻接法(NJ), 设置1 000 bootstrap重复, 构建决明IAAs和ARFs系统发育树。利用TBtools软件[21], 分析StoIAAStoARF基因的外显子-内含子结构。使用MEME在线网站(http://meme-suite.org/tools/meme) 对成员的保守基序(motifs) 进行分析。
基因重复事件与共线性分析  使用McscanX (multiple collinearity scan toolkit) 工具对决明种内IAAARF基因重复事件以及决明与拟南芥、蒺藜苜蓿、大豆种间的IAAARF基因共线性关系分别进行分析。结果均通过TBtools中Advanced Circos功能进行展示。
启动子顺式元件分析  使用TBtools软件提取CDS上游2 kb基因组DNA序列, 提交至PlantCare数据库(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) 进行顺式作用元件预测。
植物材料与处理方法  以采自陕西中医药大学药用植物园的决明种子为试材, 经陕西中医药大学张雨曲博士鉴定为药用豆科植物决明(Senna tora L.)。
选取大小均匀的决明种子, 经表面消毒处理后, 平铺于2%的水琼脂平板, 28 ℃暗培养条件下催芽24~48 h。随后将发芽的幼苗移至装有土壤混合物(珍珠岩∶蛭石体积比为2∶1) 的培养袋, 置光照培养箱(上海一恒仪器MGC-BP) 中培养(22 ℃, 光照强度9 000 lx, 16 h/8 h光暗交替), 每袋种4株, 每5~7天加入适量霍格兰营养液[22]。约15~20天, 幼苗长至4叶期, 选取长势一致的植株, 用200 mmol·L-1氯化钠+霍格兰营养液进行盐胁迫处理, 用30% PEG6000+霍格兰营养液进行干旱胁迫处理, 对照组用霍格兰营养液浇灌, 处理植株0、3、6、12和24 h后收集根; 所有样本均迅速用液氮冷冻, -80 ℃保存备用。
表达分析  决明根、叶、幼嫩种子和成熟种子全转录组数据(SAMN09657134) 下载自NCBI Sequence Read Archive (SRA) 数据库(https://www.ncbi.nlm.nih.gov/sra/), 使用TBtools-RNAseq界面化转录组数据分析功能, 先后经FastQC质控、Trimmomatic去除接头和Kallisto分析转录水平数据。
使用RNA提取试剂盒(TIANGEN, 中国) 提取决明根总RNA, SMARTScribeTM反转录酶(TaKaRa) 合成制备模板cDNA, 进行目的基因的qRT-PCR分析。使用20 μL反应体系, 包含1 μL cDNA模板、0.5 μL上下游引物、10 μL SYBR预混液及8 μL DEPC水。使用ABI 7300进行qPCR反应, 程序为: 95 ℃预变性5 min, 95 ℃变性5 s, 60 ℃退火35 s, 共进行40个循环。使用StoActin作为内参基因, 通过2-△△CT方法[23]计算表达量, 统计分析采用SPSS19.0软件进行威尔克森符号秩检验(Wilcoxon signed-rank test), 特异性引物见表 12
蛋白互作分析及酵母双杂交验证  通过STRING (https://www.string-db.org/) 在线数据库, 分析决明Aux/IAA和ARF蛋白的互作网络。由于ARF作为转录因子, 所以将候选基因StoARF的CDS全长序列与pGADT7 (active domain, AD) 载体经同源重组, 构建猎物蛋白载体StoARF-pGADT7, 转入酵母菌Y187, 同样, 将StoIAA的CDS全长序列与pGBKT7 (binding domain, BD) 经同源重组构建诱饵蛋白载体StoIAA-pGBKT7, 转入酵母菌Y2H (yeast 2 hybrid), 目的基因特异性引物见表 2, 线性化载体与目的基因PCR产物同源重组反应体系按照ClonExpress® Ⅱ One Step Cloning Kit说明书计算。两种酵母菌经杂交后筛选单克隆, 利用不同缺陷型培养基观察是否存在自激活现象, 选择无自激活现象的StoIAAs进行互作验证。将StoARF-pGADT7与StoIAA-pGBKT7共转至酵母菌株Y2H, 由于pGADT7含有亮氨酸(Leu), pGBKT7含有色氨酸(Trp), 所以融合成功的酵母菌可在SD/-Trp/-Leu二缺平板上生长, 经重组单克隆筛选, 将繁育菌液分别滴加于SD-Leu/-Trp缺陷型培养基(DDO)、SD-Leu/-Trp/-His缺陷型培养基(TDO)、SD-Leu/-Trp/-His/-Ade缺陷型培养基(QDO)+X-α-gal平板, 观察互作关系。
以拟南芥和蒺藜苜蓿IAA、ARF序列作为参考序列, 在决明基因组中搜索获得30个StoIAA和24个StoARF成员, 通过Pfam和SMART分析结构域后, 确定了28个StoIAA和24个StoARF成员, 基于染色体分布, 分别命名为StoIAA1-StoIAA28 (表 3) 和StoARF1-StoARF24 (表 4)。StoIAA氨基酸长度从85 aa (StoIAA13) 到356 aa (StoIAA7) 不等, 平均长度为229 aa; StoARF的平均氨基酸长度为802 aa, StoIAAs的预测MW从9.39 kDa到38.61 kDa不等, pI从4.81到10.00不等(表 3)。而StoARFs的预测MW从27.69 kDa到152.80 kDa, pI从5.23到8.69 (表 4)。所有StoIAAs和StoARFs预测的GRAVY为负值, 表明StoIAAs和StoARFs是亲水蛋白。
系统进化分析结果显示, 决明中Aux/IAA基因家族和ARF基因家族也分别分为10个亚家族(Ⅰ-Ⅹ) 和8个亚家族(Ⅰ-Ⅷ) (图 1AB), 与拟南芥[24]、蒺藜苜蓿[25]和大豆[26, 27]相同。在Aux/IAA的10个亚家族中, Ⅷ和Ⅸ包含以上3个物种Aux/IAA成员, 其他8个亚家族中均包含以上所有4个物种的Aux/IAA成员(图 1A), 表明Aux/IAA成员分化早于物种分化。
ARF的8个亚家族中, 绝大多数StoARFs含有由一个植物特异性B3结构域和一个ARF结构域共同组成的保守DBD。根据中间区域(MR) 的氨基酸序列特征, 亚家族Ⅲ-Ⅴ中的StoARF3、4、7、8、10、11、19、20、24的MD区域富含谷氨酰胺(Q)、丝氨酸(S) 和亮氨酸(L), 而亚家族Ⅰ中的StoARF2、5、6、12、13、16、22的MR则多含有丝氨酸、原氨酸(P)、亮氨酸和甘氨酸(G)。从系统发育树可以看出, 除了较为特殊的亚家族Ⅷ以外, StoARFs与StoIAAs类似, 在系统发育关系上与MtARFs和GmARFs更加靠近。
StoIAAsStoARFs的外显子/内含子分布结果显示, 除了StoIAA13StoIAA17只含有1个内含子外, 其他StoIAAs含有2~5个内含子。而StoARFs则含有1~20个内含子, 其中StoARF18仅含1个内含子, StoARF17含有2个内含子, StoARF14StoARF21含有3个内含子, StoARF9含有4个内含子, StoARF10含有7个内含子, StoARF1StoARF11含有9个内含子, 其他StoARFs均含有10~20个内含子。
保守基序分析表明, 大多数StoIAAs (24个) 含有4个保守motif, 其他StoIAAs (4个) 只含有2个motif, 其中StoIAA10StoIAA27缺少motif 1和motif 4, StoIAA13StoIAA25缺少motif 3和motif 4。在StoARFs中共鉴定到10个保守motif, motif 3和7构成B3结构域, motif 1、5、6、9构成ARF结构域, CTD结构域则由motif 4和10组成。B3结构域和ARF结构域共同构成了一个保守的DBD结构。有18个ARF成员含有全部的3个保守结构域, StoARF10缺少ARF结构域, StoARF11缺少B3结构域, StoARF1StoARF9StoARF18StoARF23均缺少CTD结构域。
进一步分析StoIAAsStoARFs的共线关系, 图 2结果表明, 部分StoIAAsStoARFs基因在决明基因组中存在基因重复事件。在Aux/IAA基因家族中, 共存在12对片段重复基因对, 分别是StoIAA1/18StoIAA2/17StoIAA3/15StoIAA7/19StoIAA8/19StoIAA14/16StoIAA14/20StoIAA10/27StoIAA11/26StoIAA12/25StoIAA13/24StoIAA16/20ARF基因家族中则存在StoARF1/23StoARF5/6StoARF14/17StoARF14/21StoARF17/21StoARF18/21共6对片段重复基因对, 决明Aux/IAAARF基因家族中均未发现基因串联重复事件, 说明两个家族的基因复制方式是由片段重复事件主导的。
在拟南芥中分析得到35对与决明Aux/IAA存在共线性关系的同源基因对, 而决明Aux/IAA基因家族与同为豆科的大豆有74对共线性基因, 为拟南芥的2倍, 与蒺藜苜蓿间也存在37对同源基因对。而对于决明ARF基因家族而言, 在蒺藜苜蓿和大豆中分别存在31对和61对同源基因对, 远远高于拟南芥中的19对(图 3)。
为了解StoIAAStoARF的潜在调节作用, 对二者上游2 kb的启动子序列进行分析。从图 4可以看出, 响应光照(Box 4)、厌氧环境(ARE)、脱落酸(ABRE)、赤霉素(gibberellin)、茉莉酸甲酯(CGTCA-motif) 等生长激素反应元件在StoIAAStoARF启动子区大量富集, 同时, 响应逆境胁迫和参与生长发育相关的顺式元件如干旱(MBS)、低温(LTR)、分生组织生长(CAT-box)、昼夜节律(circadian) 广泛分布于多个成员的启动子区。18个StoIAAs (64.3%) 和16个StoARFs (66.7%) 含有与响应干旱相关的顺式元件, 13个StoIAAs (46.4%) 和12个StoARFs (50%) 含有与低温胁迫相关的顺式元件。
利用决明根、叶、幼嫩种子和成熟种子4个组织的转录组数据分析StoIAAsStoARFs的时空表达模式(图 5)。总体而言, StoIAAsStoARFs在根和叶中的表达偏高。StoIAA8StoIAA17StoIAA21StoIAA24StoARF2StoARF5StoARF22在所有检测组织中表达均偏高。个别基因具有明显的组织表达特异性, 如StoIAA2StoIAA11StoIAA12StoIAA26以及StoARF9仅在叶片中表达, StoARF7只在根中表达。另外, 成熟种子中表达的基因均在幼嫩种子中表达, 且大多数在幼嫩种子中的表达水平高于成熟种子。
在模拟盐胁迫和干旱条件下, 处理后30天的植株侧根生长受到明显抑制。两个基因家族所有成员在根中的表达分析表明(图 6), 在干旱条件下, 大多数的StoIAA在处理后3~12 h表达显著上调, 如StoIAA8在处理后3 h转录达到峰值, 随后稍有下降, 但24 h内一直保持高于对照组6倍以上的转录水平。StoIAA16StoIAA22转录水平在处理后6 h达到峰值。StoARF5StoARF6StoARF8等在干旱处理后3~24 h也表现出高于对照组6~8倍的转录水平, 表明这些基因能够在干旱胁迫下迅速表达。相较于干旱胁迫, 较少的StoIAAsStoARFs参与了盐胁迫的响应, 值得注意的是, 个别基因在盐胁迫条件下, 表现出与干旱胁迫相同的表达模式, 如Aux/IAA家族中的StoIAA8StoIAA16和ARF家族中的StoARF5StoARF6StoARF8StoARF10
蛋白互作网络分析结果表明, 16个StoIAAs和12个StoARFs蛋白之间均存在互作关系。其中, StoARF8、10、19、24作为转录激活子与大部分StoIAAs之间存在强烈互作关系。多个蛋白如StoARF10与5个StoIAAs之间存在很高的协同表达相关性、StoIAA12与2个StoIAAs和1个StoARF之间也存在很高的协同表达相关性。
为了进一步验证候选蛋白的互作关系, 依据STRING蛋白网络互作预测结果, 锁定转录激活蛋白StoARF8、StoARF10, 酵母双杂交结果显示, StoARF8和StoARF10蛋白与10个StoIAAs间存在不同程度的互作关系(图 7)。在20个两两杂交结果中, 11对StoARFs-StoIAAs组合间存在弱互作, 如StoIAA5、StoIAA10、StoIAA13、StoIAA27、StoIAA28与StoARF8和StoARF10的相互作用均较弱。另有9对组合间存在强相互作用, 其中StoARF8与StoIAA2、StoIAA17、StoIAA21之间, StoARF10与StoIAA2、StoIAA8、StoIAA21之间的互作更为显著。
生长素在调控植物生长发育和生理过程中发挥的作用至关重要, 而Aux/IAA与ARF蛋白正是生长素信号传导过程中调节下游生化反应的关键[3]。不同植物中Aux/IAAARF的成员数量也存在差异, 如拟南芥中含有29个IAAs和23个ARFs[14, 24], 水稻含有31个IAAs和25个ARFs[28, 12], 蒺藜苜蓿中鉴定到25个IAAs和40个ARFs基因[25], 而大豆中则分别存在63个IAAs和55个ARFs成员[26, 27]。本研究在药用豆科植物决明中分别鉴定到28个StoIAAs和24个StoARFs成员, 同一亚家族的StoIAAsStoARFs成员在基因结构、理化性质等方面存在一定的差异性, 如ORF长度、分子质量、等电点等特性, 不过这种基因家族内的差异在水稻[12, 28]、蒺藜苜蓿[24]和二穗短柄草[29]等许多物种中同样存在。这种家族内成员间广泛的差异性表明在多变的微环境条件下, 不同的StoIAAs和StoARFs蛋白可能会共同发挥作用。
典型的Aux/IAA蛋白通常包含四个保守结构域Ⅰ-Ⅳ[2]。决明StoIAAs蛋白均含有这4个结构域, 而StoIAA10和StoIAA27缺少结构域Ⅰ和Ⅳ, StoIAA13和StoIAA25缺少结构域Ⅰ和Ⅱ。缺少结构域Ⅰ则无法与TPL蛋白结合, 从而失去对下游生长素调节基因的抑制作用, 而缺少结构域Ⅱ则无法被TIR1/AFB蛋白识别, 则这些成员属于非典型Aux/IAA蛋白, 其在植物组织中的含量太低, 几乎无法影响植物的生长发育过程[30]。组织表达结果证实缺失结构域Ⅱ的StoIAA10StoIAA13StoIAA25StoIAA27在所有检测组织中的表达水平远远低于其他典型Aux/IAA基因。非典型Aux/IAA较常见, 水稻OsIAA4OsIAA27OsIAA28OsIAA29缺少结构域Ⅰ和Ⅱ[28], 大豆GmIAA5GmIAA6GmIAA31GmIAA35GmIAA60等成员缺少结构域Ⅱ[31]。同时缺少结构域Ⅰ和Ⅱ的StoIAA10StoIAA27, 它们可能既不发挥转录抑制作用, 也不会在生长素信号传导过程中被迅速降解, 推测可能参与了生长素调节的其他生物过程。
蛋白结构分析是解析基因在调节植物发育过程和响应环境胁迫发挥的潜在作用的重要基础。典型的ARF蛋白包含DBD、MR和CTD三个结构域, 其CTD结构域与Aux/IAA基因启动子区结合, 并受到TIR1/AFB受体的调节[2]。基于拟南芥ARFs关于转录调节区(TRR) 的分析, 含有QSL-富集MR的StoARFs可能为转录激活子, 而MR含有Q、S、L、G的StoARFs一般则是转录抑制子。研究证实, 缺失CTD结构域的拟南芥AtARF3并非与传统TIR1/AFB信号通路中的元件结合, 而是独立于TIR1/AFB受体发挥功能[32], 推测StoARFs中缺失CTD的成员, 尤其是与AtARF3同属同一进化分支并互为同源基因对的StoARF1可能也以不依赖生长素的方式发挥重要作用。
共线同源性结果显示Aux/IAA家族中分别存在35对AtIAA-StoIAA、37对MtIAA-StoIAA和74对GmIAA-StoIAA共线同源基因对, ARF家族中分别存在19对AtARF-StoARF、31对MtARF-StoARF和61对GmARF-StoARF共线同源基因对, 该结果表明决明与同属豆科植物的大豆和蒺藜苜蓿的亲缘关系比拟南芥更为密切。另外, 决明种内分别存在12对StoIAAs和6对StoARFs片段重复基因对, 表明决明在进化过程中不仅与其他豆科植物共享基因复制事件, 也不断进行自身的基因复制过程[33]
多数StoIAAsStoARFs在四个检测组织中都表现出不同的表达模式, 也有一些基因表现出了组织表达的特异性, 如StoIAA15StoARF12StoARF9主要在根中表达, StoIAA4StoIAA12StoIAA23主要在叶片中表达。另外, 不论是IAAs还是ARFs, 互为片段重复基因对的基因虽然表达水平高低有别, 但组织表达模式基本一致, 比如StoIAA1在叶片中具有丰富的表达, 但在其他三个检测的组织中表达量几乎可以忽略, 而与StoIAA1具有最近同源关系且互为片段重复基因对的StoIAA18同样特异性地在叶片组织中表达, 其他重复基因对StoIAA3StoIAA15StoIAA11StoIAA26StoIAA12StoIAA25StoARF1StoARF23StoARF5StoARF6StoARF1StoARF23等也表现出非常相似的组织表达模式, 这也表明这些基因复制很可能源自基因组重复事件。
StoIAAsStoARFs的启动子区存在许多与激素调节或胁迫响应相关的顺式元件。盐胁迫条件下, 决明根中StoARF2StoARF8StoARF10StoARF19StoARF22以及超过一半的StoIAA在处理24 h内表达显著上调; 干旱处理下, StoARF4StoARF8StoARF10StoARF17StoIAA2StoIAA5StoIAA12StoIAA13StoIAA17StoIAA23StoIAA24StoIAA25等表达在处理前中期上调明显。这些基因中多含有与胁迫响应相关的顺式元件MBS、TC-rich repeat等。值得注意的是, 决明与蒺藜苜蓿很多存在同源进化关系的IAAsARFs基因在响应这两种胁迫时表达趋势具有高度的相似性[25], 说明启动子区的保守性也与进化关系有统一的联系。除此之外, 许多其他物种的IAAsARFs在干旱和盐等其他胁迫下也被显著诱导表达, 比如拟南芥AtIAA7[34], 水稻OsIAA20OsARF16[35], 二穗短柄草BdARF5BdARF12[29], 高粱SbIAA1SbIAA26SbARF3[36]等。总之, StoIAAsStoARFs响应干旱、盐胁迫的表达结果充分证明了这些基因在参与决明应对和适应非生物胁迫过程中发挥了重要作用。
蛋白质相互作用对于研究许多生理过程十分重要, 如信号转导和基因表达调控。植物生长素响应是由ARF和Aux/IAA蛋白之间的相互作用介导的[4]。在拟南芥Aux/IAA和ARF大规模的互作研究中, 发现所有蛋白成员之间均存在着复杂的互作关系[37]。本研究通过蛋白互作网络分析, 得到了16个StoIAAs和12个StoARFs之间152条特异性互作关系。结合前期的结构分析、转录水平结果, 选择了StoARF8和StoARF10这两个转录激活子, 通过酵母双杂交技术, 验证它们与10个不存在自激活作用的StoIAAs蛋白的互作关系。在20个组合中鉴定到10个较强的StoARFs-StoIAAs互作, 10个互作较弱, 与拟南芥中的研究具有一致性[37], 说明不同的StoARFs和StoIAAs的互作强度不同。StoARF8和StoARF10均含有CTD结构域, 为其与StoIAAs互作提供结构基础, 但StoIAA10和StoIAA27缺少结构域Ⅰ和Ⅳ, 即PB1结构域中只含有结构域Ⅲ, 却也可以与StoARF8、StoARF10发生互作, 说明PB1结构域缺失甚至不含有PB1结构域的Aux/IAA蛋白, 也可能具有与ARF互作的功能, 这种现象在山核桃Aux/IAA和ARF家族蛋白互作关系鉴定中同样存在[38], 具体机制尚不清楚。
环境和遗传是中药材品质形成的两大重要因素, 遗传因素通过决定“代谢特质”与“形态特征”对药材品质形成发挥主导作用, 环境因子通过调控基因时空表达间接影响药材品质[39]。药用植物生长发育过程自身涉及一系列细胞分化、物质转运、激素信号转导及转录调控等生命活动。本文基于决明全基因组注释和结构信息, 对决明Aux/IAAARF家族的功能基因进行发掘和鉴定, 为后续研究决明次生代谢产物合成与积累相关基因的调控网络奠定基础。
作者贡献: 冯昭是本研究的实验设计执行人并撰写论文初稿; 刘世鹏、吕瑞华负责实验和数据分析; 吕蕊花、胡晓晨、张明英参与数据分析和作图; 张岗和毛仁俊是项目的构思者及负责人, 指导实验、论文写作和修改。全体作者都已阅读并同意最终的文本。
利益冲突: 无任何利益冲突。
  • 陕西省教育厅专项科研计划项目(23JK0401)
  • 陕西省教育厅专项科研计划项目(21JK0989)
  • 陕西中医药大学校级科研课题(2021GP07)
  • 陕西省自然科学基金(2021JQ-636)
  • 陕西中医药大学学科创新团队(2019-QN01)
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2024年第59卷第3期
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doi: 10.16438/j.0513-4870.2023-0666
  • 接收时间:2023-05-25
  • 首发时间:2025-11-28
  • 出版时间:2024-03-12
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  • 收稿日期:2023-05-25
  • 修回日期:2023-08-15
基金
陕西省教育厅专项科研计划项目(23JK0401)
陕西省教育厅专项科研计划项目(21JK0989)
陕西中医药大学校级科研课题(2021GP07)
陕西省自然科学基金(2021JQ-636)
陕西中医药大学学科创新团队(2019-QN01)
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, 陕西省中医药管理局"秦药"研发重点实验室, 陕西 咸阳 712046
    3.延安大学生命科学学院, 陕西 延安 716000

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*毛仁俊, Tel: 86-911-2332030, E-mail: ;
张岗, Tel: 86-29-38185165, E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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