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Article(id=1198624306746982753, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198624302414263267, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-0764, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1655913600000, receivedDateStr=2022-06-23, revisedDate=1658937600000, revisedDateStr=2022-07-28, acceptedDate=null, acceptedDateStr=null, onlineDate=1763703904091, onlineDateStr=2025-11-21, pubDate=1676131200000, pubDateStr=2023-02-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763703904091, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763703904091, creator=13701087609, updateTime=1763703904091, updator=13701087609, issue=Issue{id=1198624302414263267, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='2', pageStart='235', pageEnd='468', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763703903058, creator=13701087609, updateTime=1763704055811, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1198624943157116946, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198624302414263267, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198624943161311251, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198624302414263267, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=454, endPage=464, ext={EN=ArticleExt(id=1198624307048972655, articleId=1198624306746982753, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Superoxide dismutase (SOD) is a key enzyme that scavenge superoxide anion free radical (O2·-) in vivo, and plays an important role in plant growth and development and stress. In this study, according to the genome and transcriptome data of Salvia miltiorrhizae, 9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed, including 5 Cu/Zn-SOD, 2 Fe-SOD and 2 Mn-SOD. On the basis of proteomic analysis, combined with transcriptome data, one full-length cDNA of Mn-SOD gene, namely SmMSD2 was cloned from Salvia miltiorrhizae. The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase (Mn-SOD) subfamily, and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain "DVWEHAYY". The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E. coli BL21 expressing strain, and the target recombinant protein was successfully induced and its enzymatic properties were analyzed. Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues, indicating that SmMSD2 gene was constitutively expressed at a stable level. Real-time quantitative PCR indicated that drought (15% PEG6000), abscisic acid (ABA) and indole-3-acetic acid (IAA) could induce the expression of SmMSD2 gene, suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought, as well as the signaling pathways of phytohormone ABA and IAA. These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.

, correspAuthors=Hua-sheng PENG, Ye SHEN, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2023 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=Jia-ming PENG, Ren-jun QU, Shi-wei WANG, Xin-xin WANG, Liang-ping ZHA, Hua-sheng PENG, Ye SHEN), CN=ArticleExt(id=1198624310127591927, articleId=1198624306746982753, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=丹参超氧化物歧化酶SmMSD2基因的克隆与表达分析, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

超氧化物歧化酶(superoxide dismutase, SOD) 是清除生物体内超氧阴离子自由基(O2·-) 的关键酶, 在植物生长发育和逆境胁迫中发挥重要的作用。本研究基于丹参基因组和转录组数据, 共挖掘出9条SOD基因, 分别为5个Cu/Zn-SOD、2个Fe-SOD和2个Mn-SOD, 并对9条基因进行了生物信息学和基因表达模式分析。结合蛋白质组学和转录组数据, 从丹参中克隆得到SmMSD2的全长cDNA, 根据氨基酸多重序列比对和系统进化树分析, SmMSD2属于超氧化物歧化酶的锰离子亚族, 且C端含有保守的金属结合结构域“DVWEHAYY”。构建原核表达重组载体pMAL-c2X-SmMSD2, 成功诱导表达出重组蛋白, 并对其进行了酶学性质分析。时空表达分析表明, 该基因在各个组织中均有表达, 为组成表达型基因。实时荧光定量PCR结果显示模拟干旱(15% PEG6000)、脱落酸(abscisic acid, ABA) 和吲哚-3-乙酸(indole-3-acetic acid, IAA) 均能够诱导丹参SmMSD2基因表达, 推测SmMSD2可能参与丹参响应干旱等非生物胁迫, 以及植物激素ABA和IAA的信号传导途径, 为进一步阐明超氧化物歧化酶参与丹参逆境响应及有效活性成分的积累奠定了基础。

, correspAuthors=彭华胜, 申业, authorNote=null, correspAuthorsNote=
*彭华胜, E-mail: ;
申业, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2023, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=JkIfdQGt4jGloa//DhLpGQ==, magXml=RcrFKiin7Op/ksrCb9TeDA==, pdfUrl=null, pdf=WloXYyUfVjs3Up5jtapH+A==, pdfFileSize=4147573, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=If+/yyU+8XqGVV7ei/FyWw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=IB2HYasYwOf2sI60R/Pz7A==, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=彭佳铭, 屈仁军, 王世威, 王新新, 查良平, 彭华胜, 申业)}, authors=[Author(id=1198702066299400457, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1198702066458784030, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, authorId=1198702066299400457, language=EN, stringName=Jia-ming PENG, firstName=Jia-ming, middleName=null, lastName=PENG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
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School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
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Biomolecules, 2020, 10: 281., articleTitle=Auxin-abscisic acid interactions in plant growth and development, refAbstract=null)], funds=[Fund(id=1198702077485608981, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, awardId=CI2021A04102, language=CN, fundingSource=中国中医科学院科技创新工程重大攻关项目(CI2021A04102), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1198702065934495974, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, xref=null, ext=[AuthorCompanyExt(id=1198702065938690281, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, companyId=1198702065934495974, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China), AuthorCompanyExt(id=1198702065951273196, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, companyId=1198702065934495974, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.安徽中医药大学药学院, 安徽 合肥 230012)]), AuthorCompany(id=1198702066081296630, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, xref=null, ext=[AuthorCompanyExt(id=1198702066089685239, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, companyId=1198702066081296630, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2. State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China), AuthorCompanyExt(id=1198702066098073848, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, companyId=1198702066081296630, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.中国中医科学院中药资源中心, 道地药材国家重点实验室培育基地, 北京 100700)])], figs=[ArticleFig(id=1198702073698153271, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=8oR2Ok3KAZHH2TVz6dOkuA==, figureFileBig=bPCg/dp+yQnF86rIdjYMGQ==, tableContent=null), ArticleFig(id=1198702073811399491, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 1, caption= Neighbor-joining phylogenetic tree, conserved domain and motif analysis of SmSOD proteins. A: The phylogenetic tree was generated based on the amino acid sequences of SmSOD proteins; B: Conserved domain analysis of SmSOD proteins; C: Conserved motif analysis of SmSOD proteins. Different color boxes represent different types of motifs and domains; D: Amino acid sequences of conserved motif , figureFileSmall=8oR2Ok3KAZHH2TVz6dOkuA==, figureFileBig=bPCg/dp+yQnF86rIdjYMGQ==, tableContent=null), ArticleFig(id=1198702074004337491, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=w8ZxGKEM0j7ZPjL61AZh7w==, figureFileBig=ztCsCElRf4G7g3SvG0RA3g==, tableContent=null), ArticleFig(id=1198702074167915359, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 2, caption= The heat maps of <i>SmSOD</i> gene expression in <i>Salvia miltiorrhiza</i> in response to YE+Ag<sup>+</sup> (A), YE and MeJA (B) , figureFileSmall=w8ZxGKEM0j7ZPjL61AZh7w==, figureFileBig=ztCsCElRf4G7g3SvG0RA3g==, tableContent=null), ArticleFig(id=1198702074331493232, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=LBlw1uZd59SgKJUfsyiHkA==, figureFileBig=3qUltCe65Thi/KmuhtVP0w==, tableContent=null), ArticleFig(id=1198702074499265399, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 3, caption= Alignment of the deduced amino acid sequence of SmMSD2 with MSDs from other plants. Black shading indicates amino acid identities, red and blue shading indicates amino acid with different similarity. The conserved domain of SmMSD2 protein are shown with red boxes. At: <i>Arabidopsis thaliana</i>; AtMSD1: AT3G10920. Os: <i>Oryza sativa</i>; OsMSD: NP001389439. Zm: <i>Zea mays</i>; ZmMSD: NP00110 5742 , figureFileSmall=LBlw1uZd59SgKJUfsyiHkA==, figureFileBig=3qUltCe65Thi/KmuhtVP0w==, tableContent=null), ArticleFig(id=1198702074608317320, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=fjCbm3ZWye8JlR1Be642dg==, figureFileBig=h5TS9K3vX/7+1eb9S7GITQ==, tableContent=null), ArticleFig(id=1198702074797061017, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 4, caption= Phylogenetic analysis of SOD proteins from plants. At: <i>Arabidopsis thaliana</i>; Os: <i>Oryza sativa</i>; Zm: <i>Zea mays</i>; Gh: <i>Gossypium hirsutum</i>; Mp: <i>Marchantia paleacea</i>; Pt: <i>Populus trichocarpa</i> , figureFileSmall=fjCbm3ZWye8JlR1Be642dg==, figureFileBig=h5TS9K3vX/7+1eb9S7GITQ==, tableContent=null), ArticleFig(id=1198702074918695841, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=w5+qi6SGD5FULwaT04bQMQ==, figureFileBig=v0C2BOxhmxIReYj08rVsCA==, tableContent=null), ArticleFig(id=1198702075115828142, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 5, caption= (A) The predicted three-dimensional structure of SmMSD2 protein and (B) detail of the active sites. Single Mn<sup>2+</sup> ion (purple sphere) made polar contacts with histidine 50 (H50), histidine 98 (H98), aspartic acid (D191) and histidine 195 (H195) , figureFileSmall=w5+qi6SGD5FULwaT04bQMQ==, figureFileBig=v0C2BOxhmxIReYj08rVsCA==, tableContent=null), ArticleFig(id=1198702075304571839, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=pVN3ig6YPaMtDne0/DmIhQ==, figureFileBig=zmHMxXb7PC28VJs3QywG7w==, tableContent=null), ArticleFig(id=1198702075514287050, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 6, caption= SDS-PAGE analysis of MBP-SmMSD2 recombinant protein. M: Protein Marker; 1: Uninduced <i>E. coli</i> containing pMAL-c2X; 2-4: Induced <i>E. coli</i> containing pMAL-c2X for 2, 4 and 6 h, respectively; 5: Uninduced <i>E. coli</i> containing MBP-SmMSD2; 6-8: Induced <i>E. coli</i> containing MBP-SmMSD2 for 2, 4 and 6 h, respectively; 9: Supernatant from the induced <i>E. coli</i> containing MBP-SmMSD2 , figureFileSmall=pVN3ig6YPaMtDne0/DmIhQ==, figureFileBig=zmHMxXb7PC28VJs3QywG7w==, tableContent=null), ArticleFig(id=1198702075686253525, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=HjCIyS+AQc6Z09iOdqdvsg==, figureFileBig=kaVhyzw2Ep/OzklGYkLk0g==, tableContent=null), ArticleFig(id=1198702076898407396, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 7, caption= Effects of temperature and pH on SmMSD2 activity, and thermal stability analysis of SmMSD2. A: The temperature profiles were determined by assaying the activity of SmMSD2 at temperatures ranging from 30 to 80 ℃. The activity at the optimum temperature was defined as 100%; B: The activity of SmMSD2 was evaluated in buffers ranging from pH 2.0 to 12. The activity at the optimum pH was defined as 100%; C: Thermal stability analysis of SmMSD2. The activity of non -heated SmMSD2 was defined as 100%. Each point represents the mean ± SD (<i>n</i> = 3) , figureFileSmall=HjCIyS+AQc6Z09iOdqdvsg==, figureFileBig=kaVhyzw2Ep/OzklGYkLk0g==, tableContent=null), ArticleFig(id=1198702077032625136, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=EN, label=null, caption=null, figureFileSmall=q+uwG1sUWVUK0ME+Kw0l8w==, figureFileBig=lEZW8/ALTBdt27OsBoWKdw==, tableContent=null), ArticleFig(id=1198702077179425792, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198624306746982753, language=CN, label=Figure 8, caption= A: Relative expression level of <i>SmMSD2</i> gene in difference tissues. The expression level of <i>SmMSD2</i> gene in root was set to 1, with <i>Actin1</i> used as the internal standard control. B, C, D: Relative expression level of <i>SmMSD2</i> gene treated with 15% PEG6000 (B), abscisic acid (ABA) (C), indole-3-acetic acid (IAA) (D) in <i>S. miltiorrhiza</i> seedlings, respectively. The expression level of <i>SmMSD2</i> gene at 0 h was set to 1, with <i>Actin1</i> used as the internal standard control. Data are shown as the mean ± SD (<i>n</i> = 3), <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01, <sup>***</sup><i>P</i> < 0.001, <sup>****</sup><i>P</i> < 0.000 1 <i>vs</i> control , figureFileSmall=q+uwG1sUWVUK0ME+Kw0l8w==, figureFileBig=lEZW8/ALTBdt27OsBoWKdw==, tableContent=null)], attaches=null, journal=Journal(id=1189982048455397383, delFlag=0, nameCn=药学学报, nameEn=Acta Pharmaceutica Sinica, nameHistory1=null, nameHistory2=null, issn=0513-4870, eissn=null, cn=11-2163/R, coden=null, periodic=0, language=CN, oaType=null, ccby=null, superviseOffice=null, ownerOffice=null, pubOffice=null, editorOffice=null, officeType=null, aims=null, clcCode=null, officeProv=null, officeCity=null, officeAddr=null, officeZip=null, officeEmail=null, officePhone=null, editDirector=null, officeDirector=null, officeDirectorPhone=null, officeStaffNum=null, officeEmpNum=null, coverPicUrl=BTxjudbJDVO4PqdBR6On6Q==, journalPrice=null, startedYear=null, abbrevIsoEn=null, journalRemark=null, 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丹参超氧化物歧化酶SmMSD2基因的克隆与表达分析
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彭佳铭 1, 2 , 屈仁军 2 , 王世威 2 , 王新新 2 , 查良平 1 , 彭华胜 1, 2, * , 申业 2, *
药学学报 | 研究论文 2023,58(2): 454-464
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药学学报 | 研究论文 2023, 58(2): 454-464
丹参超氧化物歧化酶SmMSD2基因的克隆与表达分析
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彭佳铭1, 2, 屈仁军2, 王世威2, 王新新2, 查良平1, 彭华胜1, 2, * , 申业2, *
作者信息
  • 1.安徽中医药大学药学院, 安徽 合肥 230012
  • 2.中国中医科学院中药资源中心, 道地药材国家重点实验室培育基地, 北京 100700

通讯作者:

*彭华胜, E-mail: ;
申业, E-mail:
Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza
Jia-ming PENG1, 2, Ren-jun QU2, Shi-wei WANG2, Xin-xin WANG2, Liang-ping ZHA1, Hua-sheng PENG1, 2, * , Ye SHEN2, *
Affiliations
  • 1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
  • 2. State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
出版时间: 2023-02-12 doi: 10.16438/j.0513-4870.2022-0764
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超氧化物歧化酶(superoxide dismutase, SOD) 是清除生物体内超氧阴离子自由基(O2·-) 的关键酶, 在植物生长发育和逆境胁迫中发挥重要的作用。本研究基于丹参基因组和转录组数据, 共挖掘出9条SOD基因, 分别为5个Cu/Zn-SOD、2个Fe-SOD和2个Mn-SOD, 并对9条基因进行了生物信息学和基因表达模式分析。结合蛋白质组学和转录组数据, 从丹参中克隆得到SmMSD2的全长cDNA, 根据氨基酸多重序列比对和系统进化树分析, SmMSD2属于超氧化物歧化酶的锰离子亚族, 且C端含有保守的金属结合结构域“DVWEHAYY”。构建原核表达重组载体pMAL-c2X-SmMSD2, 成功诱导表达出重组蛋白, 并对其进行了酶学性质分析。时空表达分析表明, 该基因在各个组织中均有表达, 为组成表达型基因。实时荧光定量PCR结果显示模拟干旱(15% PEG6000)、脱落酸(abscisic acid, ABA) 和吲哚-3-乙酸(indole-3-acetic acid, IAA) 均能够诱导丹参SmMSD2基因表达, 推测SmMSD2可能参与丹参响应干旱等非生物胁迫, 以及植物激素ABA和IAA的信号传导途径, 为进一步阐明超氧化物歧化酶参与丹参逆境响应及有效活性成分的积累奠定了基础。

丹参  /  超氧化物歧化酶  /  多组学分析  /  表达分析  /  非生物胁迫

Superoxide dismutase (SOD) is a key enzyme that scavenge superoxide anion free radical (O2·-) in vivo, and plays an important role in plant growth and development and stress. In this study, according to the genome and transcriptome data of Salvia miltiorrhizae, 9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed, including 5 Cu/Zn-SOD, 2 Fe-SOD and 2 Mn-SOD. On the basis of proteomic analysis, combined with transcriptome data, one full-length cDNA of Mn-SOD gene, namely SmMSD2 was cloned from Salvia miltiorrhizae. The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase (Mn-SOD) subfamily, and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain "DVWEHAYY". The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E. coli BL21 expressing strain, and the target recombinant protein was successfully induced and its enzymatic properties were analyzed. Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues, indicating that SmMSD2 gene was constitutively expressed at a stable level. Real-time quantitative PCR indicated that drought (15% PEG6000), abscisic acid (ABA) and indole-3-acetic acid (IAA) could induce the expression of SmMSD2 gene, suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought, as well as the signaling pathways of phytohormone ABA and IAA. These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.

Salvia miltiorrhiza  /  superoxide dismutase  /  multiple omics analysis  /  expression analysis  /  abiotic stress
彭佳铭, 屈仁军, 王世威, 王新新, 查良平, 彭华胜, 申业. 丹参超氧化物歧化酶SmMSD2基因的克隆与表达分析. 药学学报, 2023 , 58 (2) : 454 -464 . DOI: 10.16438/j.0513-4870.2022-0764
Jia-ming PENG, Ren-jun QU, Shi-wei WANG, Xin-xin WANG, Liang-ping ZHA, Hua-sheng PENG, Ye SHEN. Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza[J]. Acta Pharmaceutica Sinica, 2023 , 58 (2) : 454 -464 . DOI: 10.16438/j.0513-4870.2022-0764
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生物体在胁迫或非胁迫状态时, 细胞都能产生活性氧(reactive oxygen species, ROS), 活性氧物质是氧代谢的副产物, 包括超氧阴离子O2·-、单线态氧1O2、羟基自由基·OH和过氧化氢H2O2。通常生物体内完善的防御系统会限制ROS形成或将其去除, 如果胁迫产生过量的ROS没有被有效的消除, 会引发一系列生化反应, 导致DNA损伤、蛋白质失活和生物膜被破坏, 严重时导致细胞功能紊乱甚至凋亡[1]。作为生物体中酶类抗氧化防御系统(antioxidant defense system) 中的第一个酶——超氧化物歧化酶(superoxide dismutase, SOD) 广泛存在于动物、植物及微生物中, 主要催化有害的超氧自由基(O2·-) 通过歧化反应转化为分子氧(O2) 和过氧化氢(H2O2) 来保护细胞[1, 2]。SOD作为一种金属酶, 根据其结合金属辅因子的不同可分为4种类型, 分别为Cu/Zn-SOD、Fe-SOD、Mn-SOD及Ni-SOD; 其中, Cu/Zn-SOD、Fe-SOD和Mn-SOD存在于植物体内, 而Ni-SOD尚未在植物体内发现, 但在某些原核生物中存在[3]。不同种类的SOD定位于细胞的不同区室, Fe-SOD主要存在叶绿体、过氧化物酶体和线粒体中, Mn-SOD主要存在于线粒体内和过氧化物酶体中, 而Cu/Zn-SOD存在叶绿体、胞质或细胞外间隙。SOD酶的数量、类型和在细胞中的定位依据物种、发育阶段和环境条件的不同而变化[4]。
Mn-SODs是由约23 kDa的亚基组成同源二聚体或同源四聚体, 每个亚基只能结合一个Mn原子, 如果没有Mn原子, Mn-SODs就没有活性。Mn-SODs活性不能被氰化物或H2O2抑制, 且Mn-SOD基因在所有类型的细胞中都有表达[4]。Mn-SODs通常定位于线粒体, 而线粒体是呼吸链的氧化还原中心, 不可避免地产生ROS, 但与叶绿体和过氧化物酶体相比, 线粒体产生的ROS数量相当少, 微量的ROS在氧化还原信号中发挥作用以调节植物代谢和发育, 线粒体定位的Mn-SODs在线粒体, 乃至细胞中的氧化还原内稳态中的作用仍有待阐明[5]。近年来从许多植物中克隆Mn-SOD基因, 研究表明Mn-SOD与植物抗逆性密切相关[6]。硬质小麦的TdMnSOD异源转化到拟南芥中, 转TdMnSOD的拟南芥在盐、渗透、氧和重金属胁迫后, 总SOD、Mn-SOD、CAT和POD等酶的活性都不同程度地比野生型要高, 且表现出对盐、干旱、氧和重金属胁迫的抗性[7]。过表达棉花TaMnSOD基因株系在干旱胁迫下, 显示了比野生型高的SOD和POD酶活性, 同时, 能维持较低的MDA含量(MDA含量, 即丙二醛含量, 表示膜脂过氧化程度), 从而提高了转基因棉花的耐旱性[8]。
中药丹参为唇形科鼠尾草属植物丹参Salvia miltiorrhiza Bge.的干燥根和根茎, 其有效成分为脂溶性的丹参酮二萜类化合物和水溶性的丹酚酸类化合物, 具有抗炎、免疫调节和抗氧化活性, 临床上用于治疗冠心病、心肌梗死、高血压、高血脂等心血管疾病[9, 10]。对于营固生生活的植物, 胁迫会不同程度的影响其生长和发育, 而适度的胁迫又会引起植物次生代谢物质的积累。生物胁迫诱导子(yeast extract, YE) 和非生物胁迫诱导子(Ag+) 诱导丹参毛状根, 会引起丹参酮ⅡA、二氢丹参酮、隐丹参酮等含量不同程度增加[11-13], 比较蛋白质组分析结果推测丹参酮类物质增加的原因可能是YE和Ag+引起了ROS的爆发, 激活了Ca2+/钙调蛋白信号传导通路, 最后使碳流转到合成次生代谢产物丹参酮的生物合成[14]。生物体内微量的ROS物质能参与体内的信号通路传导, 调控体内生理生化变化; 然而过量的ROS会引起细胞损伤, 严重的会导致死亡。本课题组在YE+Ag+诱导蛋白质组学中, 检测到2个不同表达模式的SOD, 推测它们可能行使了不同的功能。全基因组分析发现丹参SOD基因家族有8个成员, 表达谱分析显示丹参的SODs可能参与了对冷、盐、干旱等非生物胁迫和脱落酸(abscisic acid, ABA)、茉莉酸甲酯(methyl jasmonate, MeJA) 等植物激素的响应[15]。不同浓度盐(NaCl) 处理丹参后, 检测叶片中SOD酶的活性, 发现随着NaCl浓度的增加, SOD酶的活性是下降的[16]。胁迫诱导后, 丹参SOD家族在基因表达模式和蛋白质层面不同的研究结果, 引起了进一步探究超氧化物歧化酶在丹参有效成分积累和非生物胁迫响应过程中的功能的兴趣。
实验室前期在YE+Ag+诱导丹参毛状根蛋白质组中发现SOD蛋白在诱导后大量积累, 结合丹参基因组和转录组数据, 本研究挖掘了丹参SOD家族基因, 共发现了9个SODs基因, 对丹参SOD家族基因进行了生物信息学分析; 依据已经发表的转录组数据, 对丹参SOD家族基因的表达模式进行了分析; 克隆了丹参SmMSD2全长cDNA, 并预测了其蛋白结构; 构建了pMAL-c2X-SmMSD2原核表达的重组载体, 转化大肠杆菌BL21中并成功诱导表达出重组蛋白, 并对其进行了酶学性质分析; 通过实时荧光定量PCR方法检测了丹参SmMSD2的器官特异性表达, 以及模拟干旱非生物胁迫、外源植物激素ABA和吲哚-3-乙酸(indole-3-acetic acid, IAA) 诱导下的表达情况, 为进一步研究SmMSD2在参与丹参胁迫防御和活性成分积累的机制奠定基础。
材料及方法
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植物材料  丹参种苗采自北京市延庆区艾草园, 由中国中医科学院中药资源中心袁庆军研究员鉴定后, 采集其根、茎、叶及花提取总RNA, 检测SmMSD2基因在不同器官中的特异性表达。紫花丹参种子为本实验室保存, 于2021年8月播种萌发, 整个过程在人工气候室培养, 出苗后选取长势相同的丹参幼苗, 分别经15% PEG6000、50 μmol·L-1 ABA和2 μmol·L-1 IAA处理0、2、4、8、12、24 h后, 取根, 液氮保存, 提取RNA作为样品, 检测SmMSD2基因在各种处理下的表达差异。
菌株、载体和试剂  原核表达载体pMAL-c2X载体为本实验室保存; 所用菌株为大肠杆菌感受态细胞Trans1-T1和BL21、胶回收试剂盒EasyPure® Quick Gel Extraction Kit、T载体pEASY®-Blunt Zero Cloning Kit、无缝拼接试剂盒pEASY®-Uni Seamless Cloning and Assembly Kit均购自北京全式金生物技术有限公司; RNAprep Pure植物总RNA提取试剂盒购于天根生化科技(北京) 有限公司; 反转录试剂盒PrimeScriptTM Ⅱ 1st Strand cDNA Synthesis Kit、PrimeScriptTM RT reagent Kit with gDNA Eraser (Prefect Real Time) 及荧光定量试剂盒TB Green® Premix Ex TaqTM Ⅱ、PrimeSTAR® HS DNA Polymerase高保真酶购自TaKaRa公司; 限制性核酸内切酶EcoRⅠ购于NEB公司; SOD活性检测试剂盒购自北京索莱宝科技有限公司; 卡那青霉素、氨苄青霉素、β-巯基乙醇、异丙基-β-D-硫代半乳糖苷(IPTG) 等化学试剂购自Sigma公司; 其他生化试剂均为国产分析纯; 引物合成由上海生工生物工程股份有限公司完成。
总RNA的提取及cDNA的合成  按照TIANGEN RNAprep Pure植物总RNA提取试剂盒说明书进行丹参总RNA提取, 利用NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) 检测总RNA的浓度及A260 nm/A280 nm, 同时采用1%琼脂糖凝胶电泳检测总RNA的完整性和质量; 使用TaKaRa反转录试剂盒将丹参总RNA反转录为cDNA, 并保存于-20 ℃冰箱。
丹参SmMSD2基因序列全长克隆  用Primer Premier 5.0软件设计基因特异性引物, 上游引物SmMSD2-F: 5'-ATGGCGCTCCGAACCC-3', 下游引物SmMSD2-R: 5'-CTAATTCGCATTCTCTTTATCAAAA GCATC-3'。以反转录的cDNA为模板, 按照下列反应体系对丹参中SmMSD2基因cDNA进行扩增: 2 μL cDNA, 10 μL 5×PS Buffer, 4 μL dNTP Mix, 0.5 μL PrimeSTAR高保真酶, 10 μmol·L-1引物各1 μL, 终体积为50 μL。PCR反应程序: 98 ℃预变性2 min; 然后98 ℃变性10 s, 55 ℃退火15 s, 72 ℃延伸1 min, 进行35个循环, 72 ℃终延伸反应7 min, 16 ℃保存。1%的琼脂糖凝胶电泳检测PCR产物, 目的片段回收和纯化按照EasyPure® Quick Gel Extraction Kit胶回收试剂盒进行。将回收的特异性扩增片段克隆pEASY®-Blunt Zero载体中, 转化至大肠杆菌Trans1-T1感受态细胞中, 在卡那抗性的平板上进行筛选, 挑取单克隆菌斑进行菌落PCR验证, 将阳性克隆菌液送至北京睿博兴科生物技术有限公司测序。
丹参SmMSD2生物信息学分析  利用NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) 进行SmSOD蛋白质的保守结构域分析; MEME Suit (https://meme-suite.org/meme/) 进行蛋白质保守基序分析; 利用ExPASy Proteomics Server在线工具ProtParam (https://web.expasy.org/protparam/) 进行SmSOD基因编码蛋白的理化性质分析; 利用ProtComp 9.0 (http://linux1.softberry.com) 进行蛋白亚细胞定位分析; 采用SWISS-MODEL (https://swissmodel.expasy.org) 进行SmMSD2蛋白质三维建模分析; 将所获得的SmMSD2基因编码的氨基酸序列在GenBank数据库中进行Blast P比对分析, 利用DNAMAN对其他物种Mn-SOD基因编码的氨基酸序列进行同源性分析; 通过MEGA 11.0软件构建Neighbor-joining系统进化树, 进化距离的计算采用泊松校正法, Bootstrap重复次数为1 000次。
丹参SmMSD2原核表达载体的构建  采用无缝拼接的方法构建pMAL-c2X-SmMSD2原核表达载体, 以EcoRI限制性内切酶作为酶切位点, 设计引物, 上游引物pMAL-c2X-SmMSD2-F: 5'-gaaggatttcagaattcATG GCGCTCCGAAC-3' (小写字母划线处为EcoRI酶切位点); 下游引物pMAL-c2X-SmMSD2-R: 5'-gtcgactct agaggatccCTAATTCGCATTCTC-3'。以pEASY-Blunt-SmMSD2质粒为模板进行扩增, 切胶回收PCR产物, 用EcoRI对表达载体pMAL-c2X质粒进行酶切, 并切胶回收, 将回收后的目的片段与表达载体采用全式金pEASY®-Uni Seamless Cloning and Assembly Kit无缝拼接试剂盒50 ℃连接30 min, 将连接产物转化至大肠杆菌Trans1-T1感受态细胞中, 在含氨苄青霉素的LB培养板(工作浓度为100 μg·L-1) 上, 37 ℃培养过夜。挑取单克隆进行菌液PCR检测、测序及提取质粒pMAL-c2X-SmMSD2。
丹参SmMSD2的原核表达  将原核表达载体pMAL-c2X-SmMSD2质粒转化至大肠杆菌BL21中, 挑取单菌落接种于含有氨苄青霉素的LB液体培养基中, 37 ℃活化过夜; 然后按照1∶100比例稀释到新鲜的含有氨苄青霉素的LB液体培养基中, 37 ℃振荡培养至A600 nm为0.4~0.6, 加入IPTG (终浓度为0.5 mmol·L-1) 后37 ℃振荡培养2、4、6 h。取相应量的菌液4 ℃、1 000 ×g离心1 min弃上清, 加入50 μL的1× SDS-PAGE上样缓冲液, 混匀, 获得含重组SmMSD2蛋白的全菌点样液; 剩余菌液离心去上清收集菌体, 加入4 mL PBS缓冲液重悬菌体, 将重悬后的菌体置于冰水浴中, 利用超声破碎仪破碎细胞, 然后4 ℃、1 000 ×g离心20 min获得SmMSD2的蛋白上清, 取20 μL上清液, 加入5 μL的5× SDS-PAGE上样缓冲液, 混匀, 获得SmMSD2上清点样液。将上述点样液沸水煮5 min后离心, 进行SDS-PAGE蛋白电泳检测。
丹参SmMSD2酶活力及酶学性质分析  利用SOD活性检测试剂盒进行酶活力实验, 本试剂盒采用黄嘌呤氧化酶法测定超氧化物歧化酶的活力, 黄嘌呤及黄嘌呤氧化酶反应系统产生超氧阴离子(O2-), O2-还原氮蓝四唑生成的蓝色甲臜在560 nm处有吸收, 可用可见分光光度计测其吸光度, 将最高的酶活力定义为100%。将含重组SmMSD2蛋白的菌液离心后弃上清, 加入相应体积的提取液, 超声破碎, 4 ℃、8 000 ×g离心10 min, 取上清, 置冰上待测。在不同温度梯度下(30、40、50、60、70、80 ℃) 作用10 min, 分别测定酶活力, 以确定SmMSD2蛋白的最适温度。将酶液置于不同温度条件(50、60、70 ℃) 下作用不同时间(0、10、20、40、60、80 min), 分别测SmMSD2相对剩余酶活力, 以此检测该酶的热稳定性。不同pH条件下25 ℃反应30 min后测定相对剩余酶活力, 检测pH值对该酶活性的影响, 缓冲液pH值范围从2.0到12, 柠檬酸-柠檬酸钠缓冲液pH 2.0~6.0, 磷酸氢二钾-磷酸二氢钾缓冲液pH 7.0, Tris-HCl缓冲液pH 8.0, 甘氨酸-氢氧化钠缓冲液pH 9.0~10, 磷酸氢二钠-氢氧化钠缓冲液pH 11, 氯化钾-氢氧化钠缓冲液pH 12。
丹参SmMSD2基因在不同器官和不同胁迫处理下的表达分析  利用实时荧光定量PCR (quantitative real-time PCR, qRT-PCR) 的方法检测丹参SmMSD2基因在不同器官和不同胁迫处理下的表达情况。使用TB Green® Premix Ex TaqTMⅡ试剂盒, 在Light Cycle® 480Ⅱ实时荧光定量PCR仪(美国罗氏医学仪器公司) 上进行。选取丹参Actin1 (GeneBank Number: MW650863) 作为目标基因定量表达的内参基因, 设计内参基因引物, 上游引物SmActin-F: 5'-ATGCTCCAA GGGCT GTCT-3', 下游引物SmActin-R: 5'-CTCGTCG CCAACATAAGC-3'。利用Primer Premier 5.0软件设计丹参SmMSD2基因实时荧光定量PCR引物, 上游引物SmMSD2-qF: 5'-TGTTGGTGGTGGTGAGCC-3', 下游引物SmMSD2-qR: 5'-CCCA TCCTGAACCCTGTA-3'。每个样品设3个生物学重复, 反应体系中含有10 μL TB Green Premix Ex TaqⅡ, 上下游引物(10 μmol·L-1) 各0.4 μL, cDNA模板2 μL, 剩余用水补齐, 总体系为20 μL。反应程序是: 95 ℃预变性30 s; 95 ℃变性5 s, 60 ℃退火/延伸30 s, 40个循环; 95 ℃变性5 s, 60 ℃ 1 min, 95 ℃持续。根据融解曲线判断RT-PCR产物的特异性, 采用2-ΔΔCt方法分析SmMSD2的相对表达量。
结果与分析
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1 蛋白质组结合基因组筛选丹参SOD家族基因
1.1 丹参SOD家族基因的鉴定
实验室前期采用2-D二维蛋白质组学方法对YE+Ag+诱导丹参毛状根进行比较蛋白质组学分析, 发现有5种抗氧化蛋白响应YE+Ag+胁迫, 其中Isotig12128和Isotig17786为SOD家族蛋白[14]。依据2016年版的丹参基因组数据[17], 发现丹参基因组中含有8个SOD基因[15]。又对最新版2021年版丹参基因组数据[18]进行挖掘, 发现了9条序列被注释为SOD基因, 其中新挖掘出两个Cu/Zn-SOD基因, 将丹参9个SOD基因命名为SmCSD1-5、SmFSD1、SmFSD2、SmMSD1和SmMSD2, 因此丹参中的SOD家族含有5个Cu/Zn SOD、2个Fe-SOD和2个Mn-SOD基因。
1.2 丹参SOD家族蛋白保守基序预测、保守结构域和系统进化树分析
由NCBI Batch CD-Search分析结果(图 1) 可见, 5个SmCSDs均包含保守的Sod_Cu结构域(Pfam: PF00080), 此外SmCSD3还存在一个重金属相关保守结构域HMA (Pfam: PF00403), 每个HMA结构域结合一个Cu2+, 对Cu2+的结合与转运至关重要; SmFSDs和SmMSDs均含有N-末端Fe/Mn SOD-α-发夹结构域(Pfam: PF00081) 和C-末端Fe/Mn SOD结构域(Pfam: PF02777), 说明它们属于Fe/Mn-SOD亚家族, 且SmFSDs和SmMSDs在系统进化树中聚在同一分支, 进一步表明其亲缘关系较近。
采用MEME Suit网站分析SmSOD蛋白的保守基序(图 1C, D), 共鉴定了10个保守Motif, 其中Motif 1和6存在于所有的SmCSD蛋白中, 推测Motif 1和6可能与Cu/Zn保守结构域(PF00080) 相关。Motif 2、3、4、7和9存在于两个SmFSD蛋白中, Motif 2、3、5、8和10存在于两个SmMSD蛋白中, Fe-SOD和Mn-SOD均含有Motif 2和3, 推测Motif 2和3与C-末端Fe/MnSOD结构域(PF02777) 有关, 并且Motif 2包含保守的金属结合结构域“DVWEHAYY”; Motif 10存在于SmFSD2、SmMSD1和SmMSD2中, 但是SmFSD1没有, SmFSD1、SmFSD2和SmMSD2均包含Motif 4, 但是SmMSD1没有。
1.3 丹参SOD家族基因的生物信息学分析
运用在线工具ExPASy Proteomics对SmSOD家族基因编码蛋白的理化性质进行分析, 丹参9个SmSOD蛋白的分子质量为15.25~34.65 kDa, 理论等电点为5.23~8.65, 不稳定系数Ⅱ为18.42~42.70, SmCSD1-5、SmFSD1和SmMSD2均属于稳定蛋白, 蛋白质亚细胞定位软件ProtComp 9.0 server预测表明, SmCSD5蛋白定位于叶绿体, 其余4个Cu/Zn-SOD蛋白定位于细胞质, 2个Mn-SOD蛋白定位于线粒体, 2个Fe-SOD蛋白存在于叶绿体中。
2 转录组分析丹参SOD家族基因的表达模式
为了进一步研究SmSOD家族基因的表达模式, 首先分析了YE+Ag+诱导丹参毛状根转录组[11]中SOD的表达模式, 发现YE+Ag+诱导后SmSOD的表达水平均出现了变化, 但表达模式不同, SmCSD1、SmCSD3和SmCSD5的表达模式相似, 表达水平呈现先升高后降低的趋势, 在S2期表达水平最高; 在诱导后S1期SmCSD2、SmCSD4、SmFSD2、SmMSD1和SmMSD2表达下调, 但S1期后表达量呈现递增的趋势(图 2A)。接着, 分析了YE和MeJA诱导的丹参毛状根转录组[19], 发现2个SmMSD基因经MeJA诱导表达下调, SmFSD1和SmFSD2的表达模式不同, SmFSD1表达水平降低而SmFSD2表达水平先降低后升高; YE诱导1 h内6个SmSOD基因SmMSDs、SmFSDs、SmCSD1和SmCSD3的表达量降低, 随后表达量快速增加, 而SmCSD2、SmCSD4和SmCSD5表达下调(图 2B)。这些结果表明YE、YE+Ag+和MeJA均诱导SmSOD基因家族的表达。
3 丹参SmMSD2基因全长cDNA的克隆和氨基酸序列同源比对分析
在YE+Ag+诱导的丹参毛状根比较蛋白质组学分析中, 发现了2个SOD蛋白, 1个是SmCSD1, 它随YE+Ag+诱导时间增加, 含量逐渐降低, 而另一个SmMSD2在诱导24 h含量增加到最高, 随后含量逐渐降低, 推测SmMSD2可能参与了丹参对YE+Ag+胁迫的反应。本研究参照SmMSD2序列设计特异性扩增引物, 以丹参cDNA为模板进行RT-PCR扩增, 获得SmMSD2的全长开放阅读框。测序结果进行序列比对, 丹参SmMSD2开放阅读框为696 bp, 编码231个氨基酸。丹参SmMSD2基因序列已提交NCBI, 登录号是ON814503。为预测丹参SmMSD2的生物学功能, 利用DNAMAN软件将丹参SmMSD2的氨基酸序列与已经报道的拟南芥、水稻和玉米进行多重序列比对(图 3), 发现蛋白序列具有高度的保守性, 在C端均具有金属结合结构域“DVWEHAYY”, 且与金属Mn2+结合的组氨酸(H) 和天冬氨酸(D) 残基也相对保守, 说明SmMSD2属于Mn-SOD亚家族。
4 丹参SmMSD2系统进化树分析
与丹参SmMSD2氨基酸序列具有同源性的其他植物来源Mn-SOD蛋白的氨基酸序列以及Fe-SOD和Cu/Zn-SOD蛋白的氨基酸序列一起构建系统进化树, 进化树分析显示(图 4), 植物中的SOD蛋白分为3种类型, 第一类为Mn-SOD类蛋白, 丹参SmMSD2与拟南芥AtMSD1 (GenBank number: AT3G10920)、陆地棉GhMSD (GenBank number: ABA00455)、粗裂地钱MpMSD (GenBank number: BAD13494) 和毛果杨PtMSD (GenBank number: XP002319332) 聚类到一起, 说明其亲缘关系较近, 进一步证明SmMSD2属于Mn-SOD亚族; 第二类为Fe-SOD类蛋白, 进化树显示Mn-SOD和Fe-SOD蛋白在进化和结构上都密切相关, 这与之前的报道相一致, 即Mn-SOD和Fe-SOD起源于同一祖先; 第三类为Cu/Zn-SOD类蛋白。
5 丹参SmMSD2蛋白的三维结构预测
Mn-SOD通常形成同源二聚体或同源四聚体, 将丹参SmMSD2的氨基酸序列通过SWISS-MODEL Workspace在线分析软件建立了SmMSD2蛋白的三维结构模型, 选择拟南芥的AtMSD1蛋白的晶体结构模型为模板, 其与模板的相似性为82.59%, 预测三维结构如图 5所示, 丹参SmMSD2蛋白能形成同源四聚体, 每个亚基在N端以α-螺旋结构为主, 而C端通常由3个β-折叠组成的, 且每个亚基通过H50、H98、D191和H195结合1个Mn2+。
6 丹参SmMSD2原核表达分析
将原核表达载体pMAL-c2X-SmMSD2重组质粒转化大肠杆菌BL21后, 挑选阳性单克隆, 进行液体培养, 至A600 nm为0.6左右时, 加入0.5 mmol·L-1 IPTG于37 ℃诱导2、4、6 h。SDS-PAGE凝胶电泳分离重组蛋白, 经IPTG诱导后, 含有MBP-SmMSD2重组蛋白的全菌在68 kDa处存在明显的特异蛋白质条带, 麦芽糖结合蛋白(MBP) 的分子质量为42.5 kDa, SmMSD2蛋白的分子质量为25.5 kDa, 表明MBP-SmMSD2重组蛋白成功诱导表达, 且重悬、超声裂解、离心后收集上清进行SDS-PAGE电泳检测分析, 发现在相同位置68 kDa处均出现特异蛋白质目的条带, 且证明该蛋白为可溶性蛋白(图 6)。
7 丹参SmMSD2酶活力及酶学性质分析
7.1 丹参SmMSD2的最适反应温度
为了比较丹参SmMSD2在不同温度下的酶活力变化情况, 将提取的待测酶液在不同温度梯度下(30、40、50、60、70、80 ℃) 温育10 min后, 分别测定其酶活力。实验结果显示(图 7A), 30~60 ℃范围内相对酶活性随温度的升高而增加, 在60 ℃时酶活性达到最大, 随后随温度的升高而降低, 80 ℃时酶活性仅为60 ℃的31.3%, 表明该酶的最适温度为60 ℃。
7.2 丹参SmMSD2的最适pH值
为了检测酸碱性对丹参SmMSD2酶活性的影响, 在不同pH值的缓冲液中测定SmMSD2相对剩余酶活性。结果显示(图 7B), 在pH 3.0~5.0的范围内该酶的活性相对较高, 保持有75%以上的酶活性; 当pH值大于9.0时, 酶活力丢失较为严重, pH值为9.0时, 酶活性仅相当于pH为3.0时的13.47%, 当pH值为2.0时, 酶活性相当于pH为3.0时的59.76%, 表明该酶不适宜过碱环境, 过碱会使SOD酶迅速失活, 且该酶的最适pH为3.0。
7.3 丹参SmMSD2的热稳定性分析
为了进一步检测SmMSD2的热稳定性, 将提取的待测酶液在不同温度梯度下(50、60、70 ℃) 作用不同时间, 测定相对剩余酶活性(图 7C)。SmMSD2的活性随着时间而降低, 但在80 min时仍保持50%以上的相对酶活性; 即使在70 ℃下保温80 min, 酶的活性损失仅为32.74%, 表明该酶具有较强的热稳定性。
8 丹参SmMSD2基因时空表达模式分析
8.1 丹参SmMSD2基因器官特异性表达分析
为研究SmMSD2在丹参不同器官中的表达模式, 利用荧光定量PCR检测SmMSD2基因在根、茎、叶和花4个器官中的转录水平, 结果显示SmMSD2基因在所有的器官中均有表达, 只是SmMSD2基因在叶中表达量相对较高, 这表明SmMSD2基因在丹参中呈现组成型表达(图 8A)。
8.2 丹参SmMSD2基因对干旱胁迫的表达模式分析
为验证丹参SmMSD2基因在植物非生物胁迫中的作用, 对丹参幼苗进行模拟干旱(15% PEG6000) 处理, 以相同生长情况下未进行任何处理的幼苗作为对照, 0、1、2、4、8、12、24 h不同时间点取样, 提取根部RNA, 进行实时荧光定量PCR分析, 检测SmMSD2基因的表达水平。实验结果显示(图 8B), 干旱胁迫下, SmMSD2的表达水平呈现先降低后升高的趋势, 在12 h达到最高, 是对照该基因表达量的约1.6倍, 表明在干旱(15% PEG6000) 胁迫的条件下, 能诱导SmMSD2基因表达。
8.3 丹参SmMSD2基因对外源植物激素IAA和ABA的表达模式分析
植物生长激素ABA、IAA对丹参幼苗处理不同时间后, SmMSD2基因的表达分析结果显示, 与相同生长情况下未进行任何处理的对照相比, 在ABA处理下(图 8C), SmMSD2的表达水平呈下降的趋势, 8 h后其表达量显著上升, 12 h表达量达到高峰, 是对照表达量的1.8倍, 随后表达量下降, 说明SmMSD2能够响应ABA的应答; 在IAA处理下(图 8D), 2 h内SmMSD2的表达水平快速升高, 随后其表达量急剧下降但呈现递增的趋势, 表明SmMSD2快速响应IAA的诱导表达。
讨论
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正常生长条件下, 植物体内活性氧自由基的产生和清除始终保持着动态平衡, 当植物遭受逆境时, 这种动态平衡就会被打破, 从而导致活性氧ROS迅速积累, 当活性氧累积超过植物自身调节能力时, 就会发生膜脂过氧化并引发连锁反应, 最终导致植物生长迟缓, 作物产量下降[20]。SOD是植物ROS信号通路中的关键酶, 是清除活性氧自由基的第一道防线。
依据2016年版的丹参基因组数据[17], 发现丹参基因组中含有8个SOD基因[15]。依据2021年版丹参基因组数据[18], 发现了9个SOD基因。Han等[15]鉴定的丹参8个SOD基因的序列中SmMSD1和SmMSD3序列都位于丹参基因组scaffold177: 260484-261033处, 本课题组认为SmMSD1和SmMSD3是同一个基因SmMSD1, 另外本研究新挖掘出2个Cu/Zn-SOD基因。因此丹参SOD家族含有: 5个Cu/Zn-SOD、2个Fe-SOD和2个Mn-SOD基因。超氧化物歧化酶家族在基因组中是相对小的基因家族, 拟南芥(Arabidopsis thaliana) 中有9个SODs (4个Cu/Zn SOD、3个Fe-SOD和2个Mn-SOD基因)[21]、水稻(Oryza sativa) 中有8个SODs (5个Cu/Zn SOD、2个Fe-SOD和1个Mn-SOD基因)[21]和陆地棉(Gossypium hirsutum) 中有18个SODs (10个Cu/Zn SOD、4个Fe-SOD和4个Mn-SOD基因)[22]。Cu/Zn SODs是SOD家族中数量相对较大的一个亚类, 它通常定位于叶绿体、胞质和细胞间隙, 而Fe SODs通常定位于叶绿体, Mn SODs通常定位于线粒体。比较这3种不同类型SODs的氨基酸序列, Mn SODs和Fe SODs是更古老的SODs类型, 很可能来自同一个祖先酶; 而Cu/Zn SODs与Mn/Fe SODs没有序列相似性, 极大可能在真核生物中是分别进化的[4]。蛋白质亚细胞定位预测表明SmMSD2位于线粒体, 且SmMSD2在丹参根、茎、花和叶中均有表达, 因此位于线粒体的SmMSD2在丹参中呈现稳定水平的组成型表达。
作为细胞内植物防御机制的重要组成部分, SODs构成了抵御非生物胁迫产生过量ROS及其反应产物的第一道防线, 高等植物中的SODs大多受发育过程中的调控, 且对外源性刺激具有高度反应。丹参SODs家族基因对冷、盐和干旱等非生物胁迫有不同程度的响应; 对重金属Cu2+, 特别是SmMSD1具有强烈的反应; 对外源植物激素ABA和MeJA反应也是不同的, 部分SODs基因受抑制, 部分SODs基因受诱导[15]。发现只加一个YE诱导子(生物胁迫), 会引起部分SODs基因表达受抑制, 部分SOD基因表达受诱导; 而在YE和Ag+ (非生物胁迫, 重金属) 混合诱导丹参毛状根后, 部分Cu SOD基因的表达量在24 h达到峰值, 而部分SOD基因的表达量在诱导12 h时最低, 随诱导时间增加表达量又增加。重金属离子(Ag+) 的加入, 并没有引起叠加效应, 预示两种以上诱导子混合会产生更加复杂的调控模式, 这种诱导机制还需要进一步研究。
Mn-SOD通常定位于线粒体, 而线粒体是呼吸链的氧化还原中心, 对植物的代谢途径有重要的影响。AtMSD1反义抑制的拟南芥植株中, 发现三羧酸(TCA) 循环中的酶(乌头酸酶和异柠檬酸脱氢酶) 被特异性抑制作用, 且在分离的线粒体中发现TCA循环的流量被抑制[5]。小麦中鉴定了一种新型的热稳定性Mn-SOD[23], 其在耐热性小麦中应对热胁迫表现出更高的活性, 表明Mn-SOD在小麦耐热性中起着至关重要的作用, 可以将其作为小麦耐热性种质资源筛选的生化标记。利用原核表达系统异源表达了丹参SmMSD2重组蛋白, 并进行了基本的酶学特性分析, 结果表明该酶具有较强的热稳定性, 因此推测该酶热稳定性较强可能由于丹参响应非生物胁迫时的氧化适应, 丹参Mn-SOD是否可以作为丹参非生物胁迫种质资源筛选的稳定生化标记还需要进一步研究。在蛋白质组学分析中, 发现SmMSD2在YE+Ag+诱导24 h蛋白的表达量是最高的, 随诱导时间增加蛋白的表达量逐渐降低; 而在YE+Ag+诱导的转录组中, SmMSD2的相对表达量在12 h最低, 随处理时间增加, 表达量逐渐增加, 在36 h达到峰值。植物激素ABA不仅调控植物的生长和发育, 更是一种与植物胁迫反应联系最紧密的激素[24]。在ABA诱导下, 2 h内SmMSD2的表达水平显著降低, 随之逐渐升高, 12 h表达量达到高峰, 表明SmMSD2响应ABA信号途径; 在IAA诱导下, 2 h内SmMSD2的表达水平快速升高, 随后其表达量急剧下降但呈现递增的趋势, 表明SmMSD2快速响应IAA的诱导表达。植物激素IAA和ABA信号途径协同或拮抗调控植物的生长发育和适应环境[25]。由此结果推测ABA信号途径和IAA信号途径在短时间内可能以拮抗的方式调控SmMSD2的表达。因此推测SmMSD2在丹参干旱胁迫、ABA和IAA诱导过程中起到重要作用。在转录水平和蛋白质水平上不同的表达模式, 预示SmMSD2受复杂的网络调控, 对其功能做进一步深入研究将有助于解析丹参应答胁迫和有效成分累积之间关系的分子机制, 对增强丹参抗逆性进而提高丹参产量和品质奠定了基础。
作者贡献: 彭佳铭执行本项工作的实验、数据分析和论文初稿的撰写; 王世威参与植物实验材料取样和RNA提取; 屈仁军、王新新、查良平和彭华胜提供实验技术支持; 申业负责实验设计、文章修改和课题资助。
利益冲突: 本文的研究内容无任何利益冲突。
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2023年第58卷第2期
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doi: 10.16438/j.0513-4870.2022-0764
  • 接收时间：2022-06-23
  • 首发时间：2025-11-21
  • 出版时间：2023-02-12
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  • 收稿日期：2022-06-23
  • 修回日期：2022-07-28
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中国中医科学院科技创新工程重大攻关项目(CI2021A04102)
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    1.安徽中医药大学药学院, 安徽 合肥 230012
    2.中国中医科学院中药资源中心, 道地药材国家重点实验室培育基地, 北京 100700

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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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