蒜芥茄SsWRKY1基因克隆及黄萎病病原菌胁迫下表达分析

【目的】克隆蒜芥茄WRKY1基因(SsWRKY1),并分析其在黄萎病病原菌胁迫下的表达模式,为探究WRKY1基因在野生蒜芥茄黄萎病抗性中的调控机制提供理论参考。【方法】利用染色体步移技术克隆SsWRKY1基因,通过在线生物信息学软件进行序列分析及构建系统发育进化树;采用GFP融合蛋白表达法对WRKY1蛋白进行亚细胞定位,利用实时荧光定量PCR(qRT-PCR)检测SsWRKY1基因在不同组织及接种黄萎病病原菌后不同时间的相对表达量。并分析9种野生茄材料接种黄萎病病原菌后的病情指数与WRKY1基因表达变化量的相关性。【结果】克隆获得的SsWRKY1基因(GenBank登录号MZ846202)ORF序列长度为1224 bp,编码407个氨基酸残基,蛋白相对分子量为44.87 kD,理论等电点(p I)为6.91,属于亲水性不稳定蛋白,亚细胞定位于细胞核,符合转录因子特征。SsWRKY1蛋白含有2个WRKY保守结构域和C2H2型锌指结构,属于Ⅰ类WRKY转录因子,其二级结构主要由α-螺旋(7.13%)、β-折叠(4.67%)、无规则卷曲(73.71%)和延伸链(14.50%)组成。SsWRKY1蛋白与番茄、野生潘那利番茄、野生番茄、马铃薯WRKY1蛋白的亲缘关系较近。SsWRKY1基因在根和茎的相对表达量显著高于叶中的相对表达量(P<0.05,下同)。黄萎病病原菌接种后不同时间,SsWRKY1基因的相对表达量均低于对照组(无菌水接种),但仅在24 h时二者差异达显著水平。9种野生茄材料接种黄萎病病原菌后的病情指数与接种前后WRKY1基因表达变化量间呈显著正相关。【结论】SsWRKY1属于第Ⅰ类WRKY转录因子,序列高度保守,在细胞核中表达,其基因具有组织表达特异性,黄萎病菌胁迫后该基因的表达下调,推测WRKY1在野生茄黄萎病抗性中起负调控作用。

Cloning and expression analysis of SsWRKY1 gene in Solanum sisymbriifolium under stress of verticillium wilt disease

【Objective】The WRKY1 gene of Solanum sisymbriifolium(SsWRKY1) was cloned and its expression pattern under the stress of verticillium wilt disease was analyzed to provide a theoretical reference for exploring the regulatory mechanism of WRKY1 gene in the resistance to verticillium wilt.【Method】SsWRKY1 gene was cloned by the genome walking technique,and its sequence was analyzed by bioinformatics software and a phylogenetic tree was constructed.The subcellular localization of WRKY1 protein was determined by its GFP fusion protein expression under its native promoter. The expression of SsWRKY1 in different tissues of S. sisymbriifolium at different times under verticillium wilt disease stress was analyzed by real-time quantitative PCR(qRT-PCR). The correlation analysis between disease index and WRKY1 gene expression of 9 wild eggplant materials inoculated with the verticillium wilt pathogen was also analyzed.【Result】The full-length open reading frame(ORF)sequence of SsWRKY1 gene(GenBank accession number:MZ846202) was 1224 bp,encoding 407 amino acids residues. Its protein molecular weight was 44.87 kD and its theoretical isoelectric point(pI)was 6.91,which indicated that SsWRKY1 was a hydrophilic and unstable protein. Its subcellular localization in the nucleus conformed to the characteristics of transcription factors. The SsWRKY1 protein contained two conserved WRKY domains and a C2H2-type zinc finger structure,indicating that SsWRKY1 was a class I WRKY transcription factor.Its secondary structure was mainly composed of α-helix(7.13%),β-bridge(4.67%),random coil(73.71%) and extended strand(14.50%). S. sisymbriifolium was closely related to S. lycopersicum,S. pennellii,S. arcanum and S. tuberosum. The relative expression of SsWRKY1 in roots and stems was significantly higher than that in leaves(P<0.05,the same below). The relative expression of SsWRKY1 at different times after the inoculation of the verticillium wilt pathogen was lower than that of the control group(sterile water inoculation),but the difference was significant at 24 h. There was a significant positive correlation between disease index and the variation in WRKY1 expression in 9 wild eggplant materials inoculated with verticillium wilt pathogen.【Conclusion】SsWRKY1 belongs to the class I of WRKY transcription factors,its sequence is highly conserved and is expressed in the nucleus. The gene expression of SsWRKY1 is tissue-specific and the expression of this gene decreased under verticillium wilt disease stress. SsWRKY1 gene may play a negative regulatory role in wild eggplant resistance to verticillium wilt.