结球甘蓝香叶基芳樟醇合酶基因的克隆及功能分析

doi:10.3785/j.issn.1008-9209.2021.12.031

浙江大学学报（农业与生命科学版）

2022, Vol. 48

Issue (5): 583-593 DOI: 10.3785/j.issn.1008-9209.2021.12.031

植物保护

结球甘蓝香叶基芳樟醇合酶基因的克隆及功能分析

汪一萍¹(

),葛洋¹,张译心¹,MUNAWAR Asim¹,张亚东¹,毛黎娟²,祝增荣¹,周文武¹(

)

^1.浙江大学农业与生物技术学院昆虫科学研究所, 农业农村部作物病虫分子生物学重点实验室, 杭州 310058
^2.浙江大学农生环测试中心, 杭州 310058

Cloning and functional analysis of geranyllinalool synthase gene from Brassica oleracea

Yiping WANG¹(

),Yang GE¹,Yixin ZHANG¹,Asim MUNAWAR¹,Yadong ZHANG¹,Lijuan MAO²,Zengrong ZHU¹,Wenwu ZHOU¹(

)

^1.Ministry of Agriculture and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
^2.Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University, Hangzhou 310058, China

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摘要：

萜类化合物在植物防御病虫害等胁迫中具有重要的生物学和生态学功能，香叶基芳樟醇合酶（geranyllinalool synthase, GES）是萜类合成途径中的关键酶。为解析结球甘蓝中GES的功能，本研究采用聚合酶链反应方法克隆了该基因，检测其在生物胁迫诱导下的表达特性，并对其蛋白质的原核表达特性及生化功能进行了研究和测定。结果表明：BoGES蛋白质的氨基酸序列在十字花科植物中高度保守；小菜蛾、丁香假单胞菌（Pst DC3000）、水杨酸、茉莉酸甲酯诱导均能上调BoGES基因的表达，说明该基因参与生物胁迫诱导的反应；该蛋白质能够以香叶基香叶基焦磷酸为底物催化合成香叶基芳樟醇。此外，用“Y”型嗅觉仪测定玉米螟赤眼蜂对不同浓度香叶基芳樟醇的行为反应，发现香叶基芳樟醇能够吸引赤眼蜂。综上所述，本研究对结球甘蓝中的BoGES基因功能进行了系统研究，为进一步研究十字花科植物中萜类合酶及萜类化合物的生物学和生态学功能提供了科学依据。

关键词： 结球甘蓝; 萜类合酶; 生物胁迫; 香叶基芳樟醇; 香叶基芳樟醇合酶

Abstract:

Terpenoids play important biological and ecological roles in plant defense against the pests’ stress, and geranyllinalool synthase (GES) is a key enzyme in the biosynthetic pathway of terpenoids. In order to understand the function of GES in Brassica oleracea, wecloned the BoGES gene by polymerase chain reaction and analyzed its expression levels under different biological stresses, after that we studied the recombinant protein by prokaryotic expression and analyzed its biochemical functions. The results showed that the amino acid sequence of BoGESprotein was highly conserved in Brassicaceae, indicating its conserved function in these plants. The expression of BoGES gene could be significantly induced by diamondback moth (DBM) damage, Pst DC3000 infection, and salicylic acid and methyl jasmonate treatments, suggesting its functions in response to the biological stresses. BoGES protein could catalyze the formation of geranyllinalool from the substrate geranylgeranyl diphosphate (GGPP). Moreover, we measured the choice response of the parasitoid wasps to different concentrations of geranyllinalool via the Y-tube olfactometer, and found that this chemical could attract the parasitoid wasps. In conclusion, this study systemically analyzed the function of BoGES gene in B. oleracea, which could provide the scientific basis for further study of the biological and ecological functions of terpene synthase and terpenoids in Brassicaceae plants.

Key words: Brassica oleracea terpene synthase biological stress geranyllinalool geranyllinalool synthase

收稿日期: 2021-12-03 出版日期: 2022-11-02

CLC:

S 635.1

基金资助: 浙江省教育厅科研项目(Y202045605);中央高校基本科研业务费专项资金(2021FZZX003-02-10);国家自然科学基金项目(31701798);浙江大学实验技术研究项目(SJS201915)

通讯作者: 周文武 E-mail: wangyiping@zju.edu.cn;wenwuzhou@zju.edu.cn

作者简介: 汪一萍（https://orcid.org/0000-0003-0668-8136），E-mail：wangyiping@zju.edu.cn

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引用本文:

汪一萍,葛洋,张译心,MUNAWAR Asim,张亚东,毛黎娟,祝增荣,周文武. 结球甘蓝香叶基芳樟醇合酶基因的克隆及功能分析[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(5): 583-593.

Yiping WANG,Yang GE,Yixin ZHANG,Asim MUNAWAR,Yadong ZHANG,Lijuan MAO,Zengrong ZHU,Wenwu ZHOU. Cloning and functional analysis of geranyllinalool synthase gene from Brassica oleracea. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(5): 583-593.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2021.12.031 或 https://www.zjujournals.com/agr/CN/Y2022/V48/I5/583

表1 引物信息

图1 BoGES 基因PCR产物电泳图1：未酶切的质粒；2：经Pvu Ⅱ酶切的质粒；M：1 kb DNA分子标志物。图中蓝色箭头所指为目的条带。

图2 结球甘蓝 BoGES 基因序列的分析A. BoGES基因起始密码子上游区域中顺式作用元件预测；B. BoGES蛋白质三级结构预测；C. BoGES与AtGES、NbGES氨基酸序列的比对结果（红色方框表示蛋白质保守结构域）。

图3 不同植物 GES 基因的系统进化树

图4 小菜蛾危害（A）和细菌 Pst DC3000侵染（B）后 BoGES 基因表达水平**、***、****分别表示相同处理时间不同处理组间在P＜0.01、P＜0.001、P＜0.000 1水平差异有统计学意义。

图5 外源MeJA（A）和SA（B）处理后 BoGES 基因表达量*、**、***分别表示相同处理时间不同处理组间在P＜0.05、P＜0.01、P＜0.001水平差异有统计学意义。

图6 BoGES重组蛋白诱导表达的SDS-PAGE检测A. 不同浓度IPTG（16 ℃）诱导过夜后BoGES重组蛋白表达的电泳图；B. BoGES重组蛋白包涵体经复性后的电泳图。1：蛋白质标志物；2：破菌上清液；3：破菌沉淀；4：破菌沉淀用洗脱液1洗涤后的上清液；5：破菌沉淀用洗脱液1洗涤后的沉淀；6：破菌沉淀用洗脱液2洗涤后的上清液；7：破菌沉淀用洗脱液2洗涤后的沉淀；8：复性蛋白。

图7 GC-MS检测BoGES重组蛋白产物A. 以GGPP为底物的酶活反应色谱图；B. 以GPP为底物的酶活反应色谱图；C～D. 主峰谱图1与香叶基芳樟醇标准谱图。

图8 玉米螟赤眼蜂对不同浓度香叶基芳樟醇的嗅觉反应*、**分别表示在P＜0.05和P＜0.01水平差异有统计学和高度统计学意义。Single asterisk (*) and double asterisks (**) indicate significant and highly significant differences at the 0.05 and 0.001 probability levels, respectively.Fig. 8‍　Olfactory reaction of T. ostriniae to different concentrations of geranyllinalool

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