茶树CsWRKY6、CsWRKY31和CsWRKY48基因的分离及表达分析

doi:10.3785/j.issn.1008-9209.2018.01.161

浙江大学学报（农业与生命科学版）

2019, Vol. 45

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

园艺学

茶树CsWRKY6、CsWRKY31和CsWRKY48基因的分离及表达分析

王鹏杰(

),岳川,陈笛,郑玉成,郑知临,林浥,杨江帆,叶乃兴(

)

1. 福建农林大学园艺学院/茶学福建省高校重点实验室，福州 350002

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

全文: PDF(3080 KB)

HTML ( ) HTML

摘要：

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

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

收稿日期: 2018-01-16 出版日期: 2019-03-28

CLC:

S 571.1

基金资助: 国家自然科学基金(31600555);福建省“2011协同创新中心”中国乌龙茶产业协同创新中心专项(闽教科〔2015〕75号);福建农林大学科技创新专项基金(CXZX2017181)

通讯作者: 叶乃兴 E-mail: 494928025@qq.com;ynxtea@126.com

作者简介: 王鹏杰（https://orcid.org/0000-0002-5444-4200）|叶乃兴（https://orcid.org/0000-0003-2955-2813）

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王鹏杰
	岳川
	陈笛
	郑玉成
	郑知临
	林浥
	杨江帆
	叶乃兴

引用本文:

王鹏杰,岳川,陈笛,郑玉成,郑知临,林浥,杨江帆,叶乃兴. 茶树CsWRKY6、CsWRKY31和CsWRKY48基因的分离及表达分析[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(1): 30-38.

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.

链接本文:

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

表1 引物序列

图1 茶树 CsWRKY6 、 CsWRKY31 和 CsWRKY48 基因的PCR扩增结果

表2 茶树CsWRKY6、CsWRKY31和CsWRKY48蛋白生物信息学分析

图2 茶树与拟南芥WRKY蛋白的系统发育树

图3 茶树CsWRKY6、CsWRKY31和CsWRKY48保守结构域序列比对

图4 茶树CsWRKY6、CsWRKY31和CsWRKY48蛋白WRKY结构域的三级结构

图5 CsWRKY6 、 CsWRKY31 和 CsWRKY48 在茶树不同组织中的表达量

图6 基于拟南芥蛋白质数据库的茶树CsWRKY6、CsWRKY31和CsWRKY48蛋白互作网络预测

图7 茶树 CsWRKY6 、 CsWRKY31 和 CsWRKY48 在3种非生物胁迫处理下的表达量

1	岳川,曹红利,周艳华,等 .茶树谷胱甘肽还原酶基因CsGRs的克隆与表达分析.中国农业科学,2014,47(16):3277-3289. YUE C , CAO H L , ZHOU Y H , et al . Cloning and expression analysis of glutathione reductase genes (CsGRs) in tea plant (Camellia sinensis). Scientia Agricultura Sinica, 2014,47(16):3277-3289. (in Chinese with English abstract)
2	ULKER B , SOMSSICH I E . WRKY transcription factors: from DNA binding towards biological function. Current Opinion in Plant Biology, 2004,7(5):491-498.
3	CIOLKOWSKI I , WANKE D , BIRKENBIHL R P , et al . Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Molecular Biology, 2008,68(1/2):81-92.
4	ISHIGURO S , NAKAMURA K . Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato. Molecular and General Genetics, 1994,244(6):563-571.
5	YODA H , OGAWA M , YAMAGUCHI Y , et al . Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants. Molecular and General Genetics, 2002,267(2):154-161.
6	LEVéE V , MAJOR I , LEVASSEUR C , et al . Expression profiling and functional analysis of Populus WRKY23 reveals a regulatory role in defense. New Phytologist, 2009,184:48-70.
7	CHEN H , LAI Z B , SHI J W , et al . Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biology, 2010,10:281.
8	YAO D X , ZHANG X Y , ZHAO X H , et al . Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton (Gossypium hirsutum L.). Genomics, 2011,98:47-55.
9	EULGEM T , RUSHTON P J , ROBATZEK S , et al . The WRKY superfamily of plant transcription factors. Trends in Plant Science, 2000,5(5):199-206.
10	HUANG S X , GAO Y F , LIU J K , et al . Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum . Molecular Genetics and GeTnomics, 2012,287(6):495-513.
11	HE H S , DONG Q , SHAO Y H , et al . Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa . Plant Cell Reports, 2012,31(7):1199-1217.
12	谷彦冰,冀志蕊,迟福梅,等 .苹果WRKY基因家族生物信息学及表达分析.中国农业科学,2015,48(16):3221-3238. GU Y B , JI Z R , CHI F M , et al . Bioinformatics and expression analysis of the WRKY gene family in apple. Scientia Agricultura Sinica, 2015,48(16):3221-3238. (in Chinese with English abstract)
13	PANDEY S P , SOMSSICH I E . The role of WRKY transcription factors in plant immunity. Plant Physiology, 2009,150(4):1648-1655.
14	CHEN L G , SONG Y , LI S J , et al . The role of WRKY transcription factors in plant abiotic stresses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2012,1819(2):120-128.
15	ZOU X L , SEEMANN J R , NEUMAN D , et al . A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway. Journal of Biological Chemistry, 2004,279(53):55770-55779.
16	蒋明,陈贝贝,管铭,等 .青花菜转录因子基因BoWRKY2的克隆与表达分析.浙江大学学报(农业与生命科学版),2015,41(2):153-159. JIANG M , CHEN B B , GUAN M , et al . Cloning and expression analysis of a transcription factor gene BoWRKY2 from broccoli. Journal of Zhejiang University (Agriculture and Life Sciences), 2015,41(2):153-159. (in Chinese with English abstract)
17	RUSHTON D L , TRIPATHI P , RABARA R C , et al . WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnology Journal, 2012,10(1):2-11.
18	WANG F , HOU X L , TANG J , et al . A novel cold-inducible gene from Pak-choi (Brassica campestris ssp. chinensis), BcWRKY46, enhances the cold, salt and dehydration stress tolerance in transgenic tobacco. Molecular Biology Reports, 2012,39(4):4553-4564.
19	MA D M, PU G B , LEI C Y , et al . Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis. Plant and Cell Physiology, 2009,50(12):2146-2161.
20	DANG F F , WANG Y N , YU L , et al . CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant Cell and Environment, 2013,36(4):757-774.
21	WU Z J , LI X H , LIU Z W , et al . Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress. Molecular Genetics and Genomics, 2016,291:255-269.
22	罗勇,陈丝,欧淑琼,等 .茶树WRKY转录因子的鉴定与分析.分子植物育种,2017,15(10):3932-3940. LUO Y , CHEN S , OU S Q, et al . Identification and analysis of the WRKY transcription factor in tea plant. Molecular Plant Breeding, 2017,15(10):3932-3940. (in Chinese with English abstract)
23	WANG Y , SHU Z , WANG W , et al . CsWRKY2, a novel WRKY gene from Camellia sinensis, is involved in cold and drought stress responses. Biologia Plantarum, 2016,60(3):443-451.
24	郭俊红,王伟东,谷星,等 .茶树WRKY转录因子基因CsWRKY57的克隆及表达分析.茶叶科学,2017,37(4):411-419. GUO J H , WANG W D , GU X , et al . Cloning and expression analysis of WRKY transcription factor gene CsWRKY57 in tea plant (Camellia sinensis). Journal of Tea Science, 2017,37(4):411-419. (in Chinese with English abstract)
25	XIA E H , ZHANG H B , SHENG J , et al . The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis. Molecular Plant, 2017,10(6):866-877.
26	姚雪倩,陈丹,岳川,等 .茶树烯醇酶基因CsENO的克隆及其在非生物胁迫中的表达分析.园艺学报,2017,44(3):537-546. YAO X Q , CHEN D , YUE C , et al . Cloning of enolase gene CsENO and its expression analysis under abiotic stress in tea plant. Acta Horticulturae Sinica, 2017,44(3):537-546. (in Chinese with English abstract)
27	王鹏杰,陈丹,俞滢,等 .茶树单萜合成酶CsTPS基因的克隆及表达分析.西北植物学报,2017,37(8):1465-1473. WANG P J , CHEN D , YU Y , et al . Clone and expression of monoterpene synthase gene CsTPS in tea plant (Camellia sinensis). Acta Botanica Boreali-Occidentalia Sinica, 2017,37(8):1465-1473. (in Chinese with English abstract)
28	孙美莲,王云生,杨冬青,等 .茶树实时荧光定量PCR分析中内参基因的选择.植物学报,2010,45(5):579-587. SUN M L , WANG Y S , YANG D Q , et al . Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis . Chinese Bulletin of Botany, 2010,45(5):579-587. (in Chinese with English abstract)
29	WU Z J , TIAN C , JIANG Q , et al . Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis). Scientific Reports, 2016,6:19748.
30	LIVAK K J , SCHMITTGEN T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001,25(4):402-408.
31	NORIKO Y , MASAHIRO O , YOSHIHIRO Y . The role of the nuclear transport system in cell differentiation. Seminars in Cell and Developmental Biology, 2009,20(5):590-599.
32	ZHENG J Y , ZOU X X , MAO Z C , et al . A novel pepper (Capsicum annuum L. WRKY Gene, CaWRKY30, is involved in pathogen stress responses. Journal of Plant Biology, 2011,54:329-337.
33	CHEN L , YANG Y , LIU C , et al . Characterization of WRKY transcription factors in Solanum lycopersicum reveals collinearity and their expression patterns under cold treatment. Biochemical and Biophysical Research Communications, 2015,464(3):962-968.
34	ZHOU Q Y , TIAN A G , ZOU H F , et al . Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal, 2008,6(5):486-503.
35	MOON S J , HAN S Y , KIM D Y, et al . Ectopic expression of CaWRKY1, a pepper transcription factor, enhances drought tolerance in transgenic potato plants. Journal of Plant Biology, 2014,57:198-207.

[1]	伍梦瑶，黄莹捷，姚燕妮，黄友谊. 勐库大叶种茶树多酚氧化酶粗酶的酶学性质[J]. 浙江大学学报(农业与生命科学版), 2017, 43(5): 579-588.
[2]	郑群艳，潘晓艺，沈锦玉，陈少波，徐洋，许婷. 罗氏沼虾谷氨酸脱氢酶基因克隆及其在MrTV 感染下的组织表达分析[J]. 浙江大学学报(农业与生命科学版), 2017, 43(5): 639-648.
[3]	侯玲，沈娴，陈琳，金恩惠，吴媛媛，屠幼英. 茶树花蛋白质碱提和酶提工艺优化及其功能性质[J]. 浙江大学学报(农业与生命科学版), 2016, 42(04): 442-450.
[4]	蒋明，陈贝贝，管铭，李金枝，黄笑梅，顾云吉. 青花菜转录因子基因BoWRKY2的克隆与表达分析[J]. 浙江大学学报(农业与生命科学版), 2015, 41(2): 153-159.
[5]	关小燕，陈丽妃，何艳军，王洁，卢钢. 番茄SlMAPK7基因的亚细胞定位与组织表达特性*[J]. 浙江大学学报(农业与生命科学版), 2014, 40(6): 598-604.
[6]	杨节，龚淑英，唐德松. 茶树中过氧化氢酶活力变化规律*[J]. 浙江大学学报(农业与生命科学版), 2014, 40(6): 647-652.
[7]	葛延松，曹嫦妤，王丽丽，李楠，江秀清，李金龙. 鸡硒蛋白T的硒代半胱氨酸插入序列元件、蛋白结构与功能及组织表达差异*[J]. 浙江大学学报(农业与生命科学版), 2014, 40(1): 9-15.
[8]	张敏1,2，陈永根3，于翠平1，潘志强1，范冬梅1，骆耀平1，王校常1. 在茶园生产周期过程中茶树群落生物量和碳储量动态估算*[J]. 浙江大学学报(农业与生命科学版), 2013, 39(6): 687-694.
[9]	陈贝贝, 蒋明, 苗立祥, 李温平. 青花菜转录因子基因BoWRKY3的克隆与表达分析[J]. 浙江大学学报(农业与生命科学版), 2012, 38(3): 243-249.
[10]	孙世利, 骆耀平. 壳聚糖对茶树抗性酶调节作用的研究[J]. 浙江大学学报(农业与生命科学版), 2009, 35(1): 84-88.
[11]	骆耀平　唐萌　任明兴　. 壳聚糖对茶树氮代谢影响[J]. 浙江大学学报(农业与生命科学版), 2007, 33(3): 298-301.
[12]	阚云超　郭泽建　李德葆. 转录因子IosWRKY基因的原核表达及其与顺式元件的结合[J]. 浙江大学学报(农业与生命科学版), 2003, 29(1): 55-58.
[13]	骆耀平　吴姗　康孟利. 二年生嫁接茶树的冬季光合特性与抗寒性[J]. 浙江大学学报(农业与生命科学版), 2002, 28(4): 397-400.
[14]	沈朝栋　黄寿波. 中国栽培茶树的生态条件及地理分布规律[J]. 浙江大学学报(农业与生命科学版), 2001, 27(4): 381-384.
[15]	骆耀平　吴姗　钱利生. 嫁接茶树的分枝习性研究[J]. 浙江大学学报(农业与生命科学版), 2001, 27(2): 215-217.

Viewed

Full text

Abstract

Cited

Shared

Discussed