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Article(id=1200500168996024385, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-1026, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1693497600000, receivedDateStr=2023-09-01, revisedDate=1711209600000, revisedDateStr=2024-03-24, acceptedDate=null, acceptedDateStr=null, onlineDate=1764151144503, onlineDateStr=2025-11-26, pubDate=1718121600000, pubDateStr=2024-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764151144503, onlineIssueDateStr=2025-11-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764151144503, creator=13701087609, updateTime=1764151144503, updator=13701087609, issue=Issue{id=1200500165426672625, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='6', pageStart='1509', pageEnd='1896', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764151143651, creator=13701087609, updateTime=1764225143180, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200810542001680840, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200810542001680841, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1854, endPage=1863, ext={EN=ArticleExt(id=1200500170556305534, articleId=1200500168996024385, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Functional study of glycosyltransferase genes CtUGT25 in the flavone biosynthesis pathway of Carthamus tinctorius L., columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

UDP glycosyltransferase (UGT) is a terminal modifying enzyme for the formation of flavonoid glycosides. In this study, we obtained two glycosyltransferase genes, CtUGT25 and CtUGT18, which are closely related to the synthesis of safflower flavonoids, through the Pierce correlation analysis of the expression of glycosyltransferase genes in the safflower corolla transcriptome database at different developmental stages with the contents of the major constituents of safflower metabolome database, and bioinformatically analyzed the gene and protein sequences of the two genes. Expression pattern analysis revealed that CtUGT25 was mainly expressed in the corolla, with the highest expression on day 3 of flowering stage; CtUGT18 was mainly expressed in the root, with the highest expression on day 1 of flowering stage. Functional validation was verified in safflower by Agrobacterium-mediated pollen-tube pathway transgenesis method, demonstrating that CtUGT25 promoted the accumulation of kaempferol-3-O-glucoside and hydroxysafflor yellow A (HSYA), and CtUGT18 promoted the accumulation of kaempferol-3-O-glucoside and orientin, both of which may be the glycosyl-modifying enzymes for the synthesis of safflower flavonoids. Meanwhile, in vitro experiments demonstrated the catalytic activity of CtUGT25 protein on naringenin, quercetin, apigenin, kaempferol, luteoin, 2-hydroxynaringenin and galangin. This study serves as reference for future advancements in regulating the quality of safflower using molecular biotechnology, particularly focuses on the industrial production of safflower exclusive component HSYA. Additionally, it offers valuable insights for researching related genes in other plants.

, correspAuthors=Yue GAO, Mei-li GUO, 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=Shu-yi QI, Lu-nuan WANG, Bei-xuan HE, Yue GAO, Mei-li GUO), CN=ArticleExt(id=1200500175564304750, articleId=1200500168996024385, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=红花黄酮生物合成途径糖基转移酶基因CtUGT25的功能研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

糖基转移酶(UDP-glycosyltransferase, UGT)是黄酮糖苷类化合物形成的末端修饰酶。本研究通过对红花花冠转录组数据库中糖基转移酶基因在四个花期的表达量与红花代谢组数据中主要成分的含量进行皮尔斯相关性分析, 获得了2个与红花黄酮类化合物合成密切相关的糖基转移酶基因CtUGT25、CtUGT18, 并对二者的基因及蛋白序列进行生物信息学分析。表达模式分析发现CtUGT25主要在花冠中表达, 在开花第3天表达量最高; CtUGT18主要在根中表达, 在开花第1天表达量最高。通过农杆菌介导的花粉管道法转基因手段在红花体内进行了功能验证, 证明CtUGT25促进了山柰酚-3-O-葡萄糖苷及羟基红花黄色素A (HSYA)的积累, CtUGT18促进了山柰酚-3-O-葡萄糖苷、荭草苷的积累, 二者可能是红花黄酮类化合物合成的糖基修饰酶。同时, 体外实验证明CtUGT25蛋白对柚皮素、槲皮素、芹菜素、山柰酚、木犀草素、2-羟基柚皮素、高良姜素均具有催化活性。本研究为下一步通过分子生物技术调控红花的品质, 特别是红花中特有成分HSYA等的工业化生产提供参考, 也为其他植物的相关基因的研究提供资料。

, correspAuthors=高越, 郭美丽, authorNote=null, correspAuthorsNote=
*高越, Tel: 13701638627, E-mail: ;
郭美丽, Tel: 13621767286, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=+D+8F9fvQyaVgrmfC3ajLA==, magXml=UvLfR7Vzn2mYE2vfl+2Fig==, pdfUrl=null, pdf=hM+gfpotxA6+WFR/aQ+UXA==, pdfFileSize=3313868, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=H4dfWZP9Po6MOzaPARDyCQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=EYNxhmvzNQiwledrZ/FDYw==, mapNumber=null, authorCompany=null, fund=null, authors=

#并列第一作者.

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Changhai Clinical Research Unit, the First Affiliated Hospital of Naval Medical University, Shanghai 200082, China), AuthorCompanyExt(id=1201118427768385971, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, companyId=1201118427743220147, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.海军军医大学第一附属医院临床研究中心, 上海 200082)])], figs=[ArticleFig(id=1201118434227614393, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=o3RExIAfHLDHgLYFXpoWmA==, figureFileBig=VWYobKtCsl+ICCFpc7EN0w==, tableContent=null), ArticleFig(id=1201118434399580870, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 1, caption= Correlation analysis between the expression of <i>CtUGT</i>s genes and the content of active ingredients in safflower obtained by screening , figureFileSmall=o3RExIAfHLDHgLYFXpoWmA==, figureFileBig=VWYobKtCsl+ICCFpc7EN0w==, tableContent=null), ArticleFig(id=1201118434558964428, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=vVOqbB0SCRpjURWZpRjTLw==, figureFileBig=jP9YTdHFgs6D4leUKfp8qA==, tableContent=null), ArticleFig(id=1201118434680599252, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 2, caption= Phylogenetic tree of amino acid sequences of <i>Ct</i>UGT18 and <i>Ct</i>UGT25 , figureFileSmall=vVOqbB0SCRpjURWZpRjTLw==, figureFileBig=jP9YTdHFgs6D4leUKfp8qA==, tableContent=null), ArticleFig(id=1201118434806428375, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=R4OumSJKlr5gwJe8ZUjEaw==, figureFileBig=EDBCvcIXEVIaUcaNc32Ntw==, tableContent=null), ArticleFig(id=1201118434907091678, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 3, caption= Relative expression levels of <i>CtUGT18</i> and <i>CtUGT25</i> in different tissues and at different developmental stages of the corolla. A: Relative expression levels of <i>CtUGT18</i> in different tissues of the plant; B: Relative expression levels of <i>CtUGT18</i> in different developmental stages of corolla; C: Relative expression levels of <i>CtUGT25</i> in different tissues of the plant; D: Relative expression levels of <i>CtUGT25</i> in different developmental stages of corolla , figureFileSmall=R4OumSJKlr5gwJe8ZUjEaw==, figureFileBig=EDBCvcIXEVIaUcaNc32Ntw==, tableContent=null), ArticleFig(id=1201118435049698023, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=jcuM3EUjMDvpVCHfkrVr5A==, figureFileBig=cUtPkCb+8RzrmhIxGizkMQ==, tableContent=null), ArticleFig(id=1201118435221664493, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 4, caption= Results of expression level measurement and determination of active flavonoid ingredient content of transgenic plants. A: Comparison between <i>Ct</i>UGT18 overexpression group (<i>n</i> = 8) and blank group (<i>n</i> = 7); B: Comparison between <i>Ct</i>UGT25 overexpression group (<i>n</i> = 7) and blank group (<i>n</i> = 7); C: Determination of active flavonoid ingredient content of <i>Ct</i>UGT18 transgenic plants; D: Determination of active flavonoid ingredient content of <i>Ct</i>UGT25 transgenic plants. <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> pMT39-blank , figureFileSmall=jcuM3EUjMDvpVCHfkrVr5A==, figureFileBig=cUtPkCb+8RzrmhIxGizkMQ==, tableContent=null), ArticleFig(id=1201118435326522097, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=6QHKeY6sSXM0JvFUZujSXw==, figureFileBig=d6SrCePgv2B7xfJM33Fxug==, tableContent=null), ArticleFig(id=1201118435406213880, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 5, caption= Molecular docking results of <i>Ct</i>UGT25 with quercetin (A), naringenin (B), apigenin (C), kaempferol (D), luteolin (E), galanign (F), 2-hydroxynaringenin (G), and <i>Ct</i>UGT18 with 2-hydroxynaringenin (H), along with the binding site prediction of <i>Ct</i>UGT25 with flavonoids (I) , figureFileSmall=6QHKeY6sSXM0JvFUZujSXw==, figureFileBig=d6SrCePgv2B7xfJM33Fxug==, tableContent=null), ArticleFig(id=1201118435544625920, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=a9A3Xmw4x7sXAjhIFzNe9A==, figureFileBig=8rNPpYfJIy/Fys9rDK9Ztw==, tableContent=null), ArticleFig(id=1201118435695620872, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 6, caption= Enzyme activation reaction of <i>Ct</i>UGT25 with naringenin as substrate. A: HPLC analysis of the enzyme activation reaction of <i>Ct</i>UGT25 with naringenin as substrate; B: MS analysis of the enzyme activation reaction of <i>Ct</i>UGT25 with naringenin as substrate. a: Naringenin; b: Naringenin mono-glycosylation product , figureFileSmall=a9A3Xmw4x7sXAjhIFzNe9A==, figureFileBig=8rNPpYfJIy/Fys9rDK9Ztw==, tableContent=null), ArticleFig(id=1201118435825644304, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=oOVIPgQsliKTxpkjjPMVUw==, figureFileBig=MYnEv1s2WkRQn/hGs6MM3g==, tableContent=null), ArticleFig(id=1201118435980833561, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Figure 7, caption= Enzymatic activity reaction of <i>Ct</i>UGT25 with galangin as substrate. A: HPLC analysis of the enzymatic activity reaction of <i>Ct</i>UGT25 with galangin as substrate; B: MS analysis of the enzymatic activity reaction of <i>Ct</i>UGT25 with galangin as substrate. a: Galangin; b: Galangin mono-glycosylation product; c: Galangin double glycosylation product , figureFileSmall=oOVIPgQsliKTxpkjjPMVUw==, figureFileBig=MYnEv1s2WkRQn/hGs6MM3g==, tableContent=null), ArticleFig(id=1201118436119245601, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequenceLength/bp
UGT18FTCAATATCACCATAACCACACAGACGG27
UGT18RCTTCCTCAACGTATTAACTACTCGCAAC28
UGT25FAATGGCTTCCTCACCTCATTTCGTA25
UGT25RTTTTCTATATTAAGCTTTGATTTC24
), ArticleFig(id=1201118436257657636, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Table 1, caption=

Design of full-length clone primers for CtUGT18 and CtUGT25

, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequenceLength/bp
UGT18FTCAATATCACCATAACCACACAGACGG27
UGT18RCTTCCTCAACGTATTAACTACTCGCAAC28
UGT25FAATGGCTTCCTCACCTCATTTCGTA25
UGT25RTTTTCTATATTAAGCTTTGATTTC24
), ArticleFig(id=1201118436425429807, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequencePositionLength /bp
U18QPFCCTCTGTGAGTCTATTCCTAAGCA27024
U18QPRCCTCTGTGAGTCTATTCCTAAGCA35622
U25QPFGAAGGTTGAGGAGATTGG1 30218
U25QPRGAAGGTTGAGGAGATTGG1 39719
), ArticleFig(id=1201118436593201976, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Table 2, caption=

Primers used for CtUGT18 and CtUGT25 qRT-PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequencePositionLength /bp
U18QPFCCTCTGTGAGTCTATTCCTAAGCA27024
U18QPRCCTCTGTGAGTCTATTCCTAAGCA35622
U25QPFGAAGGTTGAGGAGATTGG1 30218
U25QPRGAAGGTTGAGGAGATTGG1 39719
), ArticleFig(id=1201118436719031099, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequence
UGT18-pMT39-FGAGCTTTCGCGGATCCGCCACCATGGGTTCGACGGC
UGT18-pMT39-RTCCTCGCCCTTGCTCACCATGGTGGCGGCCGCATTTTTC
UGT25-pMT39-FGAGCTTTCGCGGATCCGCCACCCCTCCAAAAATGGCTTCC
UGT25-pMT39-RTCCTCGCCCTTGCTCACCATGGTGGCGGCCGCGATTCTTAGGAGGTG
), ArticleFig(id=1201118436857443143, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Table 3, caption=

CtUGT18-pMT39 and CtUGT25-pMT39 homologous clone primers

, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequence
UGT18-pMT39-FGAGCTTTCGCGGATCCGCCACCATGGGTTCGACGGC
UGT18-pMT39-RTCCTCGCCCTTGCTCACCATGGTGGCGGCCGCATTTTTC
UGT25-pMT39-FGAGCTTTCGCGGATCCGCCACCCCTCCAAAAATGGCTTCC
UGT25-pMT39-RTCCTCGCCCTTGCTCACCATGGTGGCGGCCGCGATTCTTAGGAGGTG
), ArticleFig(id=1201118438057014090, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequence
UGT18-pet28a (F)GTGGTGGTGGTGGTGCTCGAGATGGGTTCGACGGCAAAAA
UGT18-pet28a (R)CAGCAAATGGGTCGCGGATCCATTTTTCAATAAAACATCAGTAATTAACTTAT
UGT25-pet28a (F)CAGCAAATGGGTCGCGGATCCATGGCTTCCTCACCTCATTTCG
UGT25-pet28a (R)GTGGTGGTGGTGGTGCTCGAGGATTCTTAGGAGGTGTAAATCATCCA
), ArticleFig(id=1201118438199620436, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Table 4, caption=

CtUGT18-pET28a (+) and CtUGT25-pET28a (+) homologous clone primers

, figureFileSmall=null, figureFileBig=null, tableContent=
PrimerSequence
UGT18-pet28a (F)GTGGTGGTGGTGGTGCTCGAGATGGGTTCGACGGCAAAAA
UGT18-pet28a (R)CAGCAAATGGGTCGCGGATCCATTTTTCAATAAAACATCAGTAATTAACTTAT
UGT25-pet28a (F)CAGCAAATGGGTCGCGGATCCATGGCTTCCTCACCTCATTTCG
UGT25-pet28a (R)GTGGTGGTGGTGGTGCTCGAGGATTCTTAGGAGGTGTAAATCATCCA
), ArticleFig(id=1201118438296089431, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
ReceptorCtUGT18CtUGT25
Exhaustiveness8.008.00
Center_x13.82-15.82
Center_y9.901.55
Center_z24.41-48.41
Size_x67.0071.22
Size_y65.7162.51
Size_z71.4862.67
), ArticleFig(id=1201118438396752733, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500168996024385, language=CN, label=Table 5, caption=

Protein substrate docking coordinate parameters

, figureFileSmall=null, figureFileBig=null, tableContent=
ReceptorCtUGT18CtUGT25
Exhaustiveness8.008.00
Center_x13.82-15.82
Center_y9.901.55
Center_z24.41-48.41
Size_x67.0071.22
Size_y65.7162.51
Size_z71.4862.67
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红花黄酮生物合成途径糖基转移酶基因CtUGT25的功能研究
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齐书艺 1, # , 王璐暖 1, # , 何贝轩 1 , 高越 2, * , 郭美丽 1, *
药学学报 | 研究论文 2024,59(6): 1854-1863
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药学学报 | 研究论文 2024, 59(6): 1854-1863
红花黄酮生物合成途径糖基转移酶基因CtUGT25的功能研究
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齐书艺1, #, 王璐暖1, #, 何贝轩1, 高越2, * , 郭美丽1, *
作者信息
  • 1.海军军医大学药学系生药学教研室, 上海 200082
  • 2.海军军医大学第一附属医院临床研究中心, 上海 200082

通讯作者:

*高越, Tel: 13701638627, E-mail: ;
郭美丽, Tel: 13621767286, E-mail:
Functional study of glycosyltransferase genes CtUGT25 in the flavone biosynthesis pathway of Carthamus tinctorius L.
Shu-yi QI1, Lu-nuan WANG1, Bei-xuan HE1, Yue GAO2, * , Mei-li GUO1, *
Affiliations
  • 1. Department of Pharmacognosy, College of Pharmacy, Naval Medical University, Shanghai 200082, China
  • 2. Changhai Clinical Research Unit, the First Affiliated Hospital of Naval Medical University, Shanghai 200082, China
出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2023-1026
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摘要
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糖基转移酶(UDP-glycosyltransferase, UGT)是黄酮糖苷类化合物形成的末端修饰酶。本研究通过对红花花冠转录组数据库中糖基转移酶基因在四个花期的表达量与红花代谢组数据中主要成分的含量进行皮尔斯相关性分析, 获得了2个与红花黄酮类化合物合成密切相关的糖基转移酶基因CtUGT25、CtUGT18, 并对二者的基因及蛋白序列进行生物信息学分析。表达模式分析发现CtUGT25主要在花冠中表达, 在开花第3天表达量最高; CtUGT18主要在根中表达, 在开花第1天表达量最高。通过农杆菌介导的花粉管道法转基因手段在红花体内进行了功能验证, 证明CtUGT25促进了山柰酚-3-O-葡萄糖苷及羟基红花黄色素A (HSYA)的积累, CtUGT18促进了山柰酚-3-O-葡萄糖苷、荭草苷的积累, 二者可能是红花黄酮类化合物合成的糖基修饰酶。同时, 体外实验证明CtUGT25蛋白对柚皮素、槲皮素、芹菜素、山柰酚、木犀草素、2-羟基柚皮素、高良姜素均具有催化活性。本研究为下一步通过分子生物技术调控红花的品质, 特别是红花中特有成分HSYA等的工业化生产提供参考, 也为其他植物的相关基因的研究提供资料。

红花  /  黄酮类化合物  /  糖基转移酶  /  生物合成途径  /  羟基红花黄色素A

UDP glycosyltransferase (UGT) is a terminal modifying enzyme for the formation of flavonoid glycosides. In this study, we obtained two glycosyltransferase genes, CtUGT25 and CtUGT18, which are closely related to the synthesis of safflower flavonoids, through the Pierce correlation analysis of the expression of glycosyltransferase genes in the safflower corolla transcriptome database at different developmental stages with the contents of the major constituents of safflower metabolome database, and bioinformatically analyzed the gene and protein sequences of the two genes. Expression pattern analysis revealed that CtUGT25 was mainly expressed in the corolla, with the highest expression on day 3 of flowering stage; CtUGT18 was mainly expressed in the root, with the highest expression on day 1 of flowering stage. Functional validation was verified in safflower by Agrobacterium-mediated pollen-tube pathway transgenesis method, demonstrating that CtUGT25 promoted the accumulation of kaempferol-3-O-glucoside and hydroxysafflor yellow A (HSYA), and CtUGT18 promoted the accumulation of kaempferol-3-O-glucoside and orientin, both of which may be the glycosyl-modifying enzymes for the synthesis of safflower flavonoids. Meanwhile, in vitro experiments demonstrated the catalytic activity of CtUGT25 protein on naringenin, quercetin, apigenin, kaempferol, luteoin, 2-hydroxynaringenin and galangin. This study serves as reference for future advancements in regulating the quality of safflower using molecular biotechnology, particularly focuses on the industrial production of safflower exclusive component HSYA. Additionally, it offers valuable insights for researching related genes in other plants.

safflower  /  flavonoid  /  glycosyltransferase  /  biosynthetic pathway  /  hydroxysafflor yellow A
齐书艺, 王璐暖, 何贝轩, 高越, 郭美丽. 红花黄酮生物合成途径糖基转移酶基因CtUGT25的功能研究. 药学学报, 2024 , 59 (6) : 1854 -1863 . DOI: 10.16438/j.0513-4870.2023-1026
Shu-yi QI, Lu-nuan WANG, Bei-xuan HE, Yue GAO, Mei-li GUO. Functional study of glycosyltransferase genes CtUGT25 in the flavone biosynthesis pathway of Carthamus tinctorius L.[J]. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1854 -1863 . DOI: 10.16438/j.0513-4870.2023-1026
正文
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红花(Carthami Flos)是常用的传统中药, 我国最早载于经典中药古籍《开宝本草》, 有活血化瘀、祛湿止痛功效[1]。红花作为一味传统中药, 在临床应用中已有2 500年的历史[2]。红花中的多种化合物在扩张冠状动脉、改善心肌缺血、抗凝和抗血栓等方面均具有广泛的药理活性[3]。红花中黄酮类化合物为主要药效成分, 其中羟基红花黄色素A (HSYA)、红花胺(tinctormine)、carthamin、cartormin等是红花中特有的活性成分, HSYA作为红花药材质量评价的标准[4], 其生物合成途径多年来一直是研究的热点。
糖基转移酶是植物体内广泛存在的一种进行糖基化反应的转移酶, 它可以将糖基供体分子如UDP-葡萄糖、UDP-葡萄糖醛酸、UDP-木糖、UDP-鼠李糖、核苷-2-磷酸糖[5-7]上的活性糖基转移到糖基受体底物如单糖和多糖、蛋白质、脂类[8]及小分子天然化合物上, 从而改变受体的亲水性、稳定性、化学性质、亚细胞定位以及生物活性, 同时维持植物自身的代谢平衡[9]。糖基转移酶按照糖基化位点分类可以分成O-GT[10]、N-GT[11]、C-GT[12]、S-GT[13]。在红花中, Sui[14]通过体外实验发现了O-GT基因UGT88B2。Xie等[15]发现了底物杂泛性的糖基转移酶基因UGT73AE1, 进一步研究表明其具有O-GT、N-GT、S-GT催化活性。Guo等[16]在红花的转录数据库中筛选并克隆出3个UGT基因, 并初步预测了O-GT功能。但是迄今为止, 红花中C-GT基因未见报道。红花中黄酮类化合物的生物合成途径已在多种模式植物及农作物中得到广泛研究, 但仍未被完全解析。特别是红花中含有糖基的特有药用成分如HSYA[17]、红花黄色素A (safflor yellow A)[18]、红花黄色素B (safflor yellow B)[17]、cartormin[19]、carthamin[17]等, 它们生物合成途径上的相关功能基因尚不完全清楚。
本研究利用分子生物学和生物化学方法对可能影响红花黄酮类成分生物合成的糖基转移酶基因CtUGT25、CtUGT18进行了功能研究, 初步探究了其在红花黄酮类次生代谢物特别是HSYA生物合成的末端修饰过程中的作用。研究内容为下一步通过分子生物技术调控红花的品质以及工业化生产红花中重要黄酮类化合物提供了重要前提, 也为其他植物的相关基因的研究提供了资料。
材料与方法
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实验材料及处理  植物材料为课题组多代纯化的云南巍山红花品系(ZHH0119), 由海军军医大学药学系郭美丽教授鉴定为红花Carthamus tinctorius L.。红花种植条件: 温度恒定25 ± 5 ℃, 16 h光照, 8 h黑暗。采集红花组织后迅速存放于液氮并于-80 ℃冰箱中冷冻。
菌株、载体与试剂  大肠杆菌Escherichia coli T1感受态细胞、根癌农杆菌(Agrobacterium tumefaciens)GV3101感受态细胞、Rosetta (DE3)感受态细胞、植物RNA提取试剂盒、反转录试剂盒、胶回收试剂盒、质粒提取试剂盒、pEASY®-Basic Seamless Cloning and Assembly Kit无缝克隆试剂盒、qPCR Supermix试剂盒、Blunt Zero克隆载体、NcoⅠ限制性内切酶、BamHI限制性内切酶、XhoⅠ限制性内切酶购自北京全式金生物技术有限公司。2×Phanta® Flash Master Mix (Dye Plus)高保真酶购自南京诺唯赞生物科技有限公司。His标签蛋白纯化试剂盒、BCA蛋白浓度测定试剂盒购自上海碧云天生物技术有限公司。真核表达载体PMT39为课题组自存, 原核表达载体pET-28a(+)购自上海酶研生物科技有限公司。化合物标准品除2-羟基柚皮素购自成都乐美天医药科技, 其余都购自上海源叶生物科技有限公司。实验所用引物全部由上海生工生物有限公司合成。
红花黄酮类成分合成相关糖基转移酶基因筛选  基于红花花冠转录组数据库[20], 筛选得到可能的糖基转移酶基因, 根据NR注释、NT注释、COG注释、GO注释、KEGG注释等结果综合分析, 确定是否为红花中的糖基转移酶。计算得到基因的四个花期: 开花前3天(Ⅰ期)、开花当天(Ⅱ期)、开花第1天(Ⅲ期)和第3天(Ⅳ期)的表达量, 并与红花代谢组数据库中相同样本所测得的柚皮素(naringenin)、芹菜素(apigenin)、槲皮素(quercetin)、芦丁(rutin)、山柰酚(kaempferol)、木犀草素(luteolin)、carthamin、HSYA、槲皮素-3-O-葡萄糖苷(quercetin-3-O-glucoside)、山柰酚-3-O-葡萄糖苷(kaempferol-3-O-gluciside)、野黄芩素(scutellarin)、苯丙氨酸(D-phenylalanine)、山柰酚-3-O-芸香糖苷(kaempferol-3-O-rutinoside)13个主要成分的含量进行皮尔森相关性分析。根据基因表达量与有效成分含量的相关系数确定目的基因。
红花花冠RNA提取及cDNA第一链的合成  按照植物RNA提取试剂盒的说明提取红花花冠总RNA, 紫外分光光度计测量所提取RNA的吸光度, 选取A260/A280在1.8~2.0之间的样品作为反转录模板。使用TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix试剂盒合成cDNA第一链, 样品置于-80 ℃保存。
CtUGT18、CtUGT25基因全长克隆  基于数据库中的基因片段, 用Oligo软件设计特异性引物(表 1)。利用2×Phanta® Flash Master Mix (Dye Plus)高保真酶做目的基因的PCR扩增反应, 反应条件为95 ℃预变性3 min; 95 ℃变性5 s, 50~70 ℃ (设置梯度)退火15 s, 72 ℃延伸1 min, 35个循环; 72 ℃延伸5 min。PCR产物经琼脂糖电泳凝胶检测并回收, 按pEASY®-Blunt Zero Cloning Kit试剂盒说明将回收产物连接至pEASY®-Blunt Zero Cloning Vector, 转化T1大肠杆菌(E. coli)感受态细胞, 菌液PCR验证阳性单克隆菌株, 提取质粒送上海生工生物测序验证。
CtUGT18、CtUGT25基因生物信息学分析  使用NCBI网站的Open Reading Frame Finder功能模块(https://www.ncbi.nlm.nih.gov/orffinder/)和Conserved Domain Search Service功能模块(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)进行基因的ORF分析和保守域分析。使用NCBI网站的BLAST功能模块(https://blast.ncbi.nlm.nih.gov/Blast.cgi)对基因的氨基酸序列进行同源比对, 并使用MEGA7.0.14软件, 选择邻接法构建系统进化树。用Expasy在线工具(https://web.expasy.org/protparam/)分析CtUGT18和CtUGT25蛋白的理化性质, 并用Protscale (https://web.expasy.org/protscale/)模块进行蛋白的亲水性/疏水性预测。使用WoLF PSORT网站(https://wolfpsort.hgc.jp/)对CtUGT18和CtUGT25蛋白亚细胞定位预测。使用TMHMM-2.0工具(https://services.healthtech.dtu.dk/service.php?TMHMM-2.0)对蛋白进行跨膜区预测。利用SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)工具和SWISS-MODEL工具(https://swissmodel.expasy.org/interactive)对蛋白进行二级结构和三级结构预测。
表达特征分析  提取红花植株不同部位和花冠不同发育期RNA, 将提取的RNA反转录为cDNA, 所得cDNA样品于-40 ℃冰箱中保存。用Beacon design软件在CtUGT18、CtUGT25的ORF框内设计引物(表 2)。选择Ct60S (Ct60S-F: CATCCATTATCCAACAATC; Ct60S-R: AAGAGTAATCAGTCTCCA)作为内参基因。
以红花植株不同部位和花冠不同发育期组织cDNA样品为模板, 利用qPCR Supermix试剂盒和Applied BiosystemsTM QuantStudioTM 3实时定量PCR仪测定CtUGT18和CtUGT25基因的相对表达水平。
CtUGT18、CtUGT25真核表达载体构建及过表达植株获得  根据本课题组保存的pMT-39载体序列, 设计CtUGT18、CtUGT25 ORF区的同源扩增引物(表 3)。以红花cDNA第一链为模板克隆全长, 进行凝胶电泳并回收所扩增的目的片段。使用pEASY®-Basic Seamless Cloning and Assembly Kit无缝克隆试剂盒连接回收片段和线性化载体, 重连载体转化T1大肠杆菌(E. coli)感受态细胞, 测序验证后将测序正确的质粒转化根癌农杆菌GV3101感受态细胞备用。
通过花粉管道法获得过表达植株, 待T0代红花结种子后, 收集T1代转基因种子, 种下T1代植株。PCR鉴定T1代阳性植株。
转基因植株表达水平及黄酮有效成分测定  提取T1代阳性植株花冠cDNA, qRT-PCR方法检测CtUGT18和CtUGT25的转录水平。选择L-4-氯苯丙氨酸作为内标, 采用课题组前期建立的UPLC-Q-TOF/MS方法检测阳性植株中的黄酮有效成分[21]。采用GraphPad Prism8.0.1软件, 在数据框中输入各组所测得的有效成分含量, 使用t检验对结果进行统计学检验, 在“Results”模块中读取对应P值。
CtUGT18、CtUGT25原核表达载体构建及蛋白诱导表达  使用CE Design设计同源克隆引物(表 4)。按pEASY®-Basic Seamless Cloning and Assembly Kit无缝克隆试剂盒说明连接pET-28a(+)线性化载体与克隆片段, 重连载体转化T1大肠杆菌(E. coli)感受态细胞, 选取阳性单克隆菌落测序验证, 将验证正确的重连载体转化Rosetta (DE3)表达菌株。重组载体的Rosetta (DE3)菌液, 接种于100 mL LBK液体培养基(含100 mg·L-1卡那霉素的LB液体培养基)中。在摇床中37 ℃, 180 r·min-1摇至OD600为0.5左右, 使用100 mmol·L-1的IPTG在摇床中16 ℃, 90 r·min-1过夜诱导表达。将菌液离心(3 000 × g)10 min, 10 mL PBS缓冲液重悬菌液, 超声破碎仪30 kW, 4 s, 60个循环破碎细胞。超声破碎期间菌液全程浸于冰浴中, 破碎完成后菌液呈澄清状。4 ℃、17 000 ×g离心15 min破碎后的菌液, 取上清液, 使用His标签蛋白纯化试剂盒(耐还原螯合型)对目的蛋白进行纯化。纯化后的蛋白进行SDS-PAGE蛋白凝胶电泳, 并用BCA蛋白浓度测定试剂盒测定纯化蛋白浓度。
分子对接预测  为探索CtUGT25、CtUGT18的底物特异性以及与各种类型黄酮化合物的亲和力, 选择黄酮类(flavone)的芹菜素、木犀草素, 黄酮醇类(flavonol)的槲皮素、山柰酚、高良姜素, 二氢黄酮类(flavanone)的柚皮素作为底物, 同时选择植物C-糖基转移酶常见的活性底物2-羟基柚皮素与UGTs做亲和性测试。使用PyRx (v0.8)和Autodock Vina (v1.1.1)将目标分子与CtUGT18、CtUGT25进行蛋白底物对接, 对接坐标参数如表 5所示。
蛋白酶活性检测  由于几种底物分子与CtUGT25对接、2-羟基柚皮素与CtUGT18对接都具有较好的亲和力, 因此用这些底物进行体外酶活反应测定。酶促反应总体系为100 μL, 其中受体底物终浓度为0.5 mmol·L-1, 供体UDP-葡萄糖终浓度为0.5 mmol·L-1, Tris-HCl (pH 7.4)终浓度为25 mmol·L-1, 加入纯化蛋白5 μg, 使用双蒸水补足至总体积为100 μL。30 ℃过夜反应, 加入200 μL冷甲醇终止反应。4 ℃、17 000 ×g离心15 min。取20 μL上清液, 用于下一步HPLC检测, 其余样品于-80 ℃冰箱中保存。
使用串联质谱联用仪UPLC-Q-TOF/MS: Agilent 1290 Infinity LC和Agilent 6538 Accurate Mass Quadrupole Time-of-Flight。进一步检测酶活反应产物, 进样量3 μL, 色谱柱为XBridgeTM BEH C18柱(100 mm × 2.1 mm, 2.5 μm)。流动相为0.1%甲酸-水(A)和0.1%甲酸-乙腈(B)。槲皮素、柚皮素、芹菜素、山柰酚、木犀草素、高良姜素的洗脱梯度条件: 0~20 min (20%~55% A和80%~45% B), 2-羟基柚皮素的流动相比例为固定的25% A和75% B。流动相流速为0.4 mL·min-1, 柱温25 ℃。
质谱信号采集m/z为50~1 100; 正离子模式下毛细管电压4 000 V, 负离子模式下毛细管电压3 500 V; 鞘气45 psi, 离子源加热温度350 ℃, 干燥器流速11 L·min-1; 碰撞诱导解离(CID)电压20 V; 正离子模式下参比离子121.050 9和922.009 8, 负离子模式下参比离子119.036 32和966.000 725。
所测结果使用Agilent Mass Hunter B.08.00软件进行数据提取及分析。
结果
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1 红花黄酮类成分合成相关糖基转移酶基因筛选
从红花花冠转录组数据库中筛选得到58个糖基转移酶基因, 命名为CtUGT1~CtUGT58。将筛选所得的58个CtUGTs基因在4个花期的相对表达量与代谢组数据库中相同样本所测得的13个主要成分含量进行皮尔森相关分析, 分析结果如图 1所示。
通过皮尔森相关性分析发现, CtUGT18的表达量与HSYA (rHSYA = 0.80)、柚皮素(rnaringenin = 0.99)、山柰酚-3-O-芸香糖苷(rkaempferol-3-O-rutinoside = 0.87)、苯丙氨酸(rD-phenylalanine = 0.86)、carthamin (rcarthamin = 0.51)5个有效成分含量的相关系数大于0.5; CtUGT25的表达量与HSYA (rHSYA = 0.62)、槲皮素(rquercetin = 1.00)、槲皮素-3-O-葡萄糖苷(rquercetin 3-O-glucoside = 0.92)、山柰酚-3-O-葡萄糖苷(rkaempferol-3-O-glucoside = 0.89)、木犀草素(rluteolin = 0.88)、芦丁(rrutin = 0.83)、野黄芩素(rscutellarin = 0.67)、山柰酚- 3-O-芸香糖苷(rkaempferol-3-O-rutinoside = 0.54)、苯丙氨酸(rD-phenylalanine = 0.52)9个有效成分含量的相关系数大于0.5。相关系数大于0.5即可认为两者是中等相关乃至强相关的关系, 由此推测CtUGT18可能参与HSYA、山柰酚-3-O-芸香糖苷、carthamin等红花中有效成分的生物合成; CtUGT25可能参与HSYA、槲皮素-3-O-葡萄糖苷、山柰酚-3-O-葡萄糖苷、山柰酚-3-O-芸香糖苷等红花中有效成分的生物合成。选择CtUGT18、CtUGT25进行下一步实验。
2 CtUGT18、CtUGT25基因全长克隆
CtUGT18、CtUGT25分别在反应温度为65.9~70 ℃、55~57.5 ℃时, 可得到明亮条带。使用DNAMAN软件比对CtUGT18、CtUGT25测序片段与数据库中的序列, 显示测序序列正确。
3 CtUGT18、CtUGT25生物信息学分析
3.1 序列分析
CtUGT18序列全长1 738 bp, ORF区在45~1 502 bp, 编码485个氨基酸。CtUGT25序列全长1 746 bp, ORF区在11~1 483 bp, 编码490个氨基酸。
CtUGT18保守域分析显示, CtUGT18蛋白和CtUGT25蛋白都包含14个活性位点, 1个受体-底物结合口袋, 6个TDP结合位点, 属于糖基转移酶超家族。
3.2 系统进化分析
对CtUGT18、CtUGT25氨基酸序列进行系统进化分析如图 2所示, CtUGT18与洋蓟中的UDP糖基转移酶85A8类亲缘关系最近。CtUGT25与红花CtUGT90A14氨基酸序列相似度最高, 且CtUGT25和CtUGT90A14与洋蓟中的UDP糖基转移酶90A1类亲缘关系最近, UGT90A1家族在青蒿、除虫菊等植物中均有报道。
3.3 蛋白分析及预测
CtUGT18蛋白分子式为C2444H3797N645O714S33, 蛋白分子量54 697.91, 理论等电点(pI)为5.73, 为不稳定的亲水性蛋白。CtUGT25蛋白分子式为C2495H3929N669O710S20, 蛋白分子量55 298.90, pI为5.92, 为不稳定的亲水性蛋白。亚细胞定位预测显示, CtUGT18最可能定位在细胞质, CtUGT28最可能定位在细胞核。
对CtUGT25蛋白二级结构预测显示, CtUGT25氨基酸序列中有41.43%的氨基酸组成α-螺旋, 延伸链占15.10%, 无规则卷曲占36.94%, 仅有6.53%的氨基酸组成β-折叠。CtUGT18蛋白二级结构预测显示, CtUGT18氨基酸序列中有44.95%的氨基酸组成α-螺旋, 延伸链占14.85%, 无规则卷曲占33.2%, 仅有7.40%的氨基酸组成β-折叠。
4 CtUGT18、CtUGT25表达特征分析
CtUGT18在不同部位和不同发育期相对表达水平如图 3A、B所示, CtUGT18在红花植株不同部位表达水平为根 > 花冠 > 苞片 > 叶 > 茎, 主要在根中表达。CtUGT18在红花花冠各个花期的表达量为Ⅲ期 > Ⅰ期 > Ⅱ期 > Ⅳ期, 其中Ⅲ期的表达量最高。
CtUGT25在不同部位和不同发育期相对表达水平如图 3C、D所示, CtUGT25在植株不同部位表达水平为花冠 > 根 > 叶 > 茎 > 苞片, 主要在花冠中表达。CtUGT25在红花花冠各个花期的表达量为Ⅳ期 > Ⅰ期 > Ⅱ期 > Ⅲ期, 其中Ⅳ期的表达量最高。
5 真核表达载体构建与阳性植株鉴定
CtUGT18、CtUGT25与pMT-39载体相连接的菌液PCR凝胶电泳结果条带位置正确, 测序结果正确。
根据CtUGT18过表达红花样品阳性植株鉴定结果, 得到7个阳性样品CtUGT18-OVX1~7。根据CtUGT25过表达红花样品阳性植株鉴定结果, 得到8个阳性样品CtUGT25-OVX1~8。
6 阳性植株表达水平与黄酮类有效成分表达测定
阳性植株表达水平测定结果如图 4A、B所示, 与pMT39空载体处理的空白组相比, CtUGT18-OVX组的目的基因表达量提高了1.78倍(P < 0.05), CtUGT25-OVX组的目的基因表达量提高了2.17倍(P < 0.01)。
内标法测量CtUGT18转基因植株黄酮有效成分, 结果显示CtUGT18过表达植株中山柰酚-3-O-芸香糖苷含量降低了55.15% (P < 0.05), 而山柰酚-3-O-葡萄糖苷含量提高了1.62倍(P < 0.05, 图 4C)。峰面积归一化法计算CtUGT18转基因植株中荭草苷含量, 显示CtUGT18过表达组荭草苷含量升高了2.28倍(P < 0.01)。
内标法测量CtUGT25转基因植株黄酮有效成分, 结果显示CtUGT25过表达植株中山柰酚-3-O-葡萄糖苷提高了1.61倍(P < 0.05), HSYA含量提高了47.9% (P < 0.05), 苯丙氨酸含量提高了1.88倍(P < 0.01, 图 4D)。
7 CtUGT18、CtUGT25原核表达载体构建及蛋白诱导表达
CtUGT18-pET28a(+)、CtUGT25-pET28a(+)转入大肠杆菌菌液PCR结果正确, 测序结果正确。
CtUGT18蛋白纯化结果显示, 在预测的70×103 Da (CtUGT18与His蛋白共表达)左右有较为明显的纯化条带。CtUGT25蛋白纯化结果显示, 在预测的70×103 Da (CtUGT25与His蛋白共表达)左右有较为明显的纯化条带。所得纯化蛋白浓缩后进行酶活检测。
8 AutoDock Vina分子对接预测
CtUGT25与槲皮素、柚皮素、芹菜素、山柰酚、木犀草素、高良姜素、2-羟基柚皮素的结合亲和力分别为-8.2、-7.8、-7.9、-7.9、-8.1、-7.7、-8.3 kJ·mol-1, 对接结果如图 5A~G所示。预测CtUGT25与上述化合物都具有较好的酶活反应潜力。
CtUGT18与2-羟基柚皮素结合亲和力为-7.8 kJ·mol-1, 对接结果如图 5H所示, 预测CtUGT18与2-羟基柚皮素具有较好的酶活反应潜力。
将槲皮素、柚皮素、芹菜素、山柰酚、木犀草素、高良姜素、2-羟基柚皮素与CtUGT25进行合并, 结果如图 5I所示, 发现CtUGT25具有2个活性口袋, 其中槲皮素与2-羟基柚皮素结合于同一活性口袋中, 柚皮素、芹菜素、山柰酚、木犀草素、高良姜素结合于同一活性口袋中。
9 蛋白酶活反应结果
CtUGT25以柚皮素为底物酶活反应结果如图 6所示。与空白对照组相比, 酶活反应组在6.59 min处有新的峰产生, 二级质谱显示为单-O-葡萄糖苷, 推测CtUGT25可以催化柚皮素的单糖基化, 生成对应的单-O-葡萄糖苷产物。
CtUGT25分别以槲皮素、芹菜素、山柰酚、木犀草素、2-羟基柚皮素为底物进行酶活反应, 结果显示, 与空白对照组相比, 酶活反应组分别在6.66、6.60、6.38、6.78、6.00 min处有新的峰产生, 二级质谱都显示为单-O-葡萄糖苷。推测CtUGT25可以催化多种类型黄酮类化合物的单糖基化, 生成对应的单-O-葡萄糖苷产物。
CtUGT25以高良姜素为底物酶活实验结果如图 7所示。与空白对照组相比, 酶活反应组在7.10 min处有新的峰产生, 二级质谱显示为单-O-葡萄糖苷; 在5.52 min处有新的峰产生, 二级质谱显示为双-O-葡萄糖苷。推测CtUGT25可以同时催化高良姜素的单糖基化和双糖基化, 生成对应的单-O-葡萄糖苷和双-O-葡萄糖苷产物。
CtUGT18以2-羟基柚皮素为底物的酶活反应未检测到相应的糖基化产物。
讨论
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由糖基转移酶介导的糖苷化是天然产物如黄酮中最常见的修饰之一, 糖苷化可以增加化合物在细胞中的溶解度和稳定性[22]。糖基转移酶在红花生长发育和次生代谢中扮演着非常重要的角色[8]。迄今为止, 红花体内具体对黄酮成分起末端修饰作用的糖基转移酶仅有少数报道[15, 16, 23, 24]。
通过对一些功能尚不明确的植物基因进行时间及空间的表达特征分析, 可以在一定程度上提示该基因具有的生理功能[25]。在番茄[26]、茶树[27]、西葫芦[28]等植物的研究中均有大量报道。在红花中, Wu等[25]利用qRT-PCR对红花昼夜节律基因CtPRR1在红花体内时-空间转录水平测定, 发现了CtPRR1对红花中黄酮类化合物昼夜节律积累的负调节作用。Lu等[29]对红花花青素还原酶基因ANR进行表达模式分析, 发现其花中表达量最高, 在根和茎中表达量最低。Peng等[30]发现HD-Zip基因家族在红花不同组织中表达量不同。本研究运用qRT-PCR的方法, 对CtUGT18、CtUGT25在红花植株不同部位、花冠不同发育期中进行了表达特征分析, 推测CtUGT18参与根中的黄酮类化合物次生代谢途径, 可能与红花的生长、抗外界胁迫等生理功能相关; CtUGT25则与红花中黄酮积累模式相似, 推测其参与了花中黄酮类化合物的生物合成。
过表达技术作为植物体内进行基因功能研究的常用方法, 技术成熟应用广泛, 通过使目的蛋白在植物体内表达量升高[31], 影响相关生理网络, 获得相应的植株表型, 以探索该基因及蛋白的具体功能, 在水稻[32]、玉米[33]、拟南芥[34]、烟草[35]等植物中均有报道。本研究通过花粉管通道法进行过表达, 共获得CtUGT18过表达植株7株, CtUGT25过表达植株8株。UPLC-Q-TOF/MS测定T1代花冠黄酮类化合物含量, 推测CtUGT18主要在红花体内参与荭草苷、山柰酚-3-O-葡萄糖苷的合成; CtUGT25可能具有底物杂泛性并参与了红花中特有的查尔酮苷类HSYA等化合物的合成。但CtUGT18、CtUGT25在红花体内是怎样对底物进行糖基化的, 还有待进一步探究。
对目的基因进行体外蛋白表达及酶活性研究是红花UGT功能研究的一个非常重要的环节。Sui等[24]发现了UGT88B2可以体外催化山柰酚形成糖基化产物成山柰酚7-O-β-D葡萄糖醛酸苷; Zhang等[23]发现了3个UGT基因(UGT84A33、UGT71AE1和UGT90A14)对小檗碱类化合物的催化活性; Xie等[15]发现底物杂泛性的糖基转移酶UGT71E5可在体外催化19个化合物的糖基化反应。本研究使用AutoDock Vina分子对接预测两者的糖基转移酶功能, 结果显示CtUGT25对多种黄酮类化合物有较好的酶活反应潜力, CtUGT18对2-羟基柚皮素有较好的酶活反应潜力。基于预测结果, 进一步在体外原核表达蛋白并进行酶活反应, 结果显示CtUGT25蛋白可催化槲皮素、柚皮素、芹菜素、山柰酚、木犀草素、2-羟基柚皮素生成对应的单-O-葡萄糖苷产物, 同时可催化高良姜素生成对应的双-O-葡萄糖苷产物。但是CtUGT25对上述底物的具体糖基化位点还有待进一步的分析, 考虑通过更换载体、定点诱导等方法探究其在红花中的具体作用方式。体外酶活反应中, CtUGT18蛋白的催化结果为阴性, 考虑CtUGT18可能具有严格的底物选择性, 其特异性催化底物不在选择的底物范围内; 许多植物UGT蛋白的体内外功能并不完全一致[36], 考虑CtUGT18可能在体外利用类黄酮苷元以外的其他化合物作为底物; 此外, UGT蛋白催化的酶活反应常常由于反应条件的改变引起产物的改变[37], 因此也可能是体外酶活反应的条件未达到CtUGT18蛋白催化的适宜范围内。
本研究分析并验证了CtUGT25及CtUGT18在红花体内外的功能, 证明CtUGT25能促进山柰酚-3-O-葡萄糖苷及HSYA的积累, 可能参与红花中特有的有效成分HSYA的生物合成, CtUGT18可能参与山柰酚-3-O-葡萄糖苷、荭草苷的生物合成。CtUGT25在体外异源表达可以对多种底物进行糖基化修饰。下一步将继续致力于更多UGT基因的研究, 为最终实现HSYA等红花中高价值黄酮糖苷类成分的体外合成和工业化生产奠定坚实基础。
作者贡献: 郭美丽、高越设计实验并提供资金; 齐书艺、王璐暖参与实验及论文撰写; 何贝轩参与数据分析。
利益冲突: 所有作者均声明不存在利益冲突。
基金
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  • 国家自然科学基金项目(81973421)
  • 国家重点研发计划(2019YFC1711100)
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2024年第59卷第6期
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doi: 10.16438/j.0513-4870.2023-1026
  • 接收时间：2023-09-01
  • 首发时间：2025-11-26
  • 出版时间：2024-06-12
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  • 收稿日期：2023-09-01
  • 修回日期：2024-03-24
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国家自然科学基金项目(81973421)
国家重点研发计划(2019YFC1711100)
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    1.海军军医大学药学系生药学教研室, 上海 200082
    2.海军军医大学第一附属医院临床研究中心, 上海 200082

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*高越, Tel: 13701638627, E-mail: ;
郭美丽, Tel: 13621767286, 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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