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33
2025年4月3日 星期四
农业生物技术学报  2025, Vol. 33 Issue (2): 316-329    DOI: 10.3969/j.issn.1674-7968.2025.02.007
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
颠茄AbWRKY1基因的克隆与功能研究
潘航, 穆德会, 杨嘉, 谭艾娟, 马炳南, 许国菊, 杨立航, 强玮*
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,贵阳 550025
Cloning and Functional Study of the AbWRKY1 Gene from Atropa belladonna
PAN Hang, MU De-Hui, YANG Jia, TAN Ai-Juan, MA Bing-Nan, XU Guo-Ju, YANG Li-Hang, QIANG Wei*
College of Life Sciences/Research Institute of Agricultural Biological Engineering, Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China
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摘要 颠茄(Atropa belladonna)是《中国药典》收录的托品烷生物碱(tropane alkaloids, TAs)商业药源植物,WRKY转录因子可能调控TAs的生物合成。本研究对颠茄中WRKY转录因子基因家族进行了组织表达、系统进化和诱导表达分析,筛选出正调控TAs生物合成的基因AbWRKY1,并鉴定了其功能。AbWRKY1具有和TAs合成途径基因相同的组织和诱导表达特征,特异性地在颠茄不定根中表达,并且受到钙通道抑制剂维拉帕米(verapamil)的诱导。AbWRKY1和其他物种具有次生代谢调控功能的WRKY聚在同一簇。AbWRKY1蛋白包含326个氨基酸,定位于细胞核。酵母单杂交显示,AbWRKY1能够和莨菪碱6β-羟化酶(hyoscyamine 6β-hydroxylase, H6H)基因启动子上W-box富集区发生相互作用。在颠茄中通过病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术抑制AbWRKY1表达使东莨菪碱和莨菪碱含量分别降低了44.91%和79.33%。超表达AbWRKY1显著提高了颠茄发根中东莨菪碱、山莨菪碱和莨菪碱3种药用TAs的含量,同时上调了4个TAs合成途径基因腐胺N-甲基转移酶(putrescine N-methyltransferase, PMT)、托品酮还原酶Ⅰ(tropinone reductaseⅠ, TRⅠ)、莨菪碱脱氢酶(hyoscyamine dehydrogenase, HDH)和H6H的表达量。本研究为阐明TAs生物合成的转录调控和颠茄分子育种提供了理论基础。
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潘航
穆德会
杨嘉
谭艾娟
马炳南
许国菊
杨立航
强玮
关键词 WRKY转录因子颠茄托品烷生物碱(TAs)病毒诱导的基因沉默(VIGS)超表达    
AbstractAtropa belladonna is a commercial medicinal plant of tropane alkaloids (TAs) listed in the 《Chinese Pharmacopoeia》, and WRKY transcription factors may regulate the biosynthesis of TAs. In this study, tissue expression profile, phylogenetic tree and inducible expression analyses of the WRKY transcription factor family in A. belladonna were performed to screen out WRKY gene that positively regulated TAs biosynthesis. AbWRKY1 was identified and its function was verified at the biochemical and molecular genetic levels. AbWRKY1 and TAs synthetic pathway genes shared the same tissue and inducible expression characteristics that they were specifically expressed in the adventitious roots and were induced by the calcium channel inhibitor verapamil. AbWRKY1 clustered together with WRKYs from other species that had functions in regulating secondary metabolism. AbWRKY1 protein contained 326 amino acid residues, and localized in nuclear. Yeast one-hybrid result showed that AbWRKY1 could interact with the W-box rich region of hyoscyamine 6β-hydroxylase (H6H) gene promoter. Knockdown of AbWRKY1 expression by virus-induced gene silencing (VIGS) in A. belladonna plants reduced scopolamine and hyoscyamine levels by 44.91% and 79.33%, respectively. AbWRKY1 overexpression raised the amount of scopolamine, anisodamine, and hyoscyamine in A. belladonna's hairy roots, while the expression of the 4 TAs synthesis pathway genes, namely putrescine N-methyltransferase (PMT), tropinone reductaseⅠ(TRⅠ), hyoscyamine dehydrogenase (HDH) and H6H, were increased concomitantly. This study provides a theoretical basis for the elucidation of the regulatory mechanism of TAs biosynthesis and the molecular breeding of A. belladonna.
Key wordsWRKY transcription factor    Atropa belladonna    Tropane alkaloids (TAs)    Virus-induced gene silencing (VIGS)    Gene overexpression
收稿日期: 2024-01-29     
中图分类号: S2;Q78
基金资助:国家自然科学基金(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); 大学生创新创业训练计划项目(gzusc2023112)
通讯作者: * wayneqiang@126.com   
引用本文:   
潘航, 穆德会, 杨嘉, 谭艾娟, 马炳南, 许国菊, 杨立航, 强玮. 颠茄AbWRKY1基因的克隆与功能研究[J]. 农业生物技术学报, 2025, 33(2): 316-329.
PAN Hang, MU De-Hui, YANG Jia, TAN Ai-Juan, MA Bing-Nan, XU Guo-Ju, YANG Li-Hang, QIANG Wei. Cloning and Functional Study of the AbWRKY1 Gene from Atropa belladonna. 农业生物技术学报, 2025, 33(2): 316-329.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2025.02.007     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2025/V33/I2/316
 
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