Yang Bo, Jiang Yuanqing, Rahman Muhammad H, Deyholos Michael K, Kav Nat N V
Department of Agricultural, Food and Nutritional Science, Edmonton, Alberta, Canada.
BMC Plant Biol. 2009 Jun 3;9:68. doi: 10.1186/1471-2229-9-68.
Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.
In this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.
We identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.
植物WRKY转录因子家族成员广泛参与防御反应和各种其他生理过程。对于油菜（甘蓝型油菜），尚未详细描述WRKY基因。由于这种作物的经济重要性及其与拟南芥的进化关系，我们试图在病原体和激素反应的背景下对油菜WRKY基因的一个子集进行表征。
在本研究中，我们通过挖掘表达序列标签（EST）数据库从油菜中鉴定出46个WRKY基因，并克隆了38个BnWRKYs的cDNA序列。使用保守的WRKY结构域氨基酸序列构建了系统发育树，结果表明BnWRKYs可分为三大类。我们进一步将BnWRKYs与来自拟南芥的72个WRKY基因和来自水稻的91个WRKY基因进行比较，鉴定出46个AtWRKY基因的假定直系同源物。我们使用绿色荧光蛋白（GFP）检测了四种BnWRKY蛋白的亚细胞定位，仅在细胞核中观察到绿色荧光信号。通过实时定量PCR（qRT-PCR）分析了16个选定的BnWRKY基因对两种真菌病原体核盘菌和芸苔链格孢的反应。13个BnWRKY基因的转录本丰度在病原体攻击后发生了显著变化：10个WRKYs的转录本丰度增加，2个WRKY转录本在感染后减少，1个在感染后12小时减少但随后（72小时）增加。我们还观察到13/16个BnWRKY基因的转录本丰度对一种或多种激素有反应，包括脱落酸（ABA）、细胞分裂素（6-苄基氨基嘌呤，BAP）以及防御信号分子茉莉酸（JA）、水杨酸（SA）和乙烯（ET）。我们将这些转录本表达模式与先前在拟南芥和水稻中描述的这些基因的假定直系同源物的表达模式进行了比较，观察到表达模式既有相似之处也有差异。
在检测的16个BnWRKY基因中，我们鉴定出一组13个对真菌病原体和激素处理均有反应的BnWRKY基因，这表明这些反应存在共同的信号传导机制。本研究表明，大量BnWRKY蛋白参与了防御相关基因在响应真菌病原体和激素刺激时的转录调控。
