棉花CRVW的克隆与抗黄萎病功能分析

doi:10.3864/j.issn.0578-1752.2019.11.002

摘要/Abstract

摘要：

目的黄萎病（Verticillium wilt）是棉花生产上的重要病害,严重影响棉花的产量和品质。棉花基因组测序工作的完成为抗病基因挖掘提供了重要的信息资源。通过对一个尚未有功能注释的陆地棉基因CRVW（cotton resistance to Verticillium wilt）进行克隆与抗病功能验证,为棉花基因组信息完善、抗病机制解析和分子育种等方面奠定基础。方法根据参考基因组序列设计引物,同源克隆陆地棉（Gossypium hirsutum）农大601（ND601）中CRVW的开放读码框（open reading frame,ORF）。利用在线工具ProtParam预测蛋白氨基酸组成、分子量、理论等电点、不稳定指数和总平均亲水性等性质;应用PSIPRED v3.3预测蛋白二级结构;在线工具ProtComp v. 9.0进行亚细胞定位预测;PlantCARE在线软件分析顺式作用元件。构建CRVW与绿色荧光蛋白基因融合表达载体,通过基因枪介导法转化洋葱表皮细胞,观察CRVW的表达位置。利用qRT-PCR检测CRVW在棉花不同组织、黄萎病菌胁迫条件下不同抗、感品种间,以及水杨酸（salicylic acid,SA）诱导处理条件下的表达模式。构建CRVW沉默载体,应用病毒诱导的基因沉默（virus-induced gene silencing,VIGS）技术进一步验证该基因在棉花中的抗病功能。检测CRVW沉默后一些与植物抗病调控相关标志基因的表达变化,分析其介导的抗病通路。结果从陆地棉品种ND601中克隆到CRVW的ORF,其全长780 bp,编码259个氨基酸残基,分子量约为30.2 kD,理论等电点9.59;蛋白二级结构含69.50%不规则卷曲、17.76% α-螺旋、11.20%延伸链和1.54% β-卷曲。综合生物信息学预测和荧光观察结果,显示CRVW主要存在于植物细胞膜和细胞质。CRVW在棉花根、茎和叶中都有表达,但在根中的表达量最高。CRVW的ORF上游序列（CRVW-P）中包括响应乙烯（ethylene）、SA、生长素（auxin）和脱落酸（abscisic acid）等4种激素信号的顺式作用元件。另外,CRVW-P还包括一些与伤害、防御、胁迫、病菌、干旱和低温等相关的顺式作用元件。SA喷洒处理后,CRVW显著上调表达。黄萎病菌胁迫后,CRVW在抗病品种ND601和感病品种中棉所8号（CCRI8）中均显著上调表达,但在感病品种中上调表达的发生时间明显滞后。黄萎病菌处理20 d后,CRVW沉默组棉苗表现出比对照（CK）组更明显的黄化、萎蔫和落叶等黄萎病病症。进一步统计分析显示,CRVW沉默组病指显著高于CK组,表明CRVW沉默显著降低了棉苗对黄萎病菌的抗性。沉默CRVW后,棉苗中SA含量显著降低;ICS1（isochorismate synthase 1）、EDS1（enhanced disease susceptibility 1）、PAD4（phytoalexin deficient 4）、NPR1（nonexpresser of PR gene 1）和PR1（pathogenesis-related protein 1）等与SA积累和信号调控相关的标志基因均发生显著下调表达。结论 CRVW定位于细胞质和细胞膜,主要在棉花根部表达,可能通过SA信号通道参与棉花抗黄萎病反应过程。

关键词: 棉花, 黄萎病, CRVW, 克隆, 病毒诱导的基因沉默, 抗性

Abstract:

【Objective】 Verticillium wilt is an important disease in cotton production, and it seriously affects the yield and quality of cotton. Genome sequences of Gossypium hirsutum provide valuable information resources for searching for resistance genes. In this study, an uncharacterized gene, designed as CRVW (cotton resistance to Verticillium wilt), was cloned and identified for disease resistance. The results will lay a foundation for upgrading cotton genomic information, further studying the resistance mechanism and molecular breeding. 【Method】 The open reading frame (ORF) of CRVW was cloned from upland cotton cultivar ND601 using the primers, which were designed according to the reference genome sequence. The online software ProtParam was used to predict protein properties, including amino acid composition, molecular weight, the theoretic isoelectric point, instability index and grand average of hydropathicity. PSIPRED v3.3 was used to predict the protein secondary structure. The prediction of protein subcellular localization and cis-acting elements in the promoter was performed using ProtComp v. 9.0 and PlantCARE, respectively. To elucidate the subcellular localization of the CRVW protein, the CRVW-GFP fusion construct was transformed into onion epidermal cells by particle bombardment. qRT-PCR was performed using normal cotton tissues and tissues that were treated with exogenous application of salicylic acid (SA) and Verticillium dahliae stress. The function of CRVW involving in cotton resistance to V. dahliae was further verified by the technology of virus-induced gene silencing (VIGS). To preliminarily analyze the disease resistance pathway mediated by CRVW, the expression of some marker genes related to plant disease resistance was assayed in CRVW-silenced plants.【Result】 A 780 bp ORF of CRVW was successfully cloned from G. hirsutum ND601. CRVW encodes a putative protein of 259 amino acids with a molecular mass of 30.2 kD and an isoelectric point of 9.59. The protein secondary structure of CRVW contains 69.50% random coil, 17.76% α-helical, 11.20% extension and 1.54% β-sheet. By bioinformatics prediction and fluorescence observation, we found that CRVW was mainly located in the cell membrane and cytoplasm. CRVW was expressed in the roots, stems and leaves of cotton, but the highest expression occurred in the roots. The upstream sequence of CRVW ORF (CRVW-P) contains cis-acting elements in response to four kinds of hormones, including ethylene, SA, auxin and abscisic acid. Additionally, CRVW-P includes a few other elements relating to injury, defense, stress, disease, drought and low temperature. The expression of CRVW was significantly upregulated in the leaves sprayed with SA. After inoculated with V. dahliae, CRVW was dramatically upregulated both in resistant cultivar ND601 and susceptible cultivar CCRI8, but the upregulated expression in susceptible cultivar lagged behind in the resistant cultivar. After 20 days inoculated with V. dahliae, CRVW-silenced cotton seedlings showed more clearly chlorosis, wilting and defoliating comparing to CK. Further statistical analysis showed that CRVW-silenced cotton seedlings had higher disease index than the CK, suggesting that the silence of CRVW significantly reduced the resistance of cotton seedling to V. dahliae. Endogenous SA content in CRVW-silenced cotton seedlings was significantly lower than in CK. The expression of marker genes related to SA accumulation and signal regulation, including ICS1 (isochorismate synthase 1), EDS1 (enhanced disease susceptibility 1), PAD4 (phytoalexin deficient 4), NPR1 (nonexpresser of PR gene 1) and PR1 (pathogenesis- related protein 1), were significantly down-regulated after silencing CRVW.【Conclusion】 CRVW is located in the cytoplasm and the cell membrane, mainly expressed in cotton roots, and involved in the process of cotton resistance to Verticillium wilt, perhaps through SA-mediated defense pathway.

Key words: cotton, Verticillium wilt, CRVW, clone, virus-induced gene silencing, resistance

王秋莹,王伟巧,张艳,王国宁,吴立强,张桂寅,马峙英,杨君,王省芬. 棉花CRVW的克隆与抗黄萎病功能分析[J]. 中国农业科学, 2019, 52(11): 1858-1869.

WANG QiuYing,WANG WeiQiao,ZHANG Yan,WANG GuoNing,WU LiQiang,ZHANG GuiYin,MA ZhiYing,YANG Jun,WANG XingFen. Cloning and Functional Characterization of Gene CRVW Involved in Cotton Resistance to Verticillium Wilt[J]. Scientia Agricultura Sinica, 2019, 52(11): 1858-1869.

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名称 Name	序列 Sequence	位置 Position	功能 Function
ERE	ATTTTAAA	+175,+725,+1470,+1530	乙烯响应Ethylene responsiveness
TCA	TCATCTTCAT	+1935	水杨酸响应Salicylic acid responsiveness
AuxRR-core	GGTCCAT	+441	生长素响应Auxin responsiveness
ABRE	ACGTG	+67,-566	脱落酸响应Abscisic acid responsiveness
WUN-motif	AAATTTCTT	-305	伤害响应Wound responsiveness
WRE3	CCACCT	+1761,-1987	伤害响应Wound responsiveness
STRE	AGGGG	-911	胁迫响应Stress responsiveness
TC-rich repeats	ATTCTCTAAC	+550	防御和胁迫响应Defense and stress responsiveness
box S	AGCCACC	+1759	伤害和病菌响应Wound and pathogen responsiveness
LTR	CCGAAA	-347	低温响应Low-temperature responsiveness
MBS	CAACTG	+1494	干旱响应Drought responsiveness