Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica
Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica
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DOI：
10.1093/pcp/pcl041
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发表时间：
2007-01-01
影响因子：
4.9
通讯作者：
Sato, Fumihiko
中科院分区：
文献类型：
--
作者：
Kato, Nobuhiko;Dubouzet, Emilyn;Sato, Fumihiko
精选培养的黄连细胞产生大量的苄基异喹啉生物碱小檗碱。先前的研究表明，小檗碱的生产力是在生物合成基因的转录水平上控制的。我们利用功能基因组学，通过瞬时 RNA 干扰 (RNAi) 和候选基因的过度表达，确定了小檗碱生物合成中的转录调节因子。 24 个主要候选克隆是从 1,014 个表达序列标签 (EST) 中选出的，这些标签是从产生高水平小檗碱的 C. japonica 细胞系中获得的。对这些 EST 表达谱的进一步表征表明，五种 EST 是作为小檗碱产生调节剂的良好候选者。新开发的 C. japonica 原生质体瞬时 RNAi 系统表明 EST 克隆的双链 RNA 显着降低了 3'-羟基 N-甲基贝壳碱 4'-O-甲基转移酶的转录水平。序列分析表明该EST编码II组WRKY，我们将其命名为CjWRKY1。当详细检查CjWRKY1基因双链RNA的影响时，检测到所有参与小檗碱生物合成的基因的转录物显着减少，而与初级代谢相关的甘油醛-3-磷酸脱氢酶（GAPDH）和分支酸变位酶（CM）的转录物水平几乎没有发现影响。与对照处理相比，CjWRKY1 cDNA 在 C. japonica 原生质体中的异位表达明显增加了所有检查的小檗碱生物合成基因的转录物水平，而 GAPDH 和 CM 的水平不受影响。讨论了 CjWRKY1 作为小檗碱生物合成的特异性和综合调节剂的功能作用。
Selected cultured Coptis japonica cells produce a large amount of the benzylisoquinoline alkaloid berberine. Previous studies have suggested that berberine productivity is controlled at the transcript level of biosynthetic genes. We have identified a regulator of transcription in berberine biosynthesis using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene. The 24 primary candidate clones were selected from 1,014 expressed sequence tags (ESTs) that were obtained from a C. japonica cell line producing high levels of berberine. Further characterization of the expression profiles of these ESTs suggested that five ESTs would be good candidates as regulators of berberine production. A newly developed transient RNAi system with C. japonica protoplasts indicated that double-stranded RNA of an EST clone significantly reduced the level of transcripts of 3'-hydroxy N-methylcoclaurine 4'-O-methyltransferase. Sequence analysis showed that this EST encoded a group-II WRKY, and we named it CjWRKY1. When the effects of double-stranded RNA of the CjWRKY1 gene were examined in detail, a marked reduction in the transcripts of all genes involved in berberine biosynthesis was detected, whereas little effect was found in the transcript levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chorismate mutase (CM) that are associated with primary metabolism. Ectopic expression of CjWRKY1 cDNA in C. japonica protoplasts clearly increased the level of transcripts of all berberine biosynthetic genes examined compared with control treatment, whereas the levels of GAPDH and CM were not affected. The functional role of CjWRKY1 as a specific and comprehensive regulator of berberine biosynthesis is discussed.