Isolation and expression analysis of CsWRKY6, CsWRKY31 and CsWRKY48 genes in tea plant

doi:10.3785/j.issn.1008-9209.2018.01.161

Journal of Zhejiang University (Agriculture and Life Sciences)

2019, Vol. 45

Issue (1): 30-38 DOI: 10.3785/j.issn.1008-9209.2018.01.161

Horticulture

Isolation and expression analysis of CsWRKY6, CsWRKY31 and CsWRKY48 genes in tea plant

Pengjie WANG(

),Chuan YUE,Di CHEN,Yucheng ZHENG,Zhilin ZHENG,Yi LIN,Jiangfan YANG,Naixing YE(

)

1. College of Horticulture, Fujian Agriculture and Forestry University/Key Laboratory of Tea Science at Universities in Fujian, Fuzhou 350002, China

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Abstract

Three WRKY genes were cloned by using reverse transcription-polymerase chain reaction (RT-PCR) technique from the tea plant of ‘Tieguanyin’ cultivar and named as CsWRKY6, CsWRKY31, CsWRKY48 with the GenBank accession numbers of MG298953, MG298958 and MG298961, respectively. Their open reading frames (ORFs) were 1 734, 1 299 and 960 bp long, encoding 577, 432 and 319 amino acids, respectively. Phylogenetic tree and protein domain analysis indicated that all of them belong to class Ⅱ WRKY protein, and contained highly conserved DNA-binding domain of WRKYGQK and zinc finger structures. CsWRKY6 and CsWRKY31 shared the same zinc finger structure model of C-X₅-C-X₂₃-H-X-H, whereas CsWRKY48 belonged to C-X₄-C-X₂₃-H-X-H. The three genes were expressed in different tissues and had obvious tissue specificity in tea plant. The expression level of CsWRKY6 in old leaves was significantly higher than that in other tissues, while the expression level of CsWRKY31 was the highest in flowers and CsWRKY48 had higher expression level in roots and stems than that in leaves, flowers and fruits. Protein interaction prediction indicated that the three genes were involved in the stress response by interacting with multiple genes. Fluorescence quantitative analysis showed that low temperature treatment could significantly up-regulate their transcript abundance in tea plant leaves. Under drought stress, both CsWRKY31 and CsWRKY48 were induced to the maximum after 12 h. The expression level of CsWRKY48 was dramatically up-regulated after exogenous abscisic acid (ABA) treatment, whereas the expression level of CsWRKY6 and CsWRKY31 was repressed. It is revealed that the three genes are closely related to the resistance in tea plants.

Key words： Camellia sinensis WRKY transcription factors tissue expression adversity stress

Received: 16 January 2018 Published: 28 March 2019

CLC:	S 571.1
	Q 786

Corresponding Authors: Naixing YE E-mail: 494928025@qq.com;ynxtea@126.com

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	Pengjie WANG
	Chuan YUE
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	Yucheng ZHENG
	Zhilin ZHENG
	Yi LIN
	Jiangfan YANG
	Naixing YE

Cite this article:

Pengjie WANG,Chuan YUE,Di CHEN,Yucheng ZHENG,Zhilin ZHENG,Yi LIN,Jiangfan YANG,Naixing YE. Isolation and expression analysis of CsWRKY6, CsWRKY31 and CsWRKY48 genes in tea plant. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1): 30-38.

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http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2018.01.161 OR http://www.zjujournals.com/agr/Y2019/V45/I1/30

茶树CsWRKY6、CsWRKY31和CsWRKY48基因的分离及表达分析

以茶树品种‘铁观音’的芽叶为供试材料，采用逆转录-聚合酶链式反应（reverse transcription-polymerase chain reaction, RT-PCR）技术克隆获得3个WRKY基因CsWRKY6（GenBank登录号：MG298953）、CsWRKY31（MG298958）和CsWRKY48（MG298961）。它们的开放阅读框长度分别为1 734、1 299和960 bp，分别编码577、432和319个氨基酸。系统发育树及蛋白结构域分析表明，3个茶树WRKY基因均属于第Ⅱ类WRKY蛋白，都含有高度保守的DNA结合域（WRKYGQK）和锌指结构组成的WRKY结构域，其中Ⅱb亚类的CsWRKY6和CsWRKY31锌指结构模式为C-X₅-C-X₂₃-H-X-H，Ⅱc亚类的CsWRKY48锌指结构模式为C-X₄-C-X₂₃-H-X-H。3个基因在茶树不同组织中均有表达且具有明显的组织特异性，CsWRKY6在老叶中的表达量显著高于其他组织，CsWRKY31在花中的表达量最高，CsWRKY48在根和茎中的表达量显著高于叶、茶花和茶果。蛋白互作预测表明3个基因可能通过与多个基因互作来响应逆境胁迫，荧光定量表达分析显示低温处理能显著上调茶树叶片中3个基因的表达；在干旱处理下CsWRKY31和CsWRKY48表达均显著上调且在12 h后达到最大，外源脱落酸处理后CsWRKY48表达迅速上调，而CsWRKY6和CsWRKY31表达下调。推测这3个基因与茶树抗逆响应密切相关。

关键词： 茶树, WRKY转录因子, 组织表达, 逆境胁迫

Table 1 Primer sequences

Fig. 1 PCR amplification results of CsWRKY6, CsWRKY31 and CsWRKY48 genes in tea plant

Table 2 Bioinformatics analysis of CsWRKY6, CsWRKY31 and CsWRKY48 proteins in tea plant

Fig. 2 Phylogenetic tree of WRKY proteins in Camellia sinensis and Arabidopsis thaliana

Fig. 3 Alignment of conserved motifs of CsWRKY6, CsWRKY31 and CsWRKY48 in tea plant

Fig. 4 Three-dimensional structure of the WRKY domains of CsWRKY6, CsWRKY31 and CsWRKY48 proteins in tea plant

Fig. 5 Expression level of CsWRKY6, CsWRKY31 and CsWRKY48 in different tissues of tea plant

Fig. 6 Prediction of interaction networks of CsWRKY6, CsWRKY31 and CsWRKY48 in tea plant based on Arabidopsis protein database

Fig. 7 Expression level of CsWRKY6, CsWRKY31 and CsWRKY48 in tea plants under three abiotic stresses


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