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Article(id=1201124481017278555, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201124478286786612, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0939, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1691251200000, receivedDateStr=2023-08-06, revisedDate=1699718400000, revisedDateStr=2023-11-12, acceptedDate=null, acceptedDateStr=null, onlineDate=1764299992087, onlineDateStr=2025-11-28, pubDate=1710172800000, pubDateStr=2024-03-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764299992087, onlineIssueDateStr=2025-11-28, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764299992087, creator=13701087609, updateTime=1764299992087, updator=13701087609, issue=Issue{id=1201124478286786612, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='3', pageStart='493', pageEnd='788', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764299991434, creator=13701087609, updateTime=1764300490467, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1201126571420639892, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201124478286786612, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1201126571420639893, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201124478286786612, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=775, endPage=783, ext={EN=ArticleExt(id=1201124481847750781, articleId=1201124481017278555, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Enhancement of tropane alkaloids biosynthesis in Atropa belladonna hariy root by overexpression of HnCYP82M3 and DsTRI genes, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase Ⅰ (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

, correspAuthors=Wei QIANG, 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=De-hui MU, Yan-hong LIU, Piao-piao CHEN, Ai-juan TAN, Bing-nan MA, Hang PAN, Ming-sheng ZHANG, Wei QIANG), CN=ArticleExt(id=1201124484511133932, articleId=1201124481017278555, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

托品烷生物碱(TAs) 是一类临床上广泛应用的抗胆碱药物, 颠茄是提取TAs的主要药源植物, 利用代谢工程技术大量获取TAs具有重要的产业价值。本研究基于开源引流代谢工程策略, 在颠茄发根中超表达TAs合成途径分支处的细胞色素氧化酶(cytochrome oxidase, CYP82M3) HnCYP82M3 (来源于天仙子) 和托品酮还原酶Ⅰ (tropinone reductase Ⅰ, TRI) DsTRI (来源于曼陀罗) 两个酶基因, 以期通过CYP82M3催化合成托品酮(开源), TRI进而将托品酮引入TAs合成的代谢方向(引流), 大量促进3种TAs的积累。从天仙子根中克隆了HnCYP82M3基因, 其编码氨基酸与颠茄AbCYP82M3序列一致性高达91.7%。单独超表达HnCYP82M3不影响颠茄发根TAs的含量, 说明CYP82M3不是TAs合成的关键酶。同时超表达HnCYP82M3和DsTRI极大促进了3种TAs的积累, 莨菪碱、山莨菪和东莨菪碱含量分别是对照的4.97倍、2.83倍和2.19倍, 并且提高幅度大于DsTRI单表达。本研究证明分支点处酶基因共表达的开源引流策略是有效提高颠茄TAs合成的代谢工程新方法, 为工业化大量获取TAs奠定了理论和技术基础。

, correspAuthors=强玮, authorNote=null, correspAuthorsNote=
*强玮, Tel: 13678505784, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=RHPTII8Hm9Lqni0BIF8h1Q==, magXml=FbI3tCSaZvYtwHpXFgJ9ag==, pdfUrl=null, pdf=LiIZCtfxWreYdF+DV/vqLQ==, pdfFileSize=3262225, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=xTIs9PlQknraDzOv4aeMLQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=BenV8sGRe8iLRysjEiA3wA==, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=穆德会, 刘艳红, 陈漂漂, 谭艾娟, 马炳南, 潘航, 张明生, 强玮)}, authors=[Author(id=1201124484976701712, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, 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=1201124485060587801, 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A: Schematic diagram of p1305NKGFP expression vector; B: Double-gene plant expression vector. <i>HnCYP82M3</i>: Cytochrome oxidase (CYP82M3) gene from <i>Hyoscyamus niger</i>; <i>DsTRI</i>: Tropinone reductase Ⅰ (TRI) gene from <i>Datura stramonium</i> , figureFileSmall=QKL4F1JMoszjCX6rd2HlpA==, figureFileBig=AVCUiZpaE9+RkJCjLB7FDg==, tableContent=null), ArticleFig(id=1201124491985384218, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=EN, label=null, caption=null, figureFileSmall=RzRSlxNNM+LLsAi8uXaL6A==, figureFileBig=1AmkbAycpovJBXnuov9bfg==, tableContent=null), ArticleFig(id=1201124492077658913, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=CN, label=Figure 3, caption= Comparison of AbCYP82M3 and HnCYP82M3 protein sequences. Consistent amino acids are represented by white characters with black background, conserved amino acids are represented by black characters with white background, and other amino acids are represented by black characters. AbCYP82M3: Cytochrome oxidase (CYP82M3) gene from <i>Atropa belladonna</i> , figureFileSmall=RzRSlxNNM+LLsAi8uXaL6A==, figureFileBig=1AmkbAycpovJBXnuov9bfg==, tableContent=null), ArticleFig(id=1201124492278985519, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=EN, label=null, caption=null, figureFileSmall=BZhq2+kYzNHKMOfnTW+x2A==, figureFileBig=xQ1wTc/QD9gavlsZxifByw==, tableContent=null), ArticleFig(id=1201124492388037429, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=CN, label=Figure 4, caption= PCR amplification of <i>HnCYP82M3</i> and <i>DsTRI</i> gene. A: PCR amplification of <i>HnCYP82M3</i> gene; B: PCR amplification of <i>DsTRI</i> gene; M: DNA marker , figureFileSmall=BZhq2+kYzNHKMOfnTW+x2A==, figureFileBig=xQ1wTc/QD9gavlsZxifByw==, tableContent=null), ArticleFig(id=1201124492463534911, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=EN, label=null, caption=null, figureFileSmall=6Td2Em8DkkFtMSBJy35cBg==, figureFileBig=+Iqtyby77kaoAw6Ka9l8ow==, tableContent=null), ArticleFig(id=1201124492543226696, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=CN, label=Figure 5, caption= Obtainment of transgenic hairy roots of <i>A. belladonna</i>. A: Wild type <i>A. belladonna</i> aseptic plant; B, C: Inducing and culture of <i>A. belladonna</i> hairy roots; D: <i>A. belladonna</i> hairy roots cultured in MS liquid medium for one month; E: Detection of the gene (<i>HnCYP82M3</i>, <i>DsTRI</i>, <i>rolC</i> and <i>rolB</i>) by PCR. CK: Control group; TRI: <i>35S: : DsTRI</i>; CYP: <i>35S: : HnCYP82M3</i>; CT: <i>35S: : HnCYP82M3-DsTRI</i>; PC: Positive control; NC: Negative control; <i>rolB</i>: Rooting locus gene B; <i>rolC</i>: Rooting locus gene C , figureFileSmall=6Td2Em8DkkFtMSBJy35cBg==, figureFileBig=+Iqtyby77kaoAw6Ka9l8ow==, tableContent=null), ArticleFig(id=1201124492660667211, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=EN, label=null, caption=null, figureFileSmall=q5d2ARKj8dJqpGc48UPRzg==, figureFileBig=BOMzVKmItzqJFAD0acNxew==, tableContent=null), ArticleFig(id=1201124492794884948, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=CN, label=Figure 6, caption= Content and yield of tropane alkaloids. A-C: The average content of tropane alkaloids; D-F: The yield of tropane alkaloids. <i>n</i> = 15, mean ± SD. DW: Dry weight. <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> CK , figureFileSmall=q5d2ARKj8dJqpGc48UPRzg==, figureFileBig=BOMzVKmItzqJFAD0acNxew==, tableContent=null), ArticleFig(id=1201124492870382427, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=EN, label=null, caption=null, figureFileSmall=XplvowtlY3TfV9/IIWiEZw==, figureFileBig=1btvunnJxwvINrYVl3+ktA==, tableContent=null), ArticleFig(id=1201124493000405862, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=CN, label=Figure 7, caption= Expression levels of target genes in root cultures. <i>n</i> = 15, mean ± SD. <sup>**</sup><i>P</i> < 0.01 <i>vs</i> CK , figureFileSmall=XplvowtlY3TfV9/IIWiEZw==, figureFileBig=1btvunnJxwvINrYVl3+ktA==, tableContent=null), ArticleFig(id=1201124493105263471, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namePrimer sequence (5′→3′)Annealing temperature/℃
F-rolBGCTCTTGCAGTGCTAGATTT57
R-rolBGAAGGTGCAAGCTACCTCTC
F-35SACTATCCTTCGCAAGACCCT57
R-DsTRICCCATTCACAAGCCAAACTC
F-rolCTAACATGGCTGAAGACGACC57
R-rolCAAACTTGCACTCGCCATGCC
F-HnCYP82M3CACGAGATACAGTTATCAAGG55
R-35SpolyATCTGGGAACTACTAACACA
F-HnCYP82M3-OETCTATCTCTCTCGAGTCTAGACTAAGCTCTCGTTATTCTCCT56
R-HnCYP82M3-OECTCGAGCTTGTCGAGCTGCAGGGTGGAGCTTTTATTGAAGAA
F-DsTRI-OECACGGGGGACTCTTGGGATCCATGGAAGAATCAAAAGTGTCC55.5
R-DsTRI-OECGATCGGGGAAATTCGAGCTCGTGATGATAACAACTTGGAAC
Fq-DsTRICCTGCTGCTTCTTATATTACGG57
Rq-DsTRIGCCCAAGGTACAAATCACAA
Fq-HnCYP82M3AGTTATCAAGGCAACTGTATTGAGC60
Rq-HnCYP82M3ATGAGCCATGTTAGGTGAACTGC
Fq-ACTINTTGTGTTGGACTCTGGTGATGG60
Rq-ACTINCCGTTCAGCAGTGGTGGTG
Fq-PGKTCGCTCTTGGAGAAGGTTGAC59.5
Rq-PGKCTTGTCCGCAATCACTACATCAG
), ArticleFig(id=1201124493218509684, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201124481017278555, language=CN, label=Table 1, caption=

Primers of the experiment

, figureFileSmall=null, figureFileBig=null, tableContent=
Primer namePrimer sequence (5′→3′)Annealing temperature/℃
F-rolBGCTCTTGCAGTGCTAGATTT57
R-rolBGAAGGTGCAAGCTACCTCTC
F-35SACTATCCTTCGCAAGACCCT57
R-DsTRICCCATTCACAAGCCAAACTC
F-rolCTAACATGGCTGAAGACGACC57
R-rolCAAACTTGCACTCGCCATGCC
F-HnCYP82M3CACGAGATACAGTTATCAAGG55
R-35SpolyATCTGGGAACTACTAACACA
F-HnCYP82M3-OETCTATCTCTCTCGAGTCTAGACTAAGCTCTCGTTATTCTCCT56
R-HnCYP82M3-OECTCGAGCTTGTCGAGCTGCAGGGTGGAGCTTTTATTGAAGAA
F-DsTRI-OECACGGGGGACTCTTGGGATCCATGGAAGAATCAAAAGTGTCC55.5
R-DsTRI-OECGATCGGGGAAATTCGAGCTCGTGATGATAACAACTTGGAAC
Fq-DsTRICCTGCTGCTTCTTATATTACGG57
Rq-DsTRIGCCCAAGGTACAAATCACAA
Fq-HnCYP82M3AGTTATCAAGGCAACTGTATTGAGC60
Rq-HnCYP82M3ATGAGCCATGTTAGGTGAACTGC
Fq-ACTINTTGTGTTGGACTCTGGTGATGG60
Rq-ACTINCCGTTCAGCAGTGGTGGTG
Fq-PGKTCGCTCTTGGAGAAGGTTGAC59.5
Rq-PGKCTTGTCCGCAATCACTACATCAG
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超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响
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穆德会 , 刘艳红 , 陈漂漂 , 谭艾娟 , 马炳南 , 潘航 , 张明生 , 强玮 *
药学学报 | 研究论文 2024,59(3): 775-783
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药学学报 | 研究论文 2024, 59(3): 775-783
超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响
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穆德会, 刘艳红, 陈漂漂, 谭艾娟, 马炳南, 潘航, 张明生, 强玮*
作者信息
  • 贵州大学生命科学学院/农业生物工程研究院, 山地植物资源保护与种质创新教育部重点实验室, 贵州 贵阳 550025

通讯作者:

*强玮, Tel: 13678505784, E-mail:
Enhancement of tropane alkaloids biosynthesis in Atropa belladonna hariy root by overexpression of HnCYP82M3 and DsTRI genes
De-hui MU, Yan-hong LIU, Piao-piao CHEN, Ai-juan TAN, Bing-nan MA, Hang PAN, Ming-sheng ZHANG, Wei QIANG*
Affiliations
  • Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), College of Life Sciences/Institute of Agro-bioengineering, Guizhou University, Guiyang 550025, China
出版时间: 2024-03-12 doi: 10.16438/j.0513-4870.2023-0939
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摘要
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托品烷生物碱(TAs) 是一类临床上广泛应用的抗胆碱药物, 颠茄是提取TAs的主要药源植物, 利用代谢工程技术大量获取TAs具有重要的产业价值。本研究基于开源引流代谢工程策略, 在颠茄发根中超表达TAs合成途径分支处的细胞色素氧化酶(cytochrome oxidase, CYP82M3) HnCYP82M3 (来源于天仙子) 和托品酮还原酶Ⅰ (tropinone reductase Ⅰ, TRI) DsTRI (来源于曼陀罗) 两个酶基因, 以期通过CYP82M3催化合成托品酮(开源), TRI进而将托品酮引入TAs合成的代谢方向(引流), 大量促进3种TAs的积累。从天仙子根中克隆了HnCYP82M3基因, 其编码氨基酸与颠茄AbCYP82M3序列一致性高达91.7%。单独超表达HnCYP82M3不影响颠茄发根TAs的含量, 说明CYP82M3不是TAs合成的关键酶。同时超表达HnCYP82M3和DsTRI极大促进了3种TAs的积累, 莨菪碱、山莨菪和东莨菪碱含量分别是对照的4.97倍、2.83倍和2.19倍, 并且提高幅度大于DsTRI单表达。本研究证明分支点处酶基因共表达的开源引流策略是有效提高颠茄TAs合成的代谢工程新方法, 为工业化大量获取TAs奠定了理论和技术基础。

CYP82M3  /  TRI  /  托品烷生物碱  /  颠茄  /  代谢工程

Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase Ⅰ (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

CYP82M3  /  TRI  /  tropane alkaloids  /  Atropa belladonna L.  /  metabolic engineering
穆德会, 刘艳红, 陈漂漂, 谭艾娟, 马炳南, 潘航, 张明生, 强玮. 超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响. 药学学报, 2024 , 59 (3) : 775 -783 . DOI: 10.16438/j.0513-4870.2023-0939
De-hui MU, Yan-hong LIU, Piao-piao CHEN, Ai-juan TAN, Bing-nan MA, Hang PAN, Ming-sheng ZHANG, Wei QIANG. Enhancement of tropane alkaloids biosynthesis in Atropa belladonna hariy root by overexpression of HnCYP82M3 and DsTRI genes[J]. Acta Pharmaceutica Sinica, 2024 , 59 (3) : 775 -783 . DOI: 10.16438/j.0513-4870.2023-0939
正文
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托品烷生物碱(tropane alkaloids, TAs) 是一类药用茄科植物所产生的次生代谢产物, 主要包括莨菪碱(hyoscyamine)、山莨菪碱(anisodamine) 和东莨菪碱(scopolamine)。因其具有显著的抗胆碱活性, 通常用作患者麻醉前的使用药, 并用于治疗感染性休克、缓解晕动病、治疗帕金森震颤、镇痛、缓解便秘等[1, 2]。TAs是由少数的药用茄科植物产生, 野生型植物中TAs含量非常低。其中收录于中国药典的颠茄(Atropa belladonna L.), 其野生型中莨菪碱大约仅为干重的0.02%~0.17%, 东莨菪碱约为干重的0.01%~0.08%, 因此丰富和提高TAs的来源对推进中药产业化有重要意义。
Srinivasan等[2]于2020年阐明了整个TAs的合成途径。来源于鸟氨酸和精氨酸脱羧的腐胺是合成TAs的前体[3]。N-甲基-腐胺-转移酶(putrescine N-methyltransferase, PMT) 和甲基腐胺氧化酶(N-methylputrescine oxidase, MPO) 催化腐胺生成4-氨基-正丁醛[4], 该物质自发环化形成N-甲基吡咯啉阳离子, 然后依次经Ⅲ型聚酮合酶(type Ⅲ polyketide syn-thase, PYKS) 和托品酮合成酶(cytochrome oxidase, CYP82M3) 催化生成TAs特有的第一个环状中间体托品酮[5]。两种托品酮还原酶(tropinone reductase, TR) TRI和TRII均以托品酮为底物, 分别催化生成莨菪碱的合成前体托品和打碗花精的前体假托品[6]。托品与来源于苯丙氨酸转氨基、还原和糖基化的另一产物苯乳酰葡萄糖, 在海螺碱合酶(littorine synthase, LS) 的缩合下生成海螺碱[7-9], 再依次经海螺碱变位酶(cytochrome P450 enzyme, CYP80F1) 的异构和莨菪碱脱氢酶(hyoscyamine dehydrogenase, HDH) 的还原最终生成莨菪碱[10, 11]。莨菪碱在双功能酶莨菪碱6-β-羟化酶(hyoscyamine 6-β-hydroxylase, H6H) 的羟化和环化作用下依次转变为山莨菪碱和东莨菪碱(图 1)。
托品酮的合成和还原是TAs合成途径中的主要分支, 存在代谢分流。在瞬时超表达PMT、MPO、PYKS和CYP82M3的烟草叶片中, 托品酮含量(2.31 nmol·mg-1) 远低于其直接前体4-(1-甲基-2-吡咯烷基)-3-氧代丁酸(243.4 nmol·mg-1), 说明托品酮的合成可能是一个限速步骤, CYP82M3直接催化托品酮的生成, 因此可能是限速酶[5]。两个托品酮还原酶竞争托品酮底物, 因为TRII表达量和其酶活性都高于TRI, 由其生成的假托品含量也要高于托品, 且在植物中假托品不能转化为托品[6, 12], 因此TRI和TRII是调节代谢流向TAs积累的关键酶[5]。已在颠茄、三分三和铃铛子[13-15]中通过超表达TRI基因提高了TAs的产量, 证明了TRI是TAs合成中的引流关键酶基因。
基于此, 采用开源引流策略, 在颠茄中同时超表达TAs合成途径分支处的两个酶基因CYP82M3和TRI, 通过托品酮合成酶CYP82M3打破托品酮合成的限速步骤, 大量合成茛菪碱的合成前体(开源), 再通过TRI将托品酮从竞争支路引入莨菪碱合成方向(引流), 减少TRII的分流作用, 有望为TAs的合成提供大量的代谢流, 提高TAs的积累。本研究改造构建了植物双基因表达载体, 在颠茄发根中同时超表达天仙子的HnCYP82M3和曼陀罗的DsTRI基因, 获得双转基因颠茄发根, 和单基因转化相比, TAs含量提高显著, 证明了该策略的优越性, 为工业化大量获取TAs奠定了理论和技术基础。
材料与方法
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植物材料  颠茄(Atropa belladonna L.) 繁育并保存于贵州大学生命科学学院植物生理生化与分子生物学实验室。
主要仪器  高效液相色谱仪[Waters e2695, 沃特世科技(上海) 有限公司]; 实时荧光定量PCR仪(Bio-Rad CFX) 和Demo PCR仪T100 (Bio-Rad T100TM Thermal Cycler) 购买于美国Bio-Rad Laboratories公司。
试剂  植物总RNA提取试剂盒(RNAprep Pure Plant Kit-DP419, #X07131)、胶回收试剂盒(TIANgel Midi Purification Kit, #Y1316)、质粒提取试剂盒(TIAprep Mini Plasmid Kit, #X1010) 等购自天根生化科技(北京) 有限公司; 逆转录试剂盒(RNA PCRTM Kit AMV Ver 3.0, #AK92145A) 和荧光定量染料(TB Green® premix Ex TaqTM Ⅱ, #AME1402A) 购买于宝日医生物技术(北京) 有限公司; 荧光定量反转录试剂盒(PrimeScriptTM RT reagent Kit Perfect Real Time, #RR037A) 和各限制性内切酶及高保真HiFi聚合酶(TransTaq DNA Polymerase High Fidelity, #Q20916) 购买于北京全式金生物技术有限公司; 基因重组试剂盒(one step cloning kit, #L/N7E67212) 购自南京诺唯赞生物科技股份有限公司; 头孢霉素(cefradine, Cef, #323Q032) 和卡那霉素(kanamycin, Kan, #701C044) 等购买于北京索莱宝科技有限公司。本研究所用引物及测序由重庆擎科生物技术有限公司完成。其他试剂和药品皆为国产试剂(分析纯)。
p1305NKGFP植物双基因表达载体的改造  以pCAMBIA1305.1为骨架载体, 用Hind Ⅲ和EcoR Ⅰ双酶切开环获得大骨架, 同样双酶切pBI121回收CaMV35S-GUS-NOST表达框片段, T4连接酶连接这两个片段后获得双价表达载体p1305+121。接着用Hind Ⅲ和Nco Ⅰ双酶切除去Gus Plus编码区上游CaMV35S启动子, 回收载体骨架。根据质粒pBI121上的NOSp启动子序列设计扩增引物, 分别在上下游引物5′端引入含Hind Ⅲ和Nco Ⅰ位点的重组序列, 以pBI121为模板, HIFI高保真聚合酶扩增NOSp片段, 和p1305+121载体骨架进行同源重组, 将p1305+121上原CaMV35S启动子替换为NOSp启动子。同理, 根据相同方法, 通过Nco Ⅰ和BstE Ⅱ两个酶切位点, 将NOSp启动子下游的Gus Plus编码区替换为NPT Ⅱ筛选标记基因, 获得重组载体p1305NK+121。下游引物BstE Ⅱ位点前还可引入广泛使用的Sal Ⅰ内切酶的酶切位点序列, 以增加选择性。方法同上, 先Xho Ⅰ单酶切去除原Hygrr编码序列, 设计GFP编码区扩增引物, 分别在上下游引物5′端引入Xba Ⅰ和Pst Ⅰ酶切位点, PCR扩增获得GFP重组片段, 与大骨架进行重组, 获得最终的植物双基因表达载体, 命名为p1305NKGFP。
目的基因的克隆  以报道的颠茄AbCYP82M3基因序列为探针在天仙子转录组中进行序列比对, 获得一条与颠茄AbCYP82M3基因高度相似的序列, 且具有完整编码框。设计引物以天仙子根cDNA为模板进行PCR, 克隆并测序验证该基因编码序列。将验证后的HnCYP82M3基因提交NCBI (GenBank: OR253591)。在NCBI中查询DsTRI (GenBank: L20473.1) 基因序列, 设计特异引物以曼陀罗根cDNA为模板扩增目的基因。本研究所有引物序列见表 1。
植物表达载体构建  p1305NKGFP质粒用Xba Ⅰ和Pst Ⅰ双酶切切除GFP编码区, 同上述同源重组方法, 单独将HnCYP82M3定向置换入p1305NKGFP原GFP编码区位置, 构建35S: : HnCYP82M3植物表达载体。用Sac Ⅰ和BamH Ⅰ酶切载体上GUS区, DsTRI定向置换入p1305NKGFP原GUS编码区位置, 构建35S: : DsTRI植物表达载体。将其转入大肠杆菌DH5α, 送生物公司测序。在35S: : HnCYP82M3表达载体的基础上用限制性内切酶Sac Ⅰ和BamH Ⅰ进行双酶切, 切除载体中GUS基因片段。将DsTRI定向置换入GUS编码区位置, 构建35S: : DsTRI-HnCYP82M3双基因植物表达载体。将测序正确的菌液扩繁并保存, 提取质粒转化发根农杆菌C58C1, 将阳性菌液作为后续遗传转化的工程菌。
转基因颠茄发根的获得  颠茄发根的遗传转化参照Fu等[16]的方法进行, 以颠茄叶片作为外植体, 以上述C58C1工程菌浸染颠茄叶片, 浸染后的颠茄叶片于培养基(MS+100 μmol·L-1 AS) 上共培养二三天后, 将其转入培养基(MS+100 mg·L-1 Kan+200 mg·L-1 Cef) 中, 直到长出抗性发根, 提取DNA, PCR检测是否转入外源HnCYP82M3/DsTRI基因(用F-HnCYP82M3和R-35SpolyA检测CYP82M3; 用F-35S和R-DsTRI检测TRI)。
HPLC检测TAs含量  参照Lu等[17]制作生物碱标准曲线。线性回归方程分别为东莨菪碱: Y = 6 305.5X - 25 980 (R2 = 0.999 4); 山莨菪碱: Y = 4 585.7X + 5 368.5 (R2 = 0.999 4); 莨菪碱: Y = 7 928.2X - 1 874.6 (R2 = 0.999 6)。将收获的颠茄发根40 ℃烘干至恒重, 粉碎机打磨成粉末, 过60目筛。参照Ashtiania等[18]方法提取生物碱, 参照Ao等[19]高效液相色谱法测定各生物碱。截取各生物碱紫外吸收峰面积, 根据生物碱标准曲线计算各生物碱含量。再用各生物碱含量乘以每瓶发根干重得到每瓶发根的产量。
qRT-PCR检测目的基因表达量  取适量颠茄发根立即液氮速冻, 快速研磨成粉末, 用总RNA提取试剂盒(RNAprep Pure Plant Kit-DP432) 提取RNA, 超微量分光光度计测定核酸的浓度和质量, 当A260/A280 = 1.8~2.1, 表明RNA无蛋白和DNA污染。将质量合格的RNA按照反转录试剂盒说明书反转录为cDNA第一链。利用Beacon designer软件设计qRT-PCR引物(表 1), 参照Ao等[19]进行目的基因相对表达量检测, 以PGK (序列号: JX154676) 和ACTIN (序列号: JX154681) 为内参基因校准各样本的基因表达量从而进行相对定量, 采用2-ΔΔCt法计算各TAs合成途径基因的相对表达量。
统计学分析方法  采用IBM SPSS26.0版本对数据进行统计学分析, 所有数据以平均值± SD表示(mean ± SD)。组间比较采用单因素方差分析(ANOVA), 当P < 0.05时, 该差异有统计学意义。
结果
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1 p1305NKGFP植物双基因表达载体的改造
为了引入双基因遗传转化, 改造构建了植物双基因表达载体p1305NKGFP, 其结构如图 2A。p1305NKGFP载体以pCAMBIA1305.1作为骨架, 在Hind Ⅲ和EcoR Ⅰ位点间引入载体pBI121中的35S-GUS-NOST表达框, 同时将Hygrr基因替换为GFP基因, 原Gus Plus基因替换为筛选标记NPT II基因, 并将CaMV35S启动子替换为NOS启动子。改造后的p1305NKGFP拥有GUS、GFP和NPT II三个表达框, 可同时转入两个目的基因, 并以Kan作为抗性筛选标记。
2 天仙子HnCYP82M3基因的克隆与分析
基于本实验室天仙子转录组数据, 以报道[5]的颠茄AbCYP82M3基因序列为探针在天仙子转录组中进行Blant N检索, 获得一条序列高度相似的Unigene, 该序列包含一个完整的编码框。设计引物以天仙子根cDNA为模板, PCR克隆并测序验证该基因编码序列。该基因编码521个氨基酸, 与颠茄AbCYP82M3氨基酸序列一致性高达91.7%, 如图 3, 将其命名为HnCYP82M3。HnCYP82M3蛋白分析发现在5~27氨基酸处含有1个跨膜螺旋, 预测定位于内质网。
3 HnCYP82M3和DsTRI双基因表达载体的构建
以p1305NKGFP为表达载体, 设计HnCYP82M3和DsTRI基因同源重组PCR引物, 在HnCYP82M3基因片段上下游分别引入Xba Ⅰ和Pst Ⅰ限制性酶切位点, 在DsTRI基因片段上下游分别引入BamH Ⅰ和Sac Ⅰ限制性酶切位点, 扩增相应基因片段(图 4), 通过同源重组分别替换掉p1305NKGFP载体上的GUS和GFP编码基因, 得到双基因植物表达载体p1305NKGFP-HnCYP82M3-DsTRI (图 2B)。将该质粒转入发根农杆菌C58C1, 用于颠茄发根的遗传转化。
4 转基因颠茄发根的获得
以无菌颠茄叶片作为外植体(图 5A) 进行遗传转化, 15天左右分化出抗性发根后(图 5B), 剪取单克隆发根接种到固体平板除菌和扩繁(图 5C), 提取DNA进行PCR鉴定, 鉴定了十多个DsTRI单基因转化根系、HnCYP82M3单基因转化根系和HnCYP82M3-DsTRI双基因转化根系(图 5E)。剪取3根3 cm左右长的根尖接种于100 mL液体培养基中摇床培养30天, 发根表型和生物量没有显著差异(图 5D)。分别将单基因和双基因系发根各自混合, 取样用于生物碱含量和基因表达量检测分析。
5 托品烷生物碱的测定
利用HPLC对颠茄发根中托品烷生物碱含量进行检测, 结果如图 6A~C所示。HnCYP82M3-DsTRI发根(CT) 的3种生物碱(莨菪碱、山莨菪碱、东莨菪碱) 含量均显著高于对照和单基因转化, 莨菪碱含量[9 206.04 μg·g-1 dry weight (DW)] 是对照(1 851.33 μg·g-1 DW) 的4.97倍; 山莨菪碱含量(3 903.83 μg·g-1 DW) 是对照(1 378.26 μg·g-1 DW) 的2.83倍; 东莨菪碱含量(1 803.54 μg·g-1 DW) 是对照(823.48 μg·g-1 DW) 的2.19倍。DsTRI发根中莨菪碱、山莨菪碱、东莨菪碱含量分别比对照提高2.62倍、1.67倍和1.34倍。HnCYP82M3发根的生物碱含量与对照相比无显著差异。
根据TAs含量计算TAs产量, 结果如图 6D~F所示。CT发根生物碱产量均显著高于对照和单基因转化, 莨菪碱产量(每瓶7 048.76 μg) 较对照(每瓶1 191.02 μg) 提高5.92倍; 山莨菪碱产量(每瓶3 502.25 μg) 较对照(每瓶886.68 μg) 提高3.95倍; 东莨菪碱产量(每瓶1 825.78 μg) 较对照(每瓶529.77) 提高3.95倍。DsTRI发根中莨菪碱、山莨菪碱和东莨菪碱含量与对照相比, 分别提高2.31倍、1.48倍和2.25倍。HnCYP82M3的各生物碱产量较对照无显著差异。
6 基因表达量检测
采用qRT-PCR检测各样本中CYP82M3和TRI基因的相对表达量, 结果如图 7。由于在颠茄、曼陀罗和天仙子中CYP82M3和TRI基因高度保守, 序列相似性均在90%以上, 检测时很难区分内外源基因, 所以此处检测的是内外源CYP82M3和TRI总的表达水平。DsTRI单转和CT双转基因发根中, TRI的表达量分别较对照(CK) 提高3.17和5.10倍, 而HnCYP82M3单转发根的TRI表达水平无显著变化。HnCYP82M3单转和CT双转基因发根中, CYP82M3的表达量较对照分别提高2.73和2.93倍, 而DsTRI单转发根的CYP82M3表达水平无显著变化。
讨论
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TAs是来自于少数茄科植物的次生代谢产物, 因其有显著的抗胆碱活性而被广泛应用于临床医学。天然活性产物往往在药源植物中含量都较低, 需要借助现代生物技术来实现商业化生产[20], 代谢工程是最有前景的方法。颠茄TAs生物合成途径于2020年已完全解析[2], 以13个途径酶基因为靶点的分子遗传改造已很充分, 包括单基因超表达和双基因组合表达, 基本阐明了该合成通路上的限速步骤和各基因在TAs合成中的作用, 如PMT是上游的关键酶基因, 但是单独表达对促进TAs合成作用不显著, 需结合其他关键酶基因共表达才能促进TAs合成[21]。TRI和H6H分别是中游和下游的关键酶基因, 单独或组合超表达均能提高莨菪碱或东莨菪碱的积累[13, 22]。最近廖志华团队[1]在颠茄发根体系中, 通过单/双基因, 甚至四基因超表达, 系统研究了新近鉴定的4个途径基因(PYKS、CYP82M3、UGT1和LS) 在TAs合成中的作用, 发现PYKS+CYP82M3和UGT1+LS超表达组合分别可以打通托品酮和海螺碱的合成限制, 四基因超表达同时保障托品酮和海螺碱的供应可获得最高的TAs产量。尽管如此, 针对竞争支路处代谢引流策略的TAs代谢工程还未有报道涉及。颠茄TAs合成途径中游托品酮还原反应处存在严重的代谢分流, TRII无论是基因表达量还是编码的酶活力, 都显著高于TRI基因, 所以TRII竞争TRI将大量托品酮还原为假托品进而代谢合成打碗花精, 其含量是莨菪碱的2倍[13]。因此, 在充分保障托品酮供应的前提下, 通过提高TRI表达量和酶活力, 将更多代谢流引入莨菪碱合成, 预期将极大提高TAs的积累。基于此, 作者设计了同时超表达天仙子HnCYP82M3 (开源) 和曼陀罗DsTRI (引流) 基因的代谢工程策略。颠茄中TAs尤其是东莨菪碱含量低于天仙子, 这可能与AbH6H编码的蛋白质对东莨菪碱的催化效率较低所导致[23]; 另外有研究结果表明HnH6H的酶活性高于SlH6H[24], 所以推测HnCYP82M3酶活性在一定程度上可能高于颠茄的内源基因。DsTRI的酶活性较高, 已用于颠茄的转基因[13], 另外, 超表达异源同源基因也可降低基因沉默的概率, 因此HnCYP82M3和DsTRI组合效果应该高于颠茄内源基因。
CYP82M3是新进鉴定的TAs合成途径基因, 直接催化托品酮的生成, 目前仅在颠茄中进行了克隆和功能鉴定。烟草叶片瞬时表达实验发现其催化生成的产物托品酮含量远低于底物含量, 推测其是限速酶。但是本研究发现超表达HnCYP82M3的发根莨菪碱和总TAs含量与对照相比无显著差异, 这与Zeng等[1]和Liang[25]研究超表达AbCYP82M3不会促进TAs的积累的结果一致。在颠茄发根中单独超表达AbCYP82M3, 托品酮、海螺碱、莨菪碱、东莨菪碱等TAs的含量没有发生显著性改变, 仅托品的含量轻微地提高。这可能是因为紧接着的AbTRII和AbTRI催化活性较高, 导致AbCYP82M3超表达产生的托品酮被快速转化为假托品和托品。以上结果表明CYP82M3不是TAs合成中的限速酶, 或其超表达的作用会被下游分流和限速步骤抵消。
TRI位于TAs生物合成途径中的分支处, 在多个TAs资源植物中超表达TRI基因都能显著提高TAs含量。Richter等[13]最早在颠茄发根中超表达DsTRI, 使莨菪碱提高了3倍, 东莨菪碱提高了5倍, 同时TRII分支的产物打碗花精含量降低了30%~90%, 而对照颠茄中打碗花精含量是莨菪碱的2倍, 说明超表达TRI起着重要的代谢引流作用。之后, 在三分三中超表达AaTRI, 其根培养物中莨菪碱较对照提高1.87倍, 东莨菪碱提高8倍[14]。铃铛子中超表达SlTRI, 莨菪碱含量较对照提高了1.7~2.9倍[15]。以上研究均证明TRI是TAs合成的关键酶基因。本研究中DsTRI超表达颠茄的生物碱含量与对照相比也有显著提高。但提高幅度没有Richter等[13]报道的大, 这个差异除了来自实验条件的不一致外, 数据展示方式也是一个重要原因。本研究是多个转基因发根克隆系混合后测定的平均值, 而Richter等[13]报道是最高值。
HnCYP82M3超表达虽然不影响TAs的合成, 但是和DsTRI协同超表达后, 发根中生物碱含量和产量在单独超表达DsTRI的基础上又提高了1倍多, 说明HnCYP82M3在开源提高托品酮供应的前提下进一步提高了DsTRI的引流量, 证明了开源引流代谢工程策略的可行性。类似的单基因超表达无作用, 但能增强其他关键酶基因超表达效果的例子有不少的报道, 如颠茄上游的PMT基因, 其本身不影响颠茄TAs的合成, 但和下游的H6H共表达后, 极大促进了东莨菪的积累, 培育了目前最高东莨菪碱含量的颠茄新品系。三分三中共同超表达两个关键酶基因(AaPMT+AaTRI或AaH6H+AaTRI) 也有类似的效果[14, 22]。其原理是上下游两个关键酶超表达可以形成对代谢流的“推-拉”效应, 避免单一基因超表达的衰减作用。
值得注意的是, 本研究中HnCYP82M3和DsTRI双基因超表达对TAs含量提高的幅度还未达到最佳水平, 如莨菪碱仅提高4.97倍, 还有改进提高的空间。这与两个酶的亚细胞定位不同有关, AbCYP82M3已通过亚细胞定位发现定位于内质网, 而TRI是一个胞质蛋白, 这意味着内质网上合成的托品酮还需扩散到胞质去结合下一步的催化酶, 降低了代谢引流效率。后续的策略改进可将DsTRI和HnCYP82M3进行基因融合, 表达的融合蛋白可形成底物桥接和传送带式反应, 最大程度提高开源引流效率。
综上所述, 超表达TAs合成途径分支处的CYP82M3和TRI基因, 成功实现了对TAs合成的开源引流, 为颠茄TAs代谢工程提供了新策略新方法。植物代谢合成途径往往都有分支, 这种对分支点处酶基因共表达的开源引流策略, 也可应用于其他有价值的植物次生代谢产物的代谢工程生产。
作者贡献: 强玮、谭艾娟和张明生等设计了本实验的研究思路与方案; 穆德会、刘艳红和潘航完成p1305NKGFP表达载体改造; 穆德会和马炳南完成了双基因植物表达载体的构建; 穆德会、刘艳红、陈漂漂负责颠茄的遗传转化、培养、转基因材料的收获及各指标的检测等; 穆德会最终完成数据整理以及论文的写作; 强玮指导论文的写作与修改。
利益冲突: 所有作者明确声明不存在任何利益冲突。
基金
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  • 国家自然科学基金资助项目(32200225)
  • 贵州省省级科技计划项目(黔科合基础-ZK[2022]一般096)
  • 贵州大学培育项目(贵大培育[2019]51号)
  • 贵州大学引进人才科研项目(贵大人基合字[2017]58)
  • 贵州省中药材现代产业技术体系建设项目(GZCYTX-02)
  • 贵州大学大学生创新创业训练计划项目(贵大(省)创字2022(028)号)
  • 剑河县省级科技特派员试验示范项目(剑科特派合[2022]10号)
文献
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参考文献 引证文献
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2024年第59卷第3期
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doi: 10.16438/j.0513-4870.2023-0939
  • 接收时间：2023-08-06
  • 首发时间：2025-11-28
  • 出版时间：2024-03-12
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  • 收稿日期：2023-08-06
  • 修回日期：2023-11-12
基金
国家自然科学基金资助项目(32200225)
贵州省省级科技计划项目(黔科合基础-ZK[2022]一般096)
贵州大学培育项目(贵大培育[2019]51号)
贵州大学引进人才科研项目(贵大人基合字[2017]58)
贵州省中药材现代产业技术体系建设项目(GZCYTX-02)
贵州大学大学生创新创业训练计划项目(贵大(省)创字2022(028)号)
剑河县省级科技特派员试验示范项目(剑科特派合[2022]10号)
作者信息
    贵州大学生命科学学院/农业生物工程研究院, 山地植物资源保护与种质创新教育部重点实验室, 贵州 贵阳 550025

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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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