菊花萜类物质代谢关键酶FAS基因的克隆及功能分析

doi:10.16420/j.issn.0513-353x.2020-0232

园艺学报 ›› 2021, Vol. 48 ›› Issue (2): 313-324.doi: 10.16420/j.issn.0513-353x.2020-0232

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^,^*()

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/Abstract

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

中图分类号:

S682.1 ⁺1

胡昊, 杨婷, 高莉萍, MaartenA.Jongsma, 王彩云. 菊花萜类物质代谢关键酶FAS基因的克隆及功能分析[J]. 园艺学报, 2021, 48(2): 313-324.

HU Hao, YANG Ting, GAO Liping, Maarten A. Jongsma, WANG Caiyun. Cloning and Characterization of Key Synthase FAS Gene Involved in Terpenoids Pathway of Chrysanthemum morifolium[J]. Acta Horticulturae Sinica, 2021, 48(2): 313-324.

图/表 7

表1 菊花CmFAS基因克隆、载体构建及表达所用引物

Table 1 Primers for cloning,construction of vector and gene expression of CmFAS in Chrysanthemum

引物用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因ORF克隆 Cloning the full-length ORF	CmFAS LF	ATGGCATTCTGTAATAGTCTCGTAGG
基因ORF克隆 Cloning the full-length ORF	CmFAS LR	TTACTTATGTCCTTTGTATATCTTTCCG
原核表达载体构建 For prokaryotic expression vector	CmFAS_AscⅠ	TATAGGCGCGCCTGACTACGACAATGAGCAGCGA
原核表达载体构建 For prokaryotic expression vector	CmFAS_NotⅠ	TATAGCGGCCGCTTACTTATGTCCTTTGTATATCTTTCC
基因表达分析 Gene expression	CmFAS F	CCGAGGTTGGTATGACTGCTG
	CmFAS R	TCGTTGAATAGGTCCACTAGATGC
	CmActin F	CCTCTTAATCCTAAGGCTAATCAG
	CmActin R	CCAGGAATCCAGCACAATACC

图1 菊花‘1581’不同部位代谢产物成分及其相对含量

Fig. 1 The relative abundance of secondary metabolites in different tissues of Chrysanthemum‘1581’

图2 CmFAS与其他植物FDS家族进化树分析分支上的数值表示自展值,标尺表示分支长度。

Fig. 2 The phylogenetic analysis of CmFAS and FDS family with other plants The number in the tree represents boots value. The scale bar represents branch length.

图3 CmFAS与其他植物FDS家族同源蛋白的多重比对 Ⅰ ~ Ⅴ下划线：5个异戊烯基转移酶催化活性保守区域。

Fig. 3 Protein alignment of CmFAS with FDS proteins from other plant species Ⅰ-Ⅴ underline：Five conserved regions in chain elongation prenyltransferases.

图4 菊花‘1581’CmFAS及其同源基因在不同组织及发育时期的表达水平

Fig. 4 Expression levels of CmFAS and other homologous genes in different tissues and development stages of Chrysanthemum‘1581’ P < 0.05.

图5 CmFAS蛋白的功能分析 A：CmFAS表达蛋白与不同底物的GC-MS产物检测;B：CmFAS催化萜类合成的代谢途径。

Fig. 5 The catalytic function of CmFAS A：GC-MS detection of product of CmFAS protein with different substrates. B：Schematic pathway of synthesis of terpenoids catalyzed by CmFAS.

图6 菊花CmFAS酶参与赤藓糖醇磷酸代谢途径推测图

Fig. 6 The putative schematic of CmFAS involved in MEP pathway

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菊花萜类物质代谢关键酶FAS基因的克隆及功能分析

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

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菊花萜类物质代谢关键酶FAS基因的克隆及功能分析

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

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