Camptothecin (CPT) is a chemotherapeutic drug widely used to treat various cancers. Ophiorrhiza pumila is an ideal model for a camptothecin-producing plant with various advantages for studying camptothecin biosynthesis and regulation. The DNA binding protein WRKY transcription factors have a key regulatory role in secondary metabolite biosynthesis in plants. However, their involvement in camptothecin biosynthesis in O. pumila is still in its infancy. We identified 46 OpWRKY genes unevenly distributed within 11 chromosomes of O. pumila. Phylogenetic and multiple sequence alignment analyses divided the OpWRKY proteins into three subfamilies. Based on spatial and co-expression, we targeted a candidate gene, OpWRKY6. Overexpression of OpWRKY6 significantly reduced the accumulation of camptothecin compared to the control. Conversely, compared to the control, camptothecin accumulation increased in OpWRKY6 knock-out lines. Further biochemical assays showed that OpWRKY6 negatively regulates camptothecin biosynthesis, from both iridoid and shikimate pathways, by directly down-regulating the gene expression of OpGES, Op10HGO, Op7DLH, and OpTDC. Our data provide direct evidence to the involvement of WRKYs in the regulation of camptothecin biosynthesis and offer valuable information for the enrichment production of camptothecin in plant systems.

中文翻译：

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小麦冬青中参与调控抗癌药物喜树碱生物合成的WRKY转录因子的鉴定

喜树碱（CPT）是一种广泛用于治疗各种癌症的化学治疗药物。Ophiorrhiza pumila 是喜树碱生产植物的理想模型，具有研究喜树碱生物合成和调控的各种优势。DNA 结合蛋白 WRKY 转录因子在植物次生代谢物生物合成中具有关键的调节作用。然而，他们在 O. pumila 中参与喜树碱的生物合成仍处于起步阶段。我们确定了 46 个 OpWRKY 基因在 O. pumila 的 11 条染色体中分布不均。系统发育和多序列比对分析将 OpWRKY 蛋白分为三个亚家族。基于空间和共表达，我们针对候选基因 OpWRKY6。与对照相比，OpWRKY6 的过表达显着降低了喜树碱的积累。反过来，与对照相比，OpWRKY6 敲除系中喜树碱的积累增加。进一步的生化分析表明，OpWRKY6 通过直接下调 OpGES、Op10HGO、Op7DLH 和 OpTDC 的基因表达，负调节环烯醚萜和莽草酸途径的喜树碱生物合成。我们的数据为 WRKYs 参与调节喜树碱生物合成提供了直接证据，并为植物系统中喜树碱的富集生产提供了有价值的信息。