Cloning and functional analysis of geranyllinalool synthase gene from <i>Brassica oleracea</i>

doi:10.3785/j.issn.1008-9209.2021.12.031

Journal of Zhejiang University (Agriculture and Life Sciences)

2022, Vol. 48

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

Plant protection

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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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

Received: 03 December 2021 Published: 02 November 2022

CLC:

S 635.1

Corresponding Authors: Wenwu ZHOU E-mail: wangyiping@zju.edu.cn;wenwuzhou@zju.edu.cn

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	Yiping WANG
	Yang GE
	Yixin ZHANG
	Asim MUNAWAR
	Yadong ZHANG
	Lijuan MAO
	Zengrong ZHU
	Wenwu ZHOU

Cite this article:

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.

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

结球甘蓝香叶基芳樟醇合酶基因的克隆及功能分析

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

Table 1 Primer information

Fig. 1 Electrophoregram of PCR product of BoGES gene1: Undigested plasmid; 2: Plasmid digested by Pvu Ⅱ; M: 1 kb DNA marker. The blue arrow indicates the target band.

Fig. 2 Analysis of BoGES gene sequence in B. oleraceaA.Prediction of cis-elements in the upstream region of the start codon of BoGES; B. Predicted tertiary structure of BoGES protein; C. Multiple sequence alignments of BoGES with AtGES and NbGES amino acid sequences (the red box indicates the conserved domain of protein).

Fig. 3 Phylogenetic tree of GES genes in different plants

Fig. 4 BoGES gene expression levels after DBM damage (A) and Pst DC3000 infection (B)Double asterisks (**), triple asterisks (***), and quadruple asterisks (****) indicate significant differences among different treatments at the same treatment time at the 0.01, 0.001, and 0.000 1 probability levels, respectively.

Fig. 5 BoGES gene expression levels after exogenous MeJA (A) and SA (B) applicationsSingle asterisk (*), double asterisks (**), and triple asterisks (***) indicate significant differences among different treatments at the same treatment time at the 0.05, 0.01, and 0.001 probability levels, respectively.

Fig. 6 SDS-PAGE detection of BoGES recombinant protein expressionA. Electrophoretogram of BoGES recombinant protein expression induced by different concentrations of IPTG after overnight at 16 ℃‍; B. Electrophoretogram of inclusion of BoGES recombinant protein after renaturation. 1: Protein marker; 2: Supernatant of thallus lysis; 3: Bacterial lysis precipitate; 4: Supernatant of the bacterial lysis precipitate washed by washing buffer 1; 5: Precipitate of the bacterial lysis precipitate washed by washing buffer 1; 6: Supernatant of the bacterial lysis precipitate washed by washing buffer 2; 7: Precipitate of the bacterial lysis precipitate washed by washing buffer 2; 8: Protein after renaturation.

Fig. 7 GC-MS detection of the BoGES recombinant protein productsA. Chromatogram for the products taking GGPP as the substrate; B. Chromatogram for the products taking GPP as the substrate; C-D. Chromatograms of major peak 1 and the authentic standard of geranyllinalool.


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