《植物生理学报》 2017, 53(4): 609-618
通信作者：李俊杰；E-mail: guoxf@sdau.edu.cn
摘 要：
WRKY转录因子在植物休眠等生长发育过程中具有重要作用。本研究基于芍药芽转录组数据, 对芽休眠解除前后显著下调的编号为c22608.graph_c0的序列进行了qPCR验证分析, 结果表明其表达模式与转录组一致, 说明其与芍药芽休眠解除密切相关。为进一步挖掘和利用该基因, 从芍药‘大富贵’芽中克隆了该基因, 命名为PlWRKY40 (GenBank登录号: KU891820)。其cDNA全长1 347 bp, 开放阅读框882 bp, 编码293个氨基酸, 含有1个典型的WRKY结构域; 基因结构分析表明, PlWRKY40具有4个外显子和3个内含子, 其内含子具有特殊结构, 可能调控该基因表达; 以拟南芥WRKY蛋白的分类为参照, 聚类分析结果表明, PlWRKY40属于Group IIa亚族; 系统进化树分析表明, PlWRKY40与可可TcWRKY40具有最高的同源性; 亚细胞定位观测结果显示, PlWRKY40转录因子定位在细胞核上; 半定量PCR分析显示, PlWRKY40在芍药的不同组织器官中泛表达, 其中芽表达量最高。本研究为深入探索PlWRKY40的生理学功能奠定了基础。关键词：芍药; PlWRKY40; 克隆; 表达分析; 亚细胞定位
收稿：2016-11-16 修定：2017-02-20
资助：山东省自然科学基金(ZR2014CM028)。
Corresponding author: LI Jun-Jie; E-mail: guoxf@sdau.edu.cn
Abstract:
WRKY transcription factors play great roles in plant dormancy and other processes of growth and development. Based on our previous transcriptome data, the c22608.graph_c0 gene sequence which was significantly down-regulated during endodormancy release in peony buds was investigated using qRT-PCR method. The results corresponded to the transcriptome data, implying that the gene might be closely associated with peony bud dormancy release. The transcription gene, designated as PlWRKY40 (accession number KU891820), was then cloned from Paeonia lactiflora ‘Da Fugui’ for further exploration and utilization. The full length of its cDNA sequence was 1 347 bp, with an open reading frame of 882 bp, encoding a protein of 293 amino acids with a typical WRKY domain. Gene structural analysis demonstrated that PlWRKY40 contained 4 exons and 3 introns. And these introns included many cis-acting elements, which inferred meaningful in gene expression regulation. Phylogenetic analysis based on the amino acid sequences of PlWRKY40 and the WRKY family genes in Arabidopsis thaliana suggested that PlWRKY40 belong to IIa type, whereas the clustering tree of WRKY40 from peony and other plant species showed that PlWRKY40 had the highest similarity with TcWRKY40 from Theobroma cacao. Subcellular localization analysis revealed that the PlWRKY40 protein was mainly detected in the nuclear. Semi-quantitative PCR analysis showed that PlWRKY40 was expressed in each organs and extremely high in buds. This study laid an important foundation for further exploration of the physiological functions of PlWRKY40.Key words: Paeonia lactiflora; PlWRKY40; cloning; expression analysis; subcellular localization
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