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Article(id=1200860508300628048, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200860506031518620, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-1077, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1694880000000, receivedDateStr=2023-09-17, revisedDate=1698595200000, revisedDateStr=2023-10-30, acceptedDate=null, acceptedDateStr=null, onlineDate=1764237056088, onlineDateStr=2025-11-27, pubDate=1715443200000, pubDateStr=2024-05-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764237056088, onlineIssueDateStr=2025-11-27, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764237056088, creator=13701087609, updateTime=1764237056088, updator=13701087609, issue=Issue{id=1200860506031518620, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='5', pageStart='1101', pageEnd='1508', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764237055547, creator=13701087609, updateTime=1764241222263, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200877982563824311, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200860506031518620, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200877982563824312, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200860506031518620, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1494, endPage=1502, ext={EN=ArticleExt(id=1200860508778778712, articleId=1200860508300628048, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Cloning and preliminary inquiry of AlWRKY65 from Atractylodes lancea, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

WRKY transcription factor is a type of transcription factor unique to plants and plays an important role in various physiological processes of plants. This study is based on the transcriptome data of Atractylodes lancea, the correlation between the FPKM values of the AlWRKY gene and the AlTPS gene of Atractylodes lancea was analyzed. Combined with the analysis results, the candidate gene AlWRKY65 with a significant positive correlation with the expression levels of AlTPS1 and AlTPS6 genes and a relatively high FPKM value was screened. The candidate gene was cloned to obtain the open reading frame ORF of the AlWRKY65 gene, and the related information of its encoded protein was analyzed and the gene expression was studied. The results showed that AlWRKY65 contains a 681 bp open reading frame and encoding 226 amino acids. Through amino acid sequence homology analysis, it was found that AlWRKY65 amino acid sequence had high homology with several plants such as HaWRKY65 and LsWRKY65; AlWRKY65 protein had a typical WRKYGQK domain, belonging to IIe subgroup of WRKY transcription factor family; phylogenetic analysis indicated that AlWRKY65 protein had the higher homology with CcWRKY65 protein; the expression of AlWRKY65 gene in different tissues of two producing areas of Atractylodes lancea were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves and also has tissue differences. The expression level of AlWRKY65 gene was down-regulated within 48 h of methyl jasmonate (MeJA) induction; subcellular localization and transcriptional activation assay suggested that AlWRKY65 was located in the nucleus and had no transcriptional activation activity. This study provides a reference for further elucidating the biological function of AlWRKY65 in Atractylodes lancea.

, correspAuthors=Jin XIE, Liang-ping ZHA, 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=Feng-ya GUAN, Wei-wei LIU, Kai-wen CHI, Kai-ling ZENG, Jin XIE, Liang-ping ZHA), CN=ArticleExt(id=1200860512008392912, articleId=1200860508300628048, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=茅苍术转录因子AlWRKY65的克隆与功能初探, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

WRKY转录因子是植物特有的一类转录因子, 在植物多种生理过程中发挥重要的作用。本研究以茅苍术转录组数据为依据, 对茅苍术AlWRKY基因与AlTPS基因的FPKM值进行相关性分析, 结合分析结果筛选出与AlTPS1、AlTPS6基因表达量具有显著正相关性且FPKM值相对较高的候选基因AlWRKY65, 对该候选基因进行克隆, 获得AlWRKY65基因的开放阅读框(ORF), 分析其编码蛋白的相关信息并进行基因表达研究。研究结果表明, AlWRKY65包含一个681 bp的开放阅读框, 编码226个氨基酸。AlWRKY65基因编码的氨基酸序列与向日葵HaWRKY65、莴苣LsWRKY65等植物基因编码的氨基酸序列具有较高同源性; 该蛋白具有1个WRKYGQK保守结构域, 属于WRKY基因家族的第IIe组; 系统进化树分析显示, 茅苍术AlWRKY65与洋蓟CcWRKY65蛋白亲缘关系较近; 运用实时荧光定量PCR检测AlWRKY65基因在2个产地不同组织中的表达水平, 结果表明, 不同产地的茅苍术AlWRKY65基因具有组织表达差异性, 均在叶片中表达量较高; 在茉莉酸甲酯(MeJA) 诱导处理48 h内, AlWRKY65基因的表达量下调; 亚细胞定位和转录自激活活性分析显示, AlWRKY65定位于细胞核, 不具有自激活活性。这些结果为进一步研究AlWRKY65在茅苍术中的生物学功能提供参考。

, correspAuthors=谢晋, 查良平, authorNote=null, correspAuthorsNote=
*谢晋, E-mail: ;
查良平, E-mail:
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A: Correlation analysis of <i>AlWRKY</i> gene and <i>AlTPSs</i> gene; B: Expression levels of <i>AlWRKY1</i>, <i>AlWRKY22</i>, <i>AlWRKY65</i> in different tissues. FPKM: Fragments per kilobase of exon model per million mapped fragments. R: Rhizome; S: Stem; L: Leaf. <sup>**</sup><i>P</i> ≤ 0.01 <i>vs</i> AlWRKY1-R. <i>n</i> = 3, <span class="mag-xml-inline-formula"><tex-math id="M1">$ \overline{x} $</tex-math></span> ± <i>s</i> , figureFileSmall=+QoHgUvUDJ8yMnWKyU2ITw==, figureFileBig=vf1iWVoGBH4CtLPx+zfqug==, tableContent=null), ArticleFig(id=1201106661722251610, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=9mgtiMswQ2EWE44yCIJ0SA==, figureFileBig=KOB6hsZXZBBlww8hGluzOA==, tableContent=null), ArticleFig(id=1201106663068623212, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 2, caption= Bioinformatics analysis of AlWRKY65. A: Secondary structure model of AlWRKY65; B: The predicted conserved domain of AlWRKY65; C: The predicted tertiary structure of AlWRKY65 , figureFileSmall=9mgtiMswQ2EWE44yCIJ0SA==, figureFileBig=KOB6hsZXZBBlww8hGluzOA==, tableContent=null), ArticleFig(id=1201106663177675123, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=NDRJ3GT6xGO+t45L71QEqQ==, figureFileBig=BqAJv55o+3vLM3TCEpVugw==, tableContent=null), ArticleFig(id=1201106663324475773, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 3, caption= Amino acid sequence homology alignment of WRKY65s from <i>Atractylodes lancea</i>, <i>Cynara cardunculus</i> var. <i>Scolymus</i>, <i>Helianthus annuus</i> and <i>Lactuca sativa</i> , figureFileSmall=NDRJ3GT6xGO+t45L71QEqQ==, figureFileBig=BqAJv55o+3vLM3TCEpVugw==, tableContent=null), ArticleFig(id=1201106663538385291, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=ue274dLvSHwflEHU4Rprzw==, figureFileBig=wiysMo5MCBQHCjchosaq1Q==, tableContent=null), ArticleFig(id=1201106663815209376, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 4, caption= Protein-protein interaction network of AlWRKY65 protein. Black and red color characters represent <i>Arabidopsis thaliana</i> and <i>Atractylodes lancea</i>, and the thick lines represent the strength of interaction , figureFileSmall=ue274dLvSHwflEHU4Rprzw==, figureFileBig=wiysMo5MCBQHCjchosaq1Q==, tableContent=null), ArticleFig(id=1201106663991370150, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=9dHmWleLW1mAG4+lzTGwSw==, figureFileBig=AeFRUGIz2TnhAFqcoEgJ/w==, tableContent=null), ArticleFig(id=1201106664213668284, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 5, caption= Phylogenetic tree of the amino acid sequences of AlWRKY65 with some other WRKYs. Aa: <i>Artemisia annua</i>; Al: <i>Atractylodes lancea</i>; At: <i>Arabidopsis thaliana</i>; Cc: <i>Cynara cardunculus</i> var. <i>Scolymus</i>; Cj: <i>Coptis japonica</i> var. <i>Dissecta</i>; Ga: <i>Gossypium arboreum</i>; Gh: <i>Gossypium hirsutum</i>; Na: <i>Nicotiana attenuata</i>; Sm: <i>Salvia miltiorrhiza</i> , figureFileSmall=9dHmWleLW1mAG4+lzTGwSw==, figureFileBig=AeFRUGIz2TnhAFqcoEgJ/w==, tableContent=null), ArticleFig(id=1201106664347886024, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=5VbSrDSBHDDRgKt5s12NAA==, figureFileBig=11+sSSNa8TV0fZhrKJo/uQ==, tableContent=null), ArticleFig(id=1201106664465326548, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 6, caption= Expression levels of <i>AlWRKY65</i> in different origins and different tissues. R: Rhizome; S: Stem; L: Leaf; NJ: <i>A. lancea</i> in Nanjing; SY: <i>A. lancea</i> in Shiyan. <sup>*</sup><i>P</i> ≤ 0.05, <sup>**</sup><i>P</i> ≤ 0.01, <sup>***</sup><i>P</i> ≤ 0.001 <i>vs</i> NJ-R. <i>n</i> = 3, <span class="mag-xml-inline-formula"><tex-math id="M2">$ \overline{x} $</tex-math></span> ± <i>s</i> , figureFileSmall=5VbSrDSBHDDRgKt5s12NAA==, figureFileBig=11+sSSNa8TV0fZhrKJo/uQ==, tableContent=null), ArticleFig(id=1201106664624710112, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=UJu7jOJCMMy7ERcrcf3BrA==, figureFileBig=4TMao6N9W/bVanLzH2/2Sw==, tableContent=null), ArticleFig(id=1201106664813453812, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 7, caption= Expression analysis of <i>AlWRKY65</i> induced by MeJA. <sup>***</sup><i>P</i> ≤ 0.001, <sup>****</sup><i>P</i> < 0.000 1 <i>vs</i> CK. <i>n</i> = 3, <span class="mag-xml-inline-formula"><tex-math id="M3">$ \overline{x} $</tex-math></span> ± <i>s</i> , figureFileSmall=UJu7jOJCMMy7ERcrcf3BrA==, figureFileBig=4TMao6N9W/bVanLzH2/2Sw==, tableContent=null), ArticleFig(id=1201106664989614595, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=LazcV8uLZ58gfUZskFxA5Q==, figureFileBig=jQX+SFbY3Okk0JohX72/Zg==, tableContent=null), ArticleFig(id=1201106665136415241, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 8, caption= Autonomous transcriptional activation testing , figureFileSmall=LazcV8uLZ58gfUZskFxA5Q==, figureFileBig=jQX+SFbY3Okk0JohX72/Zg==, tableContent=null), ArticleFig(id=1201106665400656409, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=EN, label=null, caption=null, figureFileSmall=AadTkPz6FmHjKmNQftAhqg==, figureFileBig=WsWyUMtan2WX6vuZL8Uotg==, tableContent=null), ArticleFig(id=1201106665568428589, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200860508300628048, language=CN, label=Figure 9, caption= Subcellular localization of AlWRKY65. 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茅苍术转录因子AlWRKY65的克隆与功能初探
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管凤雅 1 , 刘巍玮 1 , 迟凯文 1 , 曾凯玲 1 , 谢晋 2, * , 查良平 1, *
药学学报 | 研究论文 2024,59(5): 1494-1502
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药学学报 | 研究论文 2024, 59(5): 1494-1502
茅苍术转录因子AlWRKY65的克隆与功能初探
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管凤雅1, 刘巍玮1, 迟凯文1, 曾凯玲1, 谢晋2, * , 查良平1, *
作者信息
  • 1.安徽中医药大学药学院, 安徽 合肥 230012
  • 2.安徽医科大学药学院, 安徽 合肥 230032

通讯作者:

*谢晋, E-mail: ;
查良平, E-mail:
Cloning and preliminary inquiry of AlWRKY65 from Atractylodes lancea
Feng-ya GUAN1, Wei-wei LIU1, Kai-wen CHI1, Kai-ling ZENG1, Jin XIE2, * , Liang-ping ZHA1, *
Affiliations
  • 1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
  • 2. School of Pharmacy, Anhui Medical University, Hefei 230032, China
出版时间: 2024-05-12 doi: 10.16438/j.0513-4870.2023-1077
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摘要
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WRKY转录因子是植物特有的一类转录因子, 在植物多种生理过程中发挥重要的作用。本研究以茅苍术转录组数据为依据, 对茅苍术AlWRKY基因与AlTPS基因的FPKM值进行相关性分析, 结合分析结果筛选出与AlTPS1、AlTPS6基因表达量具有显著正相关性且FPKM值相对较高的候选基因AlWRKY65, 对该候选基因进行克隆, 获得AlWRKY65基因的开放阅读框(ORF), 分析其编码蛋白的相关信息并进行基因表达研究。研究结果表明, AlWRKY65包含一个681 bp的开放阅读框, 编码226个氨基酸。AlWRKY65基因编码的氨基酸序列与向日葵HaWRKY65、莴苣LsWRKY65等植物基因编码的氨基酸序列具有较高同源性; 该蛋白具有1个WRKYGQK保守结构域, 属于WRKY基因家族的第IIe组; 系统进化树分析显示, 茅苍术AlWRKY65与洋蓟CcWRKY65蛋白亲缘关系较近; 运用实时荧光定量PCR检测AlWRKY65基因在2个产地不同组织中的表达水平, 结果表明, 不同产地的茅苍术AlWRKY65基因具有组织表达差异性, 均在叶片中表达量较高; 在茉莉酸甲酯(MeJA) 诱导处理48 h内, AlWRKY65基因的表达量下调; 亚细胞定位和转录自激活活性分析显示, AlWRKY65定位于细胞核, 不具有自激活活性。这些结果为进一步研究AlWRKY65在茅苍术中的生物学功能提供参考。

茅苍术  /  WRKY转录因子  /  基因克隆  /  表达分析  /  亚细胞定位

WRKY transcription factor is a type of transcription factor unique to plants and plays an important role in various physiological processes of plants. This study is based on the transcriptome data of Atractylodes lancea, the correlation between the FPKM values of the AlWRKY gene and the AlTPS gene of Atractylodes lancea was analyzed. Combined with the analysis results, the candidate gene AlWRKY65 with a significant positive correlation with the expression levels of AlTPS1 and AlTPS6 genes and a relatively high FPKM value was screened. The candidate gene was cloned to obtain the open reading frame ORF of the AlWRKY65 gene, and the related information of its encoded protein was analyzed and the gene expression was studied. The results showed that AlWRKY65 contains a 681 bp open reading frame and encoding 226 amino acids. Through amino acid sequence homology analysis, it was found that AlWRKY65 amino acid sequence had high homology with several plants such as HaWRKY65 and LsWRKY65; AlWRKY65 protein had a typical WRKYGQK domain, belonging to IIe subgroup of WRKY transcription factor family; phylogenetic analysis indicated that AlWRKY65 protein had the higher homology with CcWRKY65 protein; the expression of AlWRKY65 gene in different tissues of two producing areas of Atractylodes lancea were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves and also has tissue differences. The expression level of AlWRKY65 gene was down-regulated within 48 h of methyl jasmonate (MeJA) induction; subcellular localization and transcriptional activation assay suggested that AlWRKY65 was located in the nucleus and had no transcriptional activation activity. This study provides a reference for further elucidating the biological function of AlWRKY65 in Atractylodes lancea.

Atractylodes lancea  /  WRKY transcription factor  /  gene cloning  /  expression analysis  /  subcellular localization
管凤雅, 刘巍玮, 迟凯文, 曾凯玲, 谢晋, 查良平. 茅苍术转录因子AlWRKY65的克隆与功能初探. 药学学报, 2024 , 59 (5) : 1494 -1502 . DOI: 10.16438/j.0513-4870.2023-1077
Feng-ya GUAN, Wei-wei LIU, Kai-wen CHI, Kai-ling ZENG, Jin XIE, Liang-ping ZHA. Cloning and preliminary inquiry of AlWRKY65 from Atractylodes lancea[J]. Acta Pharmaceutica Sinica, 2024 , 59 (5) : 1494 -1502 . DOI: 10.16438/j.0513-4870.2023-1077
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WRKY转录因子是植物中特有的转录因子家族, 因其结构域中包含1个或2个高度保守的WRKY (WRKYGQK) 氨基酸基序而命名[1]。自1994年从甘薯中鉴定出第一个WRKY转录因子SPF1后[2], 越来越多的药用植物WRKY转录因子得到了广泛的研究, 如北柴胡[3]、铁皮石斛[4]、马蓝[5]等药用植物。许多研究表明, WRKY转录因子参与调节植物多种生理过程, 如促进种子萌发, 调控植物的生长发育[6-8]以及对抗生物和非生物胁迫[9]等。根据WRKY保守结构域的数目和锌指结构的种类, 通常将WRKY转录因子家族分为3组(Ⅰ、Ⅱ、Ⅲ), 其中Ⅰ家族成员包含2个WRKY结构域, 锌指结构域类型为C2H2型; Ⅱ家族成员仅包含1个WRKY结构域, 锌指结构域类型为C2H2型, 其中根据氨基酸序列的不同, 又被细分为5个亚组(Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe); Ⅲ家族成员包含1个WRKY结构域, 锌指结构域类型为C2HC型[10]。近年来, 转录因子通过调控代谢途径关键酶基因的转录来启动或限制次生代谢物合成的作用引起了广泛关注[11], 例如, 青蒿素是从药用植物黄花蒿Artemisia annua中分离的一种倍半萜内酯, 青蒿AaWRKY1转录因子能够与青蒿素生物合成过程中的关键酶基因ADS启动子区域中的顺式作用元件W-box结合, 从而激活其表达, 研究发现青蒿AaWRKY1参与调控青蒿素的生物合成[12]; 短小蛇根草OpWRKY2是抗癌化合物喜树碱生物合成的直接正调控因子[13]; 紫杉醇是红豆杉中的一种天然抗癌药物, 红豆杉TcWRKY33可与紫杉醇生物合成途径基因TcDBAT启动子中的2个W-box结合, 上调其表达水平, 研究得出红豆杉TcWRKY33正向调节紫杉醇的生物合成[14]。综上所述, WRKY转录因子在调控植物次生代谢物合成过程中发挥着重要作用。
茅苍术为菊科植物茅苍术Atractylodes lancea (Thunb.) DC.的干燥根状茎, 具有燥湿健脾、祛风散寒、明目等功效[15], 临床应用广泛。茅苍术为多年生草本, 主要分布于江苏、湖北、安徽、河南等地[16], 茅苍术喜凉爽干燥的气候, 适宜生长于丘陵山区半阴半阳的荒坡地[17]。茅苍术的根状茎中含有主要的药效成分挥发油类, 有学者对挥发油进行了系统的研究, 挥发油类主要由倍半萜类和聚乙烯炔类等成分组成, 在药理方面, 苍术具有抗癌、抗炎、抑菌、降血糖、保护心血管和神经系统等作用[18-21]。关于茅苍术萜类合成与调控的研究有助于提高茅苍术挥发油成分, 保证苍术药材质量。
目前, 本课题组已经对茅苍术萜类合成途径中的4-二磷酸胞苷-2-C-甲基-D-赤藓醇激酶基因AlCMK[22]、鲨烯合酶基因AlSQS[23]、萜类合酶基因AlTPS[24]等进行研究报道, 为茅苍术中萜类化合物的生物合成提供基础, 然而关于茅苍术萜类合成的转录调控鲜有报道, 尤其关于茅苍术WRKY转录因子的功能尚不清楚。本研究基于茅苍术转录组数据, 从茅苍术中克隆得到WRKY转录因子, 进行生物信息学分析、亚细胞定位分析、转录自激活检测、利用实时荧光定量PCR (qRT-PCR) 技术探究茅苍术2个产地、不同组织及外源性激素茉莉酸甲酯诱导的基因表达情况, 以期为深入解析其生物学功能提供参考。
材料与方法
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样品    茅苍术来源于湖北十堰、江苏南京, 经安徽中医药大学查良平副研究员鉴定为菊科植物茅苍术Atractylodes lancea (Thunb.) DC.。经鉴定后收集茅苍术根状茎、茎、叶进行后续实验。
茅苍术种子来源于湖北十堰, 培养于1/2 MS液体培养基中, 光照周期为16 h光照和8 h黑暗, 温度为25 ℃。取长势一致的5个月茅苍术无菌苗样品, 于液体培养基加入200 μmol·L-1茉莉酸甲酯进行诱导处理, 处理后分别于0、3、6、9、12、24、48 h收取茅苍术根。所有样品均于液氮中速冻后保存于-80 ℃冰箱。
试剂    大肠杆菌Trans1-T1感受态细胞(Lot: Q4611209)、pEASY-Blunt Zero Cloning Kit载体(Lot: R20512)、EasyPure® Quick Gel Extraction Kit切胶回收试剂盒(Lot: R10507)、pEASY Uni Seamless Cloning and Assembly Kit无缝拼接试剂盒(Lot: Q30915) 购于北京全式金生物技术有限公司; 根癌农杆菌(Agrobacterium tumefaciens) GV3101 (Lot: 2112#31220307A)、超快速植物RNA提取试剂盒(Lot: 2207#12)、营养缺陷型基础培养基SD/-Trp (Lot: 2204#18P)、金担子素A (AbA) (Lot: 2205#17K) 购于北京华越洋生物科技有限公司; 2×Realab Green PCR Fast mixture荧光定量试剂盒(Lot: 0202111621)、反转录试剂盒(Lot: 0202010531)、5-溴-4-氯-3-吲哚-α-D-半乳糖苷(X-α-Gal) (Lot: 0200120411) 购于北京兰博利德商贸有限公司; 酵母Y2H菌株(Lot: 20210415) 购于上海欧易生物医学科技有限公司。
仪器与设备    ETC821型PCR扩增仪(北京东胜创新生物科技有限公司); DYCP-31DN型琼脂糖水平电泳仪(北京六一生物科技有限公司); G500312-0001蓝光切胶仪(上海生工生物工程股份有限公司); Alphalmager HP型凝胶色谱成像仪(美国ProteinSimple公司); BHP-75恒温培养箱(合肥右科仪器设备有限公司); CFX96 Optics Module实时荧光定量PCR仪(美国伯乐公司); Olympus FV3000激光共聚焦显微镜(日本Olympus公司)。
AlWRKY基因与AlTPS基因FPKM值相关性分析    为了进一步推测AlWRKY基因与茅苍术萜类合酶基因的相关性, 根据筛选出的茅苍术转录组中3个WRKY Ⅱe家族成员(AlWRKY1、AlWRKY22、AlWRKY65) 结合课题组前期筛选出的茅苍术9个萜类合酶基因AlTPS[24], 选取湖北十堰茅苍术根状茎、茎、叶中的FPKM (fragments per kilobase of exon model per million mapped fragments) 值, 分别计算出基因间的皮尔森相关性系数, 运用Chiplot (https://www.chiplot.online/) 在线网站进行作图和美化。
总RNA提取与cDNA合成    使用RNA提取试剂盒提取湖北十堰、江苏南京茅苍术根状茎、茎、叶中总RNA及经MeJA处理后湖北十堰茅苍术根中总RNA。使用反转录试剂盒进行反转录, 得到茅苍术样品的cDNA。
基因克隆    以反转录后的茅苍术叶片cDNA为模板进行PCR扩增, 使用Primer Premier 5.0软件设计基因特异性引物, 上游引物AlWRKY65-F: 5′-ATGGAAGA AGGCTCCCAGTCTCCAC-3′, 下游引物AlWRKY65-R: 5′-TTAAGAAGGTTGAACAAGAACTCTC-3′, PCR扩增产物经1%琼脂糖凝胶电泳检测后, 切胶回收, 切胶回收产物与T载pEASY-Blunt Zero连接, 转化至大肠杆菌Trans1-T1感受态细胞中, 菌液PCR, 取阳性克隆菌液进行测序。
生物信息学分析    利用ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) 获取基因的开放阅读框(ORF); 使用NCBI BLAST (http://www.ncbi.nlm.nih.gov/) 在线工具进行氨基酸序列的相似性比对分析; 利用ExPASy (https://web.expasy.org/protparam/) 预测该基因编码的蛋白理化性质; 利用SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) 预测蛋白的二级结构; 利用SWISS MODEL (http://swissmodel.expasy.org/) 及PyMOL软件进行三级结构的预测; 使用CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) 进行蛋白质结构域分析; 应用Clustal W及Espript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) 进行同源氨基酸序列比对; 以拟南芥AtWRKY65转录因子为参考, 采用STRING 11.5 (https://version-11-5.string-db.org/) 在线网站进行互作蛋白网络分析, 置信度设置为0.40; 应用MEGA 6.06软件构建Neighbor-joining系统进化树, bootstrap重复次数为1 000次。
基因表达分析    采用实时荧光定量PCR法对经茉莉酸甲酯处理不同时间点的茅苍术根及湖北十堰茅苍术(根状茎、茎、叶) 和江苏南京茅苍术(根状茎、茎、叶) 中AlWRKY65基因表达模式进行分析。每个样品3株, 分别提取RNA并反转录获得cDNA。以茅苍术UBQ2基因作为内参基因, 上游引物UBQ2-F: 5′-GGTTGAG GGGAGGAATGC-3′, 下游引物UBQ2-R: 5′-AGACG AAGGACAAGGTGA-3′, 荧光定量引物使用Primer Premier 5.0软件设计, 分别为上游引物qAlWRKY65-F: 5′-AGACCCCAAGGAATCAAAACAAC-3′, 下游引物qAlWRKY65-R: 5′-GGCTCGGATGTAGCGAAAGT TAT-3′。根据荧光定量试剂盒的说明书配制20 μL的反应体系, 放置于CFX96 Optics Module实时荧光定量PCR仪中进行PCR扩增, 每个样品进行3个重复。采用2-ΔΔCt法计算目的基因的相对表达量。
转录自激活验证    以pGBKT7载体为酵母表达载体, 根据克隆成功的基因序列, 设计含有BamH I酶切位点的引物, 上游引物AlWRKY65-pGBKT7-F: 5′-GG AGGCCGAATTCCCGGGGAATGGAAGAAGGCTCC CAGTCTCCAC-3′; 下游引物AlWRKY65-pGBKT7-R: 5′-GGCCGCTGCAGGTCGACGGATTAAGAAGGTTG AACAAGAACTCTC-3′。使用BamH I限制性内切酶对载体pGBKT7线性化并与目的基因切胶回收产物进行重组, 转化至大肠杆菌Trans1-T1感受态细胞中, 菌液PCR, 经测序分析正确后提取质粒, 成功构建pGBKT7-AlWRKY65重组载体。将pGBKT7空载体与pGBKT7-AlWRKY65分别转化到Y2HGold酵母菌中, 取转化后的菌液5 µL分别在SD/-Trp, SD/-Trp/X-α-Gal, SD/-Trp/X-α-Gal/AbA平板上点样, 30 ℃培养3天。观察酵母菌落生长状态与菌落颜色。
亚细胞定位    以pRI101-eGFP载体为亚细胞定位载体, 根据克隆成功的基因序列, 设计含有BamH I酶切位点的引物, 上游引物AlWRKY65-eGFP-F: 5′- TCGACCCCGGGGGTACCGGAATGGAAGAAGGCT CCCAGTCTCCAC-3′; 下游引物AlWRKY65-eGFP-R: 5′-TCGCCCTTGCTCACCATGGAAGAAGGTTGAAC AAGAACTCTCTCC-3′。使用BamH I限制性内切酶对载体pRI101-eGFP线性化并与目的基因切胶回收产物进行重组, 转化至大肠杆菌Trans1-T1感受态细胞中, 菌液PCR, 经测序分析正确后提取质粒, 构建植物表达载体pRI101-AlWRKY65-eGFP。将pRI101-AlWRKY65-eGFP重组载体质粒和pRI101-eGFP空载体质粒分别转入根癌农杆菌GV3101中, 取阳性菌液于YEB液体培养基28 ℃, 180 r·min-1培养过夜, 离心重悬, 调整OD600至0.6~0.8, 室温静置3 h后吸取菌液注射于烟草叶片下表皮, 弱光条件下培养48 h后于激光共聚焦显微镜下观察亚细胞定位结果。
统计学分析    采用GraphPad Prism 8.0软件统计分析和作图, 实验结果以均值±标准差表示, P < 0.05被认为差异具有统计学意义。
结果与分析
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1 AlWRKY基因与AlTPS基因FPKM值相关性分析
相关性分析结果表明(图 1A), 3个WRKY Ⅱe家族成员基因表达量具均与AlTPS1、AlTPS6基因表达量具有显著正相关性, 其中AlWRKY22、AlWRKY65基因表达量与AlTPS8基因表达量呈现较弱的正相关性, 本研究选取FPKM值相对较高的AlWRKY65基因作为候选基因, 进行进一步的研究(图 1B)。
2 AlWRKY65基因的克隆及序列分析
茅苍术AlWRKY65基因来源于本实验室茅苍术转录组数据库[美国国家生物技术信息中心(NCBI) 登录号为PRJNA854065], 使用NCBI ORF Finder对基因序列进行预测分析, 结果显示, AlWRKY65基因含有1个681 bp完整的开放阅读框。以反转录得到的茅苍术叶片cDNA为模板使用特异性引物进行RT-PCR扩增, PCR产物连接至T载体, 所得测序结果与预测结果相符。
应用NCBI BLAST对其编码的氨基酸序列进行比对, 结果显示AlWRKY65氨基酸序列与GenBank中已注册植物WRKY氨基酸序列有较高的同源性, 其中茅苍术AlWRKY65与向日葵HaWRKY65 (XP_021987700.1) 相似度为72.04%, 与小飞蓬EcWRKY65 (XP_043635398.1) 相似度为71.82%, 将该基因命名为AlWRKY65 (GenBank注册号: OR343917)。
3 生物信息学分析
3.1 蛋白理化性质分析
利用在线工具ExPASy对AlWRKY65基因编码蛋白进行理化性质分析。结果显示, AlWRKY65编码226个氨基酸, 相对分子质量为26 144.98, 蛋白理论等电点为5.29, 不稳定系数为50.26, 脂肪指数为47.43, 可初步判断AlWRKY65为不稳定的酸性蛋白。
3.2 结构功能域与二级、三级结构预测
分析茅苍术AlWRKY65转录因子的蛋白结构域(图 2B), 结果显示AlWRKY65在53~109 aa含有1个WRKYGQK保守结构域, 属于WRKY super family家族。二级结构预测结果显示(图 2A), 该转录因子编码蛋白的二级结构主要由无规则卷曲(random coil)、α螺旋(alpha helix)、延伸链(extended strand) 和β-转角(β-turn) 构成, 其中无规则卷曲有154个氨基酸, 占比68.14%, α-螺旋有39个氨基酸, 占比17.26%, 延伸链有26个氨基酸, 占比11.50%, β-转角有7个氨基酸, 占比3.10%, 呈现无规则卷曲 > α-螺旋 > 延伸链 > β-转角的规律。利用SWISS-MODEL在线工具和PyMOL软件构建茅苍术AlWRKY65蛋白的三维空间模型(图 2C)。
3.3 氨基酸序列同源比对
对茅苍术AlWRKY65基因氨基酸序列与其他植物氨基酸序列进行比对分析(图 3)。结果表明, 茅苍术AlWRKY65基因氨基酸序列与其他3种植物的WRKY65基因氨基酸序列具有较高的同源性, 其中与洋蓟CcWRKY65 (XP_024981405.1) 的同源性为77.13%, 与莴苣LsWRKY65 (XP_052623230.1) 的同源性为73.68%, 与向日葵HaWRKY65 (XP_021987700.1) 的同源性为72.04%, 均具有1个典型的WRKYGQK保守结构域序列。
3.4 蛋白互作结果分析
利用STRING数据库的拟南芥关联模型搜索与AlWRKY65蛋白相互作用的其他蛋白质信息, 构建蛋白互作网络(图 4), 预测AtWRKY65 (AlWRKY65) 蛋白与SR (AT4G11640.1)、NFD1 (AT4G30930.1)、BME3 (AT3G54810.1)、GATA2 (AT2G45050.1)、ADR1 (AT1G33560.1)、ADR1-L2 (AT5G04720.1) 等11个蛋白具有互作关系, 其中ADR1和ADR1-L2作为正调控因子参与植物免疫应答反应; SR参与调控植物生长发育; BME3参与调控胚轴发育; GATA2蛋白具有限制细胞分裂活性的功能。推测AlWRKY65蛋白也同样通过与这些蛋白互作来参与调控植物的生长发育。
3.5 系统进化树分析
根据WRKY结构域数量与锌指结构域的结构特征, 基于不同物种WRKY基因的氨基酸序列, 采用邻接法构建系统进化树(图 5)。来源于不同物种的WRKY蛋白被分为3个主要类群(Ⅰ、Ⅱ和Ⅲ), 其中茅苍术AlWRKY65与洋蓟CcWRKY65聚为一支, 都属于进一步细分的Ⅱe亚类家族成员, 推测两者亲缘关系较近。
4 qRT-PCR分析2个产地、不同组织茅苍术AlWRKY65基因表达模式
对江苏南京、湖北十堰两地茅苍术AlWRKY65基因不同组织表达量进行分析(图 6)。结果显示, 在南京茅苍术中AlWRKY65基因在叶中相对表达量较高, 茎中次之, 在根状茎中的相对表达量较低; 在十堰茅苍术中AlWRKY65基因在叶中相对表达量较高, 根状茎中次之, 在茎中的相对表达量较低, 两个产地中AlWRKY65基因均在叶中表达量最高, 这表明AlWRKY65基因的表达呈现一定的组织特异性, 表达模式在不同生长环境下存在差异。
5 qRT-PCR分析MeJA诱导下茅苍术AlWRKY65的表达模式
为分析茉莉酸甲酯胁迫下茅苍术AlWRKY65基因的表达特征, 采用茉莉酸甲酯溶液处理茅苍术组培苗, 进行qRT-PCR分析(图 7)。结果显示, 在外源激素茉莉酸甲酯处理的48 h内, 与CK (0 h) 对照相比, AlWRKY65基因表达量出现显著下调。
6 转录自激活检测
如图 8所示, 与阴性对照(pGBKT7) 一致, 转入pGBKT7-AlWRKY65的酵母菌株在SD/-Trp单缺培养基上有酵母菌落正常生长, SD/-Trp/X-α-Gal培养基上有酵母菌落正常生长但没有显示蓝色, SD/-Trp/X-α-Gal/AbA培养基上未见明显生长且无颜色变化(图 8)。由此推测, pGBKT7-AlWRKY65诱饵表达载体不能激活酵母菌株营养缺陷型报告基因的表达, 无转录自激活活性。
7 亚细胞定位分析
采用烟草瞬时表达体系, 使用Olympus FV3000激光共聚焦显微镜分别在波长488和633 nm的激发波长下观察绿色荧光蛋白GFP和叶绿体自发红色荧光信号(图 9)。结果显示, 对照空载体在细胞膜、细胞核中都可以观察到清晰的GFP绿色荧光信号, pRI101-AlWRKY65-eGFP融合蛋白仅在细胞核中出现绿色荧光信号, 推测AlWRKY65蛋白定位在细胞核中, 属于核定位蛋白。
讨论
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WRKY转录因子作为目前研究广泛的转录因子家族之一, 在植物生长发育过程中发挥着不可或缺的作用, 随着基因组测序技术的不断进步与完善, 越来越多植物的WRKY转录因子家族成员的作用与机制得到深入的研究[25]。目前已发现的WRKY蛋白大多属于WRKY家族的Ⅱ家族[26]。有研究表明, 在番茄中过表达WRKY Ⅱe家族成员SlWRKY35能提高MEP途径中相关基因的表达, 从而提高番茄果实中类胡萝卜素类化合物与叶片中叶绿素的合成[27]; 有学者通过研究发现, 橡胶树中WRKY Ⅱe家族成员HbWRKY27可以正向调控天然橡胶关键生物合成基因HbFPS1的表达[28]。本课题组在分析茅苍术转录组数据时, 发现一条WRKY Ⅱe家族成员AlWRKY65, 对其与萜类合酶基因进行相关性分析, 结果表明, 它与部分萜类合酶基因有明显的正相关性, 为了进一步研究该基因的功能, 本研究对茅苍术AlWRKY65转录因子进行克隆, 得到长为681 bp、编码226个氨基酸的基因序列, 为后续深入基因功能提供依据。
WRKY基因的表达通常具有组织特异性, 这与其所调控的生命活动有关, 例如: 茶树CsWRKY17在茶树的根、茎、叶、花中均有表达, 在根部表达量最高, 具有明显的组织特异性[29]; 葡萄VvWRKY26在葡萄的花、茎、芽、卷须、幼叶、成熟叶和果实组织中均有表达, 此外, 在幼果期和转色期表达量相对较高[30]。荷花NnWRKY22在荷花的根、茎、叶、花中均有表达, 但在根中表达量最高, 推测NnWRKY22主要通过荷花根部发挥该基因功能[31]。本研究结果表明在南京与十堰两个产地的茅苍术中, AlWRKY65基因在根状茎, 茎, 叶中均有表达, 具有组织特异性, 且表达量均在叶片中相对较高, 说明其可能主要在叶片中发挥作用。茉莉酸甲酯是茉莉酸类化合物的一种重要衍生物, 在植物界中广泛分布, 在植物体营养发育、植物防御和代谢调节中发挥重要作用[32], 许多转录因子受到茉莉酸甲酯诱导, 并参与特定代谢过程的调节, 有学者研究表明经茉莉酸甲酯处理的薄荷, 部分薄荷WRKY基因表达出现上调, 部分WRKY基因表达下调[33]; 在药用植物青蒿中, AaWRKY1受到茉莉酸甲酯的强烈诱导, 并调节青蒿素生物合成的关键基因ADS的表达[12]; 甘蔗ScWRKY3在茉莉酸甲酯处理下, 表达水平逐渐降低, 受到抑制[34]。进一步的qRT-PCR结果显示, 茉莉酸甲酯影响茅苍术AlWRKY65基因的表达, 在茉莉酸甲酯处理48 h内, 显著降低了AlWRKY65基因的表达量, 但具体的响应机制仍需要后续实验的验证。
大量研究表明WRKY蛋白定位于植物细胞核, 例如枇杷EjWRKY15[35]、茶树CsWRKY17[29]、人参PgWRKY22[36]等都定位于植物的细胞核中, 本研究构建植物表达载体pRI101-AlWRKY65-eGFP, 利用烟草瞬时表达系统实验证明AlWRKY65蛋白主要定位于细胞核中, 推测AlWRKY65可能作为核蛋白发挥作用。作为转录因子, 转录激活相关基因表达是其行使功能的主要形式, 但亦有部分转录因子通过结合其他基因的启动子或形成同源二聚体发挥其功能[37], 转录因子无转录自激活活性的情况也有报道, 如甘蔗ScWRKY4[38]、桂花OfWRKY120[39]等。本研究构建pGBKT7-AlWRKY65酵母诱饵载体, 通过对比不同培养基上的酵母单菌落生长情况, 发现AlWRKY65基因无转录自激活活性, 因此推测AlWRKY65基因可能通过与其他具有自激活作用的转录因子互作来调控下游靶基因表达, 可用于后续酵母双杂交试验, 筛选与其编码蛋白相互作用的相关蛋白。
WRKY转录因子参与多种与植物胁迫应答、生长发育等有关的重要生理过程, 调控过程较为复杂。植物中萜类代谢的过程也是极其复杂, 萜类代谢相关的酶与转录因子之间可能存在相互作用, 它们相互配合, 共同促进某个特定的萜类代谢途径。本研究从茅苍术中克隆获得1个WRKY转录因子, 命名为AlWRKY65, 对其进行生物信息学分析及基因表达分析, 为后续可进一步研究该转录因子与萜类合成关键酶基因之间的调控作用奠定基础。但茅苍术在生长发育及次生代谢过程中受到多个转录因子家族的共同调控, 还需要更进一步的研究来阐明其生物学功能。
作者贡献: 查良平和谢晋设计实验并提供资金; 管凤雅和刘巍玮参与实验及撰写论文; 迟凯文和曾凯玲参与数据分析及提供实验技术支持。
利益冲突: 所有作者均声明不存在利益冲突。
基金
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  • 国家自然科学基金面上项目(82073957)
  • 安徽省自然科学基金优青项目(2208085Y30)
  • 安徽省高校杰出青年科研项目(2023AH020036)
  • 安徽省高校优秀青年人才支持计划重点项目(gxyqZD2022051)
  • 安徽高校自然科学研究重点项目(2023AH050634)
  • 中华中医药学会青年人才托举工程项目(CACM-2023-QNRC2-B23)
文献
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参考文献 引证文献
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2024年第59卷第5期
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doi: 10.16438/j.0513-4870.2023-1077
  • 接收时间：2023-09-17
  • 首发时间：2025-11-27
  • 出版时间：2024-05-12
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  • 收稿日期：2023-09-17
  • 修回日期：2023-10-30
基金
国家自然科学基金面上项目(82073957)
安徽省自然科学基金优青项目(2208085Y30)
安徽省高校杰出青年科研项目(2023AH020036)
安徽省高校优秀青年人才支持计划重点项目(gxyqZD2022051)
安徽高校自然科学研究重点项目(2023AH050634)
中华中医药学会青年人才托举工程项目(CACM-2023-QNRC2-B23)
作者信息
    1.安徽中医药大学药学院, 安徽 合肥 230012
    2.安徽医科大学药学院, 安徽 合肥 230032

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*谢晋, E-mail: ;
查良平, E-mail:
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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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