Identification and Functional Analysis of <i>WRKY</i> Gene Family of <i>Lonicera japonica</i> Thunb.

YANG Jiaotong; LI Pengfei; XIAO Qiaoqiao

doi:10.13718/j.cnki.xdzk.2023.07.008

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WRKY transcription factors play an important role in plants, and they participate in the regulation of plant growth and development, hormone and stress response. In this study, the WRKY gene family was identified based on the genomic information of Lonicera japonica, and the conserved domain, promoter characteristics, evolutionary characteristics, gene duplication and expression of WRKY transcription factors in two different transcriptomes were analyzed. Finally, 46 WRKY genes were identified from Lonicera japonica, which could be divided into three subfamilies: Ⅰ(16), Ⅱ(13), and Ⅲ(17) by evolutionary analysis. The molecular weight of WRKY proteins ranged from 15 963.55 to 80 967.23 Da, the number of amino acids ranged from 149 to 748, the isoelectric point ranged from 4.75 to 9.71, and the instability coefficient ranged from 40.97 to 67.76. Many elements involved in growth and development, and hormone response present in the promoter region of WRKY in Lonicera japonica. By analyzing two different expression profiles, we found that WRKY transcription factors might regulate the flowering process. Our research will lay a foundation for further functional studies of WRKY genes in Lonicera japonica.

Corresponding author: XIAO Qiaoqiao

Received Date: 21/10/2021

Available Online: 20/07/2023

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Abstract: WRKY transcription factors play an important role in plants, and they participate in the regulation of plant growth and development, hormone and stress response. In this study, the WRKY gene family was identified based on the genomic information of Lonicera japonica, and the conserved domain, promoter characteristics, evolutionary characteristics, gene duplication and expression of WRKY transcription factors in two different transcriptomes were analyzed. Finally, 46 WRKY genes were identified from Lonicera japonica, which could be divided into three subfamilies: Ⅰ(16), Ⅱ(13), and Ⅲ(17) by evolutionary analysis. The molecular weight of WRKY proteins ranged from 15 963.55 to 80 967.23 Da, the number of amino acids ranged from 149 to 748, the isoelectric point ranged from 4.75 to 9.71, and the instability coefficient ranged from 40.97 to 67.76. Many elements involved in growth and development, and hormone response present in the promoter region of WRKY in Lonicera japonica. By analyzing two different expression profiles, we found that WRKY transcription factors might regulate the flowering process. Our research will lay a foundation for further functional studies of WRKY genes in Lonicera japonica.

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金银花是我国常用的药用植物之一，在世界各地均有种植，主要分布在东亚的中国、韩国和日本等国家，在中国常使用金银花的花蕾和花入药，而日本常使用金银花的茎和叶入药^[1]. 金银花具有清热解毒、疏散风热等功效，在国内分布广，品种多，使用量大. 现已鉴定出金银花具有多种活性成分，包括挥发油、黄酮类、有机酸类和三萜类化合物等^[2]，可用于治疗肺结核、肺炎、急性细菌性痢疾等疾病^[3]. 金银花除了在医学方面有重要的应用外，在化妆品、食品和健康饮料方面也有广泛的应用价值^[4-5]. 此外，金银花还经常作为观赏植物呈现在大众面前^[1]. 金银花的药用、养生和观赏价值决定了其具有较大的市场开发利用前景.

WRKY转录因子是植物特有的一类转录因子，因其具有高度保守的WRKYGQK氨基酸序列而得名^[6]. WRKY基因最初在甘薯中被发现^[7]，后期的研究中发现WRKY基因家族包含3个亚家族^[8]，并且第2个亚家族又可进一步细分为5个次亚家族^[9]. 有研究表明WRKY转录因子能识别靶基因的W-box[(T)TGAC(C/T)] 序列并进行特异性结合，进而调控靶基因的功能^[10]. 高等植物的WRKY转录因子在不同组织中发挥着重要作用. WRKY基因可以调节植物的生长发育，如拟南芥的WRKY46，WRKY54和WRKY70可以通过油菜素内酯调控其生长发育过程^[11]. 此外，WRKY转录因子还参与非生物胁迫反应，如干旱、损伤、水分和盐胁迫等^[12-14]. 在拟南芥中过表达WRKY30可增强盐胁迫的抗性，在水稻中过表达WRKY47可以显著增强水稻对干旱的耐受性. WRKY蛋白在应答生物胁迫(包括细菌、真菌和病毒病原体)方面也发挥重要作用^[15-16]，如拟南芥的WRKY22能够通过水杨酸和茉莉酸信号调控其对蚜虫的易感性^[17]. 综上所述，WRKY基因在植物的生长发育、逆境应答方面发挥了重要的作用.

目前，越来越多植物的WRKY家族基因被鉴定且相关功能被报道，如刺梨^[18]、玉米^[19]、茄子^[6]、菠萝^[20]和番茄^[21]等，但金银花的WRKY基因家族鉴定还未见报道. 本研究基于WRKY家族的种子序列对金银花WRKY家族基因进行鉴定，并基于公共转录组数据对其功能进行预测，为将来开展金银花WRKY基因的功能研究奠定基础.

1. 材料与方法

1.1. 金银花WRKY基因家族成员鉴定

从中国国家生物信息中心(CNCB)收集金银花的全蛋白序列，从Pfam网站^[22]上下载WRKY蛋白的结构域序列，利用HMMER软件^[23]对金银花全蛋白序列进行评估以鉴定WRKY基因，进一步使用保守域预测软件CDD^[24]确保候选基因含有WRKY保守域.

1.2. 理化性质分析

使用ExPASy在线软件(https://web.expasy.org/protparam/)对候选WRKY基因的理化性质进行分析，指标包括分子量(Da)、氨基酸数量、等电点、不稳定指数、脂肪系数和平均亲疏水性.

1.3. 进化分析

提取金银花和拟南芥WRKY蛋白的WRKY结构域序列，使用进化树分析软件Mega7^[25]内置Clustalx进行多序列比对，然后根据NJ方法进行进化树的构建与分析，执行参数：Poission correction，pairwise deletion和bootstrap(1 000次重复).

1.4. 共线性分析

使用MCscanX软件^[26]，在默认参数下分析家族成员的复制事件. 结合家族成员的染色体位置信息，使用TBtools软件绘制基因共线性图.

1.5. 启动子顺式作用元件分析

以全基因组数据库为基础，利用TBtools^[27]获取基因上游2 000 bp的基因组序列，然后递交至PlantCARE网站进行启动子顺式作用元件分析^[28]，最后通过TBtools ^[27]对启动子功能元件进行可视化操作.

1.6. 蛋白结构域分析

根据WRKY蛋白的长度信息及结构域的定位信息，使用TBtools软件^[27]可视化金银花WRKY蛋白的结构域.

1.7. 热图分析

从金银花功能基因组数据库(LjaFGD)^[29]中获取两组不同转录组数据的表达量，用TBtools的heatmap工具绘制WRKY基因家族表达热图，分析基因表达与金银花表型的关系.

3. 讨论与结论

本研究在全基因组范围内对金银花的WRKY家族基因进行了鉴定并分析，发现其WRKY基因家族可能在生长发育、逆境应答过程中发挥着重要作用，如调控金银花花色变化、干旱和水分胁迫应答等. WRKY基因参与金银花花色调控虽然没有报道，但是在杜鹃花的研究中表明WRKY在开花后期可能参与了花色的形成^[31]；在番茄的研究中表明WRKY8参与了干旱和盐胁迫的耐受性^[32]；在大豆的研究中表明GmWRKY基因在脱水和盐胁迫下可能发挥作用^[33]. 这些分析将为更深入研究金银花的WRKY基因功能奠定重要的基础.

大量的研究已经表明高等植物的WRKY基因家族在其生长发育、逆境应答等过程中起着重要作用. 本研究根据金银花WRKY基因的序列特点将WRKY基因分成了3个不同的亚家族. 不同分支上的亚家族之间可能由于序列的差异导致功能存在一定的分化，进而产生WRKY基因功能的多样性. 有研究表明金银花在全基因组水平上发生了染色体加倍事件^[34]，本研究对WRKY基因在不同染色体上的重复性分布进行了分析，发现WRKY基因存在显著的基因组加倍情况，这种加倍情况可能是染色体加倍所致. 以上分析结果为将来开展WRKY基因功能分析提供了思路.

为了进一步分析金银花WRKY基因的潜在功能，本研究选用顺式作用元件分析与基因表达分析共同探索金银花WRKY基因存在的功能. 通过顺式作用元件分析发现许多与光应答、激素应答和生长发育相关的功能元件出现在WRKY基因家族的启动子区域，而通过分析不同光照强度的转录组数据发现了一些存在显著差异表达的WRKY基因，这些差异表达基因很可能参与了光应答过程. 此外，WRKY基因参与激素应答、生长发育等过程在植物中也有报道^[35-38]，表明该分析结果具有一定的可信度. 通过表达分析发现许多WRKY基因的表达与金银花生长发育阶段的趋势相一致，表明WRKY基因很有可能参与了金银花的生长发育过程.

本研究对全基因组范围内的金银花WRKY家族基因进行鉴定，并利用不同类型的转录组数据进行表达分析，发现WRKY基因家族可能在光应答、花发育和花色调控过程中发挥着重要作用，结果为深入开展金银花WRKY基因的功能研究奠定了基础.

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Identification and Functional Analysis of WRKY Gene Family of Lonicera japonica Thunb.

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Identification and Functional Analysis of WRKY Gene Family of Lonicera japonica Thunb.

Corresponding author: XIAO Qiaoqiao