DataSheet1_Genomic profiling of WRKY transcription factors and functional analysis of CcWRKY7, CcWRKY29, and CcWRKY32 related to protoberberine alkaloids biosynthesis in Coptis chinensis Franch.ZIP

Coptis chinensis Franch. (Huanglian in Chinese) is an important economic crop with medicinal value. Its rhizome has been used as a traditional herbal medicine for thousands of years in Asia. Protoberberine alkaloids, as the main bioactive component of Coptis chinensis, have a series of pharmacological activities. However, the protoberberine alkaloids content of C. chinensis is relatively low. Understanding the molecular mechanisms affecting the transcriptional regulation of protoberberine alkaloids would be crucial to increase their production via metabolic engineering. WRKY, one of the largest plant-specific gene families, regulates plant defense responses via the biosynthesis of specialized metabolites such as alkaloids. Totally, 41 WRKY transcription factors (TFs) related to protoberberine alkaloid biosynthesis were identified in the C. chinensis genome and classified into three groups based on phylogenetic and conserved motif analyses. Three WRKY genes (CcWRKY7, CcWRKY29, and CcWRKY32) may regulate protoberberine alkaloid biosynthesis, as suggested by gene-specific expression patterns, metabolic pathways, phylogenetic, and dual-luciferase analysis. Furthermore, the CcWRKY7, CcWRKY29, and CcWRKY32 proteins were specifically detected in the nucleus via subcellular localization. This study provides a basis for understanding the regulatory mechanisms of protoberberine alkaloid biosynthesis and valuable information for breeding C. chinensis varieties.

黄连（Coptis chinensis Franch.）是一种兼具药用价值的重要经济作物，其根茎在亚洲地区作为传统中药材的应用历史已达数千年。原小檗碱类生物碱作为黄连的主要生物活性成分，具有一系列药理活性。然而，黄连中这类生物碱的含量相对较低。解析影响原小檗碱类生物碱转录调控的分子机制，对通过代谢工程手段提升其产量至关重要。WRKY转录因子家族是植物界最大的特异基因家族之一，可通过调控生物碱等特化代谢物的生物合成参与植物防御响应的调控。本研究在黄连基因组中共鉴定出41个与原小檗碱类生物碱生物合成相关的WRKY转录因子（Transcription Factors, TFs），并通过系统发育分析与保守基序分析将其划分为3个类群。结合基因特异性表达模式、代谢通路分析、系统发育分析以及双荧光素酶实验结果，推测CcWRKY7、CcWRKY29与CcWRKY32这3个WRKY基因可能参与调控原小檗碱类生物碱的生物合成。此外，通过亚细胞定位实验发现，CcWRKY7、CcWRKY29与CcWRKY32蛋白均特异性定位于细胞核中。本研究为解析原小檗碱类生物碱的生物合成调控机制提供了理论基础，同时为黄连品种的遗传改良育种提供了有价值的参考信息。