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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2021, Vol. 47 Issue (2): 182-192    DOI: 10.3785/j.issn.1008-9209.2020.07.211
植物保护     
结球甘蓝α-法呢烯合成酶基因的鉴定与功能分析
张彤1(),汪一萍2,葛洋2,NTIRI Eric2,周文武1,2()
1.福建农林大学应用生态研究所，闽台作物有害生物生态防控国家重点实验室，福州 350002
2.浙江大学昆虫科学研究所，农业农村部作物病虫分子生物学重点实验室，杭州 310058
Identification and functional analysis of α-farnesene synthase gene from the cabbage Brassica oleracea
Tong ZHANG1(),Yiping WANG2,Yang GE2,Eric NTIRI2,Wenwu ZHOU1,2()
1.State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2.Ministry of Agriculture and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China
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摘要：

为阐明结球甘蓝α-法呢烯合成酶基因（BoTPSa）的生物学功能，本研究克隆了BoTPSa基因并分析了其在不同环境胁迫影响下的表达情况。利用大肠埃希菌原核表达系统，异源表达其蛋白和测定其生化功能，并在拟南芥中对BoTPSa的亚细胞定位进行了研究。结果表明：BoTPSa基因与十字花科特别是芸薹属植物的同源基因在碱基序列上高度相似，说明其具有较保守的结构；小菜蛾危害、蚜虫危害、高温胁迫、低温胁迫均能显著诱导BoTPSa基因的表达，并且受小菜蛾虫害诱导的基因表达变化幅度高于其他胁迫处理，说明在结球甘蓝体内BoTPSa可能参与虫害和非生物胁迫反应；BoTPSa蛋白未定位于叶绿体和细胞核中，在体外能催化合成多种萜类产物，是一个多功能酶。综上所述，本研究系统解析了结球甘蓝的α-法呢烯合成酶的功能，为十字花科作物的萜类合酶的功能研究和实践应用提供了理论基础。
关键词： 结球甘蓝;  α-法呢烯合成酶;  原核表达;  基因表达;  亚细胞定位    
Abstract:

In order to illuminate the biological function of α-farnesene synthase gene (BoTPSa) from the cabbage Brassica oleracea, the BoTPSa gene was cloned and its expression levels were measured in the plants treated by different environmental stresses. Then the protein was heterologously expressed in Escherichia coli prokaryotic expression system and its biochemical function was determined, and the subcellular localization of this protein was studied in Arabidopsis thaliana. The results showed that BoTPSa gene and its homologous genes from cruciferous plants (especially from Brassica) were quite similar in the nucleic acid sequence level, indicating that this gene had conserved structures. The stresses including the damages caused by diamond back moth (DBM), aphids, the high and low temperatures could significantly affect the expression levels of BoTPSa, and DBM damage caused the highest change of gene expression level among all the tested stresses, indicating that this gene was involved in the insect herbivore and abiotic stresses responses in cabbage. The BoTPSa protein was not localized in chloroplasts or cell nucleus, and could catalyze the formation of several terpenoids in vitro, which was a multifunctional enzyme. Above all, this study systemically characterizes the α-farnesene synthase gene (BoTPSa) from the cabbage, which could provide a basis for the functional and applied studies of terpene synthase in cruciferous plants.
Key words: Brassica oleracea    α-farnesene synthase    prokaryotic expression    gene expression    subcellular localization
收稿日期: 2020-07-21 出版日期: 2021-04-25
CLC:  Q 78  
基金资助: 国家自然科学基金(31701798);浙江省重点研发计划(2018C04G2011264)
通讯作者: 周文武     E-mail: ztnb2020@163.com;wenwuzhou@zju.edu.cn
作者简介: 张彤（https://orcid.org/0000-0003-4931-6308），E-mail：ztnb2020@163.com
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引用本文:

张彤,汪一萍,葛洋,NTIRI Eric,周文武. 结球甘蓝α-法呢烯合成酶基因的鉴定与功能分析[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(2): 182-192.

Tong ZHANG,Yiping WANG,Yang GE,Eric NTIRI,Wenwu ZHOU. Identification and functional analysis of α-farnesene synthase gene from the cabbage Brassica oleracea. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(2): 182-192.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.07.211    或    http://www.zjujournals.com/agr/CN/Y2021/V47/I2/182

图1  BoTPSa蛋白的结构域和三级结构A. BoTPSa蛋白的结构域；B. BoTPSa蛋白的三级结构。
图2  16种植物中TPS基因的系统进化树
图3  小菜蛾诱导结球甘蓝叶、茎、根BoTPSa基因的相对表达量A. 叶；B. 茎；C. 根。CK：未受小菜蛾危害的结球甘蓝组。***表示在同一处理时间不同处理间在P＜0.001水平差异有极高度统计学意义。
图4  外界因素诱导结球甘蓝叶中BoTPSa基因的相对表达量CK：未受小菜蛾危害的结球甘蓝组。*表示与对照组相比在 P＜0.05水平差异有统计学意义。
  
图6  重组BoTPSa酶催化产物的GC-MS分析A. FPP纯品的全扫描质谱图；B. 98%法呢烯混合烯烃（稀释1×104倍）的全扫描质谱图；C. 以FPP为底物的BoTPSa酶活产物的全扫描质谱图。a：草烯；b：β-法呢烯；c：α-法呢烯。
图7  BoTPSa在拟南芥原生质体中的亚细胞定位A. BoTPSa荧光与细胞核标志物荧光的位置；B. GFP与叶绿体自发荧光的位置。图a～d为含目的基因的亚细胞定位载体在不同视野下的定位结果，图e～h为不含目的基因的亚细胞定位载体在不同视野下的定位结果。图中的蓝点为细胞核标志物基因表达蛋白的荧光，红点为叶绿体的自发荧光，绿点为GFP表达产物的荧光。
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