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https://www.ahs.ac.cn/images/0513-353X/images/top-banner3.jpg|#|菊花
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园艺学报 ›› 2021, Vol. 48 ›› Issue (2): 313-324.doi: 10.16420/j.issn.0513-353x.2020-0232

• 研究论文 • 上一篇    下一篇

菊花萜类物质代谢关键酶FAS基因的克隆及功能分析

胡昊1,2, 杨婷3, 高莉萍3, MaartenA.Jongsma3, 王彩云1,2,*()   

  1. 1华中农业大学园艺植物生物学教育部重点实验室,武汉 430070
    2农业农村部华中都市农业重点实验室,武汉 430070
    3Wageningen Plant Research,Wageningen University and Research,P.O. Box 619,6700 AP,Wageningen,The Netherlands
  • 收稿日期:2020-09-22 修回日期:2020-12-07 出版日期:2021-02-25 发布日期:2021-03-09
  • 通讯作者: 王彩云 E-mail:wangcy@mail.hzau.edu.cn
  • 基金资助:
    国家自然科学基金项目(31902051);中国博士后科学基金项目(2018M640720);中央高校基本科研业务费专项(2662019FW016);国家重点研发计划项目(2019YFD1001500)

Cloning and Characterization of Key Synthase FAS Gene Involved in Terpenoids Pathway of Chrysanthemum morifolium

HU Hao1,2, YANG Ting3, GAO Liping3, Maarten A. Jongsma3, WANG Caiyun1,2,*()   

  1. 1Key Laboratory for Biology of Horticultural Plants,Ministry of Education,Huazhong Agricultural University,Wuhan 430070,China
    2Key Laboratory of Urban Agriculture in Central China,Ministry of Agriculture and Rural Affairs,P. R. China,Wuhan 430070,China
    3Wageningen Plant Research,Wageningen University and Research,P.O. Box 619,6700 AP,Wageningen,The Netherlands
  • Received:2020-09-22 Revised:2020-12-07 Online:2021-02-25 Published:2021-03-09
  • Contact: WANG Caiyun E-mail:wangcy@mail.hzau.edu.cn

摘要:

以菊花模式品种(Chrysanthemum morifolium‘1581’)为材料,通过固相微萃取(SPME)结合GC-MS气相质谱联用技术分析其叶片及子房中次生代谢挥发物成分及相对含量,发现萜类代谢物在挥发物中占主要地位。基于转录组测序结果,从萜类合成酶基因FDS基因家族中克隆得到1个菊花法尼醇合成酶(Farnesol synthase,CmFAS)基因。其ORF全长1 197 bp,编码氨基酸398个,5′端具有质体定位信号肽。进化树分析表明CmFAS属于菊科植物FDS家族,与除虫菊及艾蒿中的单萜合成酶CDS亲缘关系最近。多重比对及氨基酸序列分析证实,CmFAS具备该基因家族典型的类异戊二烯代谢催化活性保守结构域。基因时空表达分析显示CmFAS在菊花幼嫩组织中表达量最高,其次是子房与成熟叶片,但均低于菊花中另外2个FDS基因。通过大肠杆菌原核表达与酶学反应证实,CmFAS可以利用类异戊二烯代谢路径上游底物二甲基丙烯焦磷酸(DMAPP)和异戊烯基焦磷酸(IPP)以及菊基焦磷酸(CPP)分别产生法尼醇和菊基醇,同时具备了异戊烯基转移酶以及萜类合成酶的双重活性。法尼醇合成酶CmFAS是菊花中鉴定的第1个质体定位的倍半萜合成酶。

关键词: 菊花, 法尼醇合成酶, 萜类化合物, 基因表达, 原核表达

Abstract:

The secondary metabolite composition and relative abundance in leaves and ovaries of the Chrysanthemum morifolium model cultivar‘1581’were analyzed by SPME extraction and GC-MS analysis. The terpenoids represented the dominant group of volatiles. Based on conserved sequences FDS-like genes identified in a transcriptome database derived from chrysanthemum,a farnesol synthase gene(CmFAS)was cloned with an 1 197 bp open reading frame(ORF),encoding 398 amino acids and putatively located in the plastids. Phylogenetic analysis showed that CmFAS belongs to the FDS gene family in Asteraceae and has higher sequence similarity with the monoterpene CDS genes of pyrethrum (Tanacetum cinerariifolium)and Artemisia. Multiple alignment of FDS proteins of several different plant species demonstrated that CmFAS had the five typical conserved regions of chain elongation prenyltransferases. The gene expression of CmFAS was the highest in young leaves and lower in ovaries and mature leaves. The expression level of CmFAS was lower than that of CmFDS1 and CmFDS2 in chrysanthemum. By enzyme assay,it showed that CmFAS not only catalyzed DMAPP and IPP to farnesol but also converted CPP to chrysanthemol. CmFAS is a bifunctional enzyme generating both monoterpene and sesquiterpene products. CmFAS is the first sesquiterpene synthase located in plastids of chrysanthemum.

Key words: Chrysanthemum, farnesol synthase, terpenoids, gene expression, prokaryotic expression

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