Identification of WRKY22 direct targets under submergence in Arabidopsis with ChIP

To identify direct targets of WRKY22, we created a transgenic Arabidopsis line that expresses a c-myc epitope-tagged WRKY22 and used ChIP followed by microarray hybridization (ChIP-chip) to screen for candidates and validate the in vivo protein-DNA interactions with ChIP followed by quantitative PCR (ChIP-Q-PCR). The WRKY22 and c-myc epitope tag fusion construct was generated and transformed into wrky22-ko2 plants. The resulting transgenic lines should have better ChIP efficiency than the wild-type background, due to the reduced competition for WRKY22 binding sites from endogenous WRKY22. ChIP-enriched DNA fragments were identified using criteria of a window of +300 to －1200 of a gene for a promoter, a width of 4 probes or more, and a false discovery rate (FDR) < 0.1. The ChIP-chip experiments were repeated six times, i.e., six biological replicas. Candidates were defined by the presence of the promoter in three out of six biological replicas. Candidates were then classified based on their hypoxic responsiveness with a positive response defined as gene expression levels exhibiting > 2 or < 0.5-fold induction in any time point under submergence treatments in expression array data. Comparison of c-myc tagged WRKY22 transgenic plants vs wild-type (Columbia) plants. Both materials were submergence treated for 3 hours.为鉴定WRKY22的直接靶基因，我们构建了表达c-myc表位标签（c-myc epitope tag）融合WRKY22的转基因拟南芥株系，采用染色质免疫共沉淀结合芯片杂交（ChIP-chip）筛选候选靶标，并通过染色质免疫共沉淀结合定量PCR（ChIP-Q-PCR）验证体内蛋白质-DNA互作。我们构建了WRKY22与c-myc表位标签的融合表达载体，并转化至wrky22-ko2突变体植株中。相较于野生型背景，该转基因株系的ChIP效率更优，这是因为内源WRKY22对WRKY22结合位点的竞争被削弱。我们采用以下标准鉴定ChIP富集的DNA片段：启动子区域定义为基因转录起始位点上游1200 bp至下游300 bp的区间、探针数量不少于4个，且错误发现率（False Discovery Rate, FDR）<0.1。本次ChIP-chip实验共重复6次，即包含6个生物学重复。当6个生物学重复中有3个及以上检测到目标基因的启动子区域时，该基因即可被认定为候选靶标。随后我们根据候选靶标的低氧响应特性对其进行分类：在表达芯片数据中，若某基因在淹水处理的任意时间点的表达量诱导倍数大于2倍或低于0.5倍，则定义为具有正响应的低氧应答基因。本研究的表达芯片实验采用两组材料：表达c-myc标签WRKY22的转基因植株与野生型哥伦比亚（Columbia）生态型植株，两组材料均接受3小时淹水处理。