受多逆境诱导表达的GhWRKY64基因启动子克隆与功能分析

摘要

WRKY 蛋白属于锌指型转录调控因子,参与植物生长发育及耐逆响应。以陆地棉遗传标准系 TM-1为材料,克隆GhWRKY64(KF031101)基因上游1064 bp的启动子序列,并对其调控元件及功能进行分析。生物信息学分析表明,该区域含18个组织器官表达及诱导表达关键元件,分别为6个ROOTMOTIFTAPOX1根特异调控元件,4个CACTFTPPCA1叶肉特异性调控元件、4个 OSE2ROOTNODULE 病菌诱导元件、2个 GTIGMSCAM4盐调控元件和2个W-box胁迫应答响应元件。将该启动子与GUS基因融合,构建pBIW64：GUS植物表达载体,通过农杆菌介导叶盘转化法获得12个转基因烟草株系。选择GUS表达量最高的pBIW64-5进行转基因不同组织器官表达及诱导表达分析。GUS组织化学染色显示,苗期的转基因烟草植株在叶和根部均具有GUS活性,开花期在转基因烟草植株根、叶及叶柄均检测到GUS活性,特别在转基因烟草的根及根尖部分染色更深,在茎和花组织上未检测到GUS活性。对该转基因烟草幼苗进行黄萎病菌诱导处理,诱导48 h后,转基因烟草幼苗根和叶片的GUS染色比未诱导处理的对照明显加深。结果表明,GhWRKY64上游1064 bp长度的DNA序列，具有启动子的相关顺式作用元件，且为病原菌诱导型启动子。该启动子可为开展棉花抗黄萎病转基因研究提供调控元件。%WRKY proteins are members of a transcription factor family with Zinc-finger structure in higher plant, which partici-pate in various physiological processes and responses to multiple stresses. In this study we isolated a 1064 bp promoter sequence ofGhWRKY64 (KF031101) from Gossypium hirsutum acc. TM-1 and analyzed its regulatory elements and functional characteri-zation. Bioinformatics analysis revealed 18 putative tissue-specific or stress-induced regulatory motifs corresponding to known cis-elements in eukaryotic genes, including six ROOTMOTIFTAPOX1 of root-specific regulatory elements, two W-boxes, four CACTFTPPCA1 mesophyll-specific regulatory elements, four OSE2ROOTNODULE of pathogen-induced elements, and two GTIGMSCAM4 of salt-induced regulatory elements. The vector pBIW64:GUS was constructed by cloning the pGhWRKY64 promoter region into a pBI121 plasmid upstream of theβ-glucuronidase (GUS) reporter gene. Twelve transgenic tobacco plants were obtained by means ofAgrobacterium-mediated leaf-disc transformation. Transgenic line pBIW64-5 with the highestGUS expression was selected for tissue-specific expression and induced-expression analyses. Histochemical analyses indicated that the full-length promoter directed efficient expressions of the reporter gene in root and leaf of pBIW64-5 seedlings, and root, leaf and petiole of pBIW64-5 plants at flowering stage, especially in root and root tip; however, no GUS activity was detected in stem and flower in the transgenic tobacco plants. The pBIW64-5 seedlings were also inoculated withVerticilliumdahliae for 48 hours and the GUS activities in root and leaf showed the increased expressions compared to that of the untreated transgenic tobacco plants. Our results suggest that the upstream region ofGhWRKY64 with 1064 bp containscis-elements of the promoter, and is also a pathogen-induced promoter. This promoter is an efficient regulatory element in cotton transgenic research aiming at resistance to Verticillium dahliae.