Analysis of the transcriptional activators for plant defense response-related genes

在这项研究中，我们研究了从激发子处理的豌豆上胚轴中分离到的新型转录因子E84 （ERDP）的结构和功能。E84是植物特异性转录因子，在n端具有dof型DNA结合域。E84的随机位点选择实验表明，重组E84蛋白与含有aaag的DNA片段结合。利用能与E84结合的模型DNA片段BS4/37 （TCATTTAAAGTGTTTT）进行DNA结合实验，结果表明，高亲和力的结合不仅需要AAAG核心序列，还需要3′-TGT序列。E84蛋白还结合到含有AAAG序列的激发子反应基因PsCHS1启动子的DNA片段上。当我们用豌豆原生质体和含有4个重复“BS4/37”/CaMV 35S最小启动子加报告基因的报告质粒进行瞬时转染实验时，我们观察到启动子增强了报告基因的活性，这表明AAAG序列可能参与了启动子介导的转录激活。我们还观察到E84蛋白与E84基因本身的启动子序列结合，提示可能的自调节机制。这些结果表明E84蛋白及其结合序列可能参与了启动子介导的转录调控。此外，我们还发现豌豆中至少有7个Dof基因家族成员。这些结果为阐明植物防御反应相关基因的转录激活机制提供了信息。

In this study, we investigated the structure and function of novel transcription factor E84 (ERDP) that is isolated from elicitor-treated pea epicotyls. E84 is a plant specific transcription factor possessing Dof-type DNA binding domain at the N-terminus. Random site selection assay for E84 revealed that the recombinant E84 protein binds to AAAG-containing DNA fragments. DNA binding assay using a model DNA fragment, BS4/37 (TCATTTAAAGTGTTTT) that could bind to E84, revealed that not only AAAG core sequence but also 3'-TGT sequence were required for high affinity of the binding. E84 protein also bound to the DNA fragments in the promoter of the elicitor-responsive gene, PsCHS1, which contain AAAG sequence. When we carried out transient transfection assay with pea protoplasts and reporter plasmid possessing 4 repeats of "BS4/37"/CaMV 35S minimal promoter plus reporter gene, we observed that the elicitor enhanced reporter gene activity, indicating the possible involvement of AAAG sequence in elicitor-mediated transcriptional activation. We also observed that E84 protein binds to the promoter sequence of E84 gene itself, indicating possible mechanism of auto-regulation. These results show that E84 protein and its binding sequences might contribute to the elicitor-mediated transcriptional regulation. Furthermore, we also found that at least 7 members of the Dof gene family in pea. These observations provide the information to elucidate the mechanism of the transcriptional activation for plant defense-response related genes.