《植物生理学报》 2015, 51(3): 391-398

葡萄WRKY18基因的克隆及表达特性分析

肖培连, 冯睿杰, 侯丽霞, 吕晓彤, 朱丹, 刘新*

青岛农业大学生命科学学院, 山东省高校植物生物技术重点实验室, 山东青岛266109

通信作者：刘新；E-mail: liuxin6080@126.com；Tel: 0532-88030311

摘　要：

以抗性品种‘左优红’组培苗为材料, 克隆得到WRKY18基因, 测序结果显示: VvWRKY18基因片段大小为954 bp, 编码317个氨基酸序列。生物信息学分析结果显示VvWRKY18蛋白分子量约为35.2416 kDa, 等电点为8.22, 不稳定系数为48.61, 推测为不稳定蛋白; 与已知的粳稻、高粱、毛果杨及拟南芥WRKY家族蛋白高度同源; 亚细胞定位预测结果显示主要存在于细胞核中, 属于第二类WRKY转录因子家族成员。实时荧光定量PCR分析显示, VvWRKY18在葡萄不同组织中均有表达, 在花中表达量最高; 多种逆境胁迫因子如盐、干旱和低温等诱导VvWRKY18上调表达, 且在低温胁迫6 h时诱导表达量最高。另外, VvWRKY18受胁迫信号分子水杨酸、脱落酸、一氧化氮和过氧化氢诱导上调表达, 且VvWRKY18在一氧化氮处理下的表达模式与低温诱导的类似, 推测VvWRKY18可能通过调控一氧化氮信号分子代谢途径来调节葡萄对低温胁迫应答。

关键词：葡萄; WRKY18; 基因克隆; 表达特性分析

收稿：2015-01-07   修定：2015-01-30

资助：国家自然科学基金(31401844)、山东省科技攻关项目(2013-GNC11016)和山东省高等学校科技计划项目(J14LE12)。

Gene Cloning and Expression Analysis of WRKY18 in Vitis vinifera

XIAO Pei-Lian, FENG Rui-Jie, HOU Li-Xia, LÜ Xiao-Tong, ZHU Dan, LIU Xin*

Key Lab of Plant Biotechnology in Universities of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao, Shangdong 266109, China

Corresponding author: LIU Xin; E-mail: liuxin6080@126.com; Tel: 0532-88030311

Abstract:

Abiotic stress severely restrict the development of grape industry, so it will provide theoretical basis for grape breeding through gene cloning and studying its molecular mechanisms. We cloned the full-length cDNA of VvWRKY18 from leaves of Vitis vinifera cultivar ‘Zuoyouhong’ tissue culture seedling. The results showed that VvWRKY18 amplified fragment size of 954 bp, encoding a protein of 317 amino acids. Bioinformatic analysis indicated that VvWRKY18 with molecular weight 35.2416 kDa, isoelectric point 8.22 and instability coefficient 48.61, speculating it was unstable protein. Besides it shared high homology with WRKY18 in Oryza sativa Japonica Group, in Sorghum bicolor, in Populus trichocarpa and in Arabidopsis thaliana. Subcellular localization result indicated that it was located in nucleus. And it belonged to type II WRKY transcription factor. Real-time PCR analysis indicated that VvWRKY18 expressed in different tissues, especially in flower. In addition, VvWRKY18 was induced by low temperature (4 ℃), salt stress, osmotic stress, salicylic acid (SA), abscisic acid (ABA), nitric oxide (NO) and hydrogen peroxide (H₂O₂). Moreover, the expression of VvWRKY18 was highly induced at 6 h by low temperature and the expression pattern of VvWRKY18 under NO were similar to those of low temperature induction, which indicated that NO participated in the signal transduction process of grape response to low temperature stress.

Key words: Vitis vinifera; WRKY18; gene clone; expression analysis