不结球白菜WRKY家族转录因子起源进化及其非生物胁迫下表达调控机制研究

Non-heading Chinese cabbage (NHCC) is susceptible abiotic stresses, usually resulting in lower yield and poor quality. The WRKY gene family has been suggested to play important roles in the regulation of transcriptional reprogramming associated with plant stress responses. Prior to the present study, only two full-length of NHCC WRKY proteins had been reported. In this study, we conducted a genome-wide search for NHCC WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such as Arabidopsis. Quantitative RT-PCR was applied to reveal the expression patterns of BcWRKY gene family in response to abiotic stress. These studies will be useful for understanding the role of WRKY genes in plant responses to abiotic stresses. WRKY proteins also function via protein-protein interaction and autoregulation or cross-regulation is extensively recorded among WRKY genes, which help us understand the complex mechanisms of signaling and transcriptional reprogramming controlled by WRKY proteins. Furthermore, overexpression vectors were construced and transformed into NHCC by agrobacterium-mediated method. In addition,these results provide information about the relationship between evolution and functional divergence of the WRKY family.

不结球白菜在生产中频繁地遭受非生物逆境影响，导致产量和品质的降低，而WRKY家族转录因子在不结球白菜适应不良环境过程中扮演着重要的调节作用。但是关于WRKY家族转录因子在不结球白菜中的研究较少，尤其是参与非生物胁迫方面的研究则更少。本研究通过对不结球白菜全基因组分析WRKY基因，以期得到较为完整的WRKY家族转录因子，进一步明确WRKY基因的起源与进化；对WRKY基因的表达谱研究，分离并鉴定WRKY家族转录因子在非生物胁迫信号转导中的调控作用；筛选并分析与WRKY蛋白互作的逆境响应蛋白；选取对于非生物胁迫响应特异表达WRKY家族转录因子，构建过量表达载体，进行不结球白菜转化和功能研究，鉴定WRKY转录因子基因在非生物胁迫中的功能，初步阐明不结球白菜WRKY家族转录因子的调控网络，进而开发利用WRKY家族转录因子在植物抗逆反应中的巨大潜能，为植物抗逆分子育种提供有效基因。

本研究通过对不结球白菜全基因组分析WRKY 基因，采用同源克隆方法在不结球白菜白菜逆境cDNA文库，克隆并得到了56个WRKY转录因子基因；系统比较了他们与拟南芥WRKY蛋白的同源关系并命名。56个不结球白菜逆境相关WRKY蛋白与已报道植物WRKY蛋白在结构和序列上高度保守和同源。基于系统聚类关系和WRKY结构域的数目，56个BcWRKY蛋白被分为三个组：I亚族有11个成员，每个成员都有两个WRKY结构域；II和III亚族分别包含36和9个成员，且每个成员都仅有一个WRKY结构域。这些将有助进一步开展不结球白菜WRKY转录因子参与逆境应答机制的研究。利用生物信息学预测和试验验证的手段对已克隆的56个不结球白菜逆境相关WRKY基因进行亚细胞定位分析，结果发现大部分BcWRKY蛋白都具有核定位特性，对BcWRKY25，39和40的亚细胞定位检测，发现它们也都分别定位在细胞核上，这表明不结球白菜WRKY基因编码蛋白亚细胞定位于细胞核上，他们具有WRKY转录因子特性。采用qRT-PCR技术检测了56个BcWRKY基因在不结球白菜叶片中对多种非生物胁迫（如ABA，低温，高盐，高温及渗透胁迫）的响应情况。结果表明，22个差异表达的BcWRKY基因中对多种非生物胁迫存在特异性表达和共表达现象；其中BcWRKY33、40、54和70作为整个共表调控网络的关键基因，协同其他BcWRKY成员及其他逆境相关基因参与不结球白菜非生物胁迫应答响应，鉴定WRKY 转录因子基因在非生物胁迫中的功能，初步阐明不结球白菜WRKY 家族转录因子的调控网络。