樟树WRKY转录因子的克隆与表达分析

Cloning and Expression Analysis of WRKY Transcription factor Genes in Cinnamum camphora

WRKY转录因子超家族在植物生物和非生物胁迫响应、生长发育、代谢过程中起着重要的调控作用。本研究基于樟树叶高通量转录组数据设计特异引物,以芳樟醇型樟树叶片为材料,克隆获得6个CcWRKY转录因子,分别命名为CcWRKY1,CcWRKY2,CcWRKY3,CcWRKY4,CcWRKY5,CcWRKY6;序列多重比对显示,CcWRKY蛋白均含有一个保守WRKYGQK氨基酸残基;系统进化分析表明,除CcWRKY2属于第Ⅱ类,其余5个基因均属第Ⅲ类WRKY转录因子亚家族;杨树原生质体瞬时表达结果显示,6个CcWRKY蛋白均定位于细胞核;实时定量PCR检测结果揭示:6个基因在不同组织的表达模式较为相似,均是嫩叶表达量高于花蕾,嫩茎和幼果;在不同化学型中,3个基因CcWRKY1,CcWRKY2,CcWRKY4在芳樟醇型樟叶中优势表达,CcWRKY3在樟脑型叶中表达量相对较高;而CcWRKY5,CcWRKY6的表达均相对较低。推测CcWRKY1,CcWRKY2,CcWRKY4可能参与单萜类化合物芳樟醇的合成调控。

The superfamily of WRKY transcription factors plays an important regulating role in biological and abiotic stress response, growing development and metabolism process in plant. The study designed specific primers based on high throughput transcription data of Cinnamomum camphora leaves and cloned 6 WRKY transcription factors by taking Linalool-type C.camphora leaf as test materials, which were named CcWRKY1, CcWRKY2, CcWRKY3, CcWRKY4, CcWRKY5 and CcWRKY6 respectively. Multiple sequence alignments revealed that CcWRKY proteins all contained a conserved amino acid residue domain （WRKYGQK）. Phylogenetic analysis showed that Cc WRKY2 gene belonged to group Ⅱ subfamily of WRKY transcription factors and the rest 5 genes belonged to group Ⅲ. Transient expression of Populus protoplasts demonstrated that these 6 CcWRKYs proteins were all localized to the nucleus. All genes had similar expression pattern in different issues that the expressions in young leaves were all higher than that in flower bud, tender stem and young fruit according to the result from real-time quantitative PCR. In different chemical types, CcWRKY1, CcWRKY2, CcWRKY4 genes highly expressed in Linalool-type C.camphora leaf and CcWRKY3 gene had higher expression level in Camphor-type C.camphora leaf, while the expression of CcWRKY5, CcWRKY6 were both lower in all five types. It is speculated that CcWRKY1, CcWRKY2, CcWRKY4 might participate in the synthesis and regulation of monoterpenoid compound linalool.