College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
Plant Cell Rep. 2012 Nov;31(11):2109-20. doi: 10.1007/s00299-012-1321-1. Epub 2012 Jul 31.
Chinese wild grapevine Vitis pseudoreticulata accession 'Baihe-35-1' is identified as the precious resource with multiple resistances to pathogens. A directional cDNA library was constructed from the young leaves inoculated with Erysiphe necator. A total of 3,500 clones were sequenced, yielding 1,727 unigenes. Among them, 762 unigenes were annotated and classified into three classes, respectively, using Gene Ontology, including 22 ESTs related to transcription regulator activity. A novel WRKY transcription factor was isolated from the library, and designated as VpWRKY3 (GenBank Accession No. JF500755). The full-length cDNA is 1,280 bp, encoding a WRKY protein of 320 amino acids. VpWRKY3 is localized to nucleus and functions as a transcriptional activator. QRT-PCR analysis showed that the VpWRKY3 specifically accumulated in response to pathogen, salicylic acid, ethylene and drought stress. Overexpression of VpWRKY3 in tobacco increased the resistance to Ralstonia solanacearum, indicating that VpWRKY3 participates in defense response. Furthermore, VpWRKY3 is also involved in abscisic acid signal pathway and salt stress. This experiment provided an important basis for understanding the defense mechanisms mediated by WRKY genes in China wild grapevine. Generation of the EST collection from the cDNA library provided valuable information for the grapevine breeding. Key message We constructed a cDNA library from Chinese wild grapevine leaves inoculated with powdery mildew. VpWRKY3 was isolated and demonstrated that it was involved in biotic and abiotic stress responses.
中国野生葡萄品种“白河-35-1”被鉴定为具有多种抗病性的宝贵资源。我们从接种白粉菌的幼叶中构建了一个定向 cDNA 文库。共测序了 3500 个克隆,得到了 1727 个基因。其中,有 762 个基因被注释,并使用基因本体论分为三个类别,包括 22 个与转录调控活性相关的 EST。我们从文库中分离出一个新的 WRKY 转录因子,命名为 VpWRKY3(GenBank 登录号 JF500755)。全长 cDNA 为 1280 bp,编码一个 320 个氨基酸的 WRKY 蛋白。VpWRKY3 定位于细胞核,作为转录激活因子发挥作用。实时荧光定量 PCR 分析表明,VpWRKY3 特异性地积累以响应病原体、水杨酸、乙烯和干旱胁迫。在烟草中超表达 VpWRKY3 增加了对青枯菌的抗性,表明 VpWRKY3 参与防御反应。此外,VpWRKY3 还参与脱落酸信号通路和盐胁迫。该实验为理解中国野生葡萄中 WRKY 基因介导的防御机制提供了重要依据。cDNA 文库的 EST 集的生成为葡萄的育种提供了有价值的信息。