ThWRKY7转录因子参与柽柳应答镉胁迫的调控机理

WRKY transcription factor is a large family, involved in a variety of adverse stress responses. Previous researches showed that ThWRKY7 gene from Tamarix hispida may be the upstream regulatory gene of the ThVHAc1 gene to improve the ability of plant in response to cadmium stress. In this study, we will further study the regulation mechanism of ThWRKY7 gene involved in the Tamarix hispida in response to Cd stress. The promoter of ThWRKY7 will be cloned, and the shortest fragment of promoter will be identified to respond to Cd stress in Tamarix hispida. The TF-centered Y1H, Chromatin Immunoprecipitation (ChIP) and EMSA technology will be employed in studying the motif recognized by ThWRKY7. The transgenic T. hispida plants overexpressing or inhibiting ThWRKY7 gene are constructed to assay the Cd stress tolerance and physiological regulation by ThWRKY7 under the Cd stress. Furthermore, the differential expressed genes are identified between the transgenic T. hispida plants overexpressing or inhibiting ThWRKY7 by the transcriptome and gene expression profiling techniques. These differential expressed genes may be the downstream target genes regulated by ThWRKY7. The interaction proteins with ThWRKY7 protein will be also found, and the influence of these interaction proteins on the recognition of cis-elements by ThWRKY7 will be further analyzed. These results play role in understanding regulation mechanism of WRKY gene in woody plants for Cd tolerance.

WRKY转录因子是一个庞大的家族，参与多种逆境胁迫应答。前期研究结果表明柽柳ThWRKY7是ThVHAc1基因可能的上游调控因子，能提高柽柳的抗Cd胁迫能力。在此基础上，本项目拟进一步系统研究ThWRKY7基因参与的柽柳应答Cd胁迫调控机制。克隆ThWRKY7基因启动子，鉴定启动子中参与Cd胁迫应答的最短片段；利用转录因子为中心的酵母单杂交、染色质免疫共沉淀（ChIP）、EMSA等技术研究ThWRKY7识别的motif；制备ThWRKY7基因过表达和抑制表达柽柳，分析Cd胁迫下ThWRKY7基因过表达和抑制表达对柽柳生理水平的调节。此外，利用转录组及基因表达谱技术，鉴定ThWRKY7基因所调控的下游靶基因。分析ThWRKY7蛋白的互作蛋白，并找出互作蛋白对其识别顺式作用元件结合的影响。以上研究对进一步解析林木WRKY基因抗Cd调控机制具有重要意义。

WRKY转录因子是植物体内最大的转录因子家族之一，参与多种逆境胁迫应答。在前期研究中，我们发现柽柳ThWRKY7基因可能是ThVHAc1基因的上游调控基因，可能能提高柽柳的Cd胁迫耐受性。在此基础上，本项目进一步系统研究了ThWRKY7基因参与的柽柳应答Cd胁迫调控机制。结果显示，ThWRKY7能特异识别W-box、ABRE和未知元件TTAGT，调控下游CAX等靶基因的表达，增加保护酶活性，提高活性氧清除能力，从而提高刚毛柽柳耐镉胁迫能力。此外，ThWRKY8与ThWRKY7互作可能参与ThWRKY7基因在CdCl2胁迫条件下对顺式作用元件的结合。以上研究对进一步解析林木WRKY基因抗Cd调控机制具有重要意义。