目的 对北柴胡Bupleurum chinense中WRKY的全基因组分析，并鉴定积极响应水、热胁迫信号变化的关键WRKY基因，为环境调控下北柴胡质量形成的分子机制提供理论依据。方法 基于北柴胡全基因组数据，利用生物信息学方法对WRKY基因家族成员进行鉴定；并系统分析其染色体定位、系统进化关系、保守结构域、基因结构及启动子顺式作用元件；通过共线性分析筛选潜在响应水热信号的WRKY基因；采用实时荧光定量PCR技术，分析积极响应干旱、干旱低温、干旱高温环境差异下潜在的BcWRKY的表达模式，并将柴胡皂苷合成途径相关基因与潜在的BcWRKY进行蛋白互作预测分析。结果 共鉴定出90个WRKY转录因子，系统进化分析将其分为GroupⅠ～Ⅳ 4个亚群，保守基序分析表明所有成员均含有Motif1、Motif2、Motif3 3个核心基序。基因结构分析显示大多数BcWRKY基因含3～10个外显子，WRKY结构域位于编码区中后段。顺式作用元件预测发现启动子区域富含光响应、茉莉酸甲酯、水杨酸、脱落酸等激素响应元件。染色体定位显示BcWRKY基因分布不均，在ChrA01/02/03上分布较为密集。共线性分析及选择压力分析，表明WRKY基因家族的扩张主要因为全基因组复制和片段复制事件，并伴随纯化选择部分基因。RT-qPCR结果显示，BcWRKY22/42/75基因在栽培种与野生种间表达差异显著；在干旱、干旱＋高温、干旱＋低温等胁迫处理下，BcWRKY10/42/75基因在干旱＋高温条件下表达显著上调。蛋白互作预测结果中互作元件涵盖了激素响应、非生物胁迫响应及光节律信号相关类型，为进一步证明BcWRKY10/42/75作为胁迫响应核心转录因子提供依据。结论 初步筛选了潜在积极响应水、热胁迫信号变化的关键WRKY基因，为后续功能验证和分子育种提供了潜在的靶点，也为深入理解北柴胡药材品质形成的分子机制奠定了理论基础。

Objective This study aimed to conduct a genome-wide analysis of the WRKY transcription factor family in Bupleurum chinense and identify key WRKY genes that actively respond to water and heat stress signals. The findings are expected to provide a theoretical basis for elucidating the molecular mechanisms underlying the quality formation of B. chinense under environmental regulation. Methods Based on the whole-genome sequencing data of B. chinense, bioinformatics approaches were employed to identify members of the WRKY gene family. Systematic analyses were performed, including chromosomal localization, phylogenetic relationship reconstruction, conserved domain analysis, gene structure characterization, and promoter cis-acting element prediction in promoter regions. Collinearity analysis was utilized to screen potential WRKY genes responsive to water and heat signals. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was further applied to analyze the expression patterns of candidate BcWRKY genes under different stress conditions, including drought, drought combined with low temperature, and drought combined with high temperature. Protein-protein interactions between candidate BcWRKY proteins and proteins encoded by genes involved in saikosaponin biosynthesis were predicted. Results A total of 90 WRKY transcription factor genes were identified from the B. chinense genome, which were phylogenetically classified into four subgroups (Group Ⅰ–Ⅳ). Conserved motif analysis revealed that all identified BcWRKY proteins contained three core motifs (Motif 1, Motif 2, and Motif 3). Gene structure analysis indicated that most BcWRKY genes contained 3—10 exons, with the WRKY domain predominantly located in the middle-late region of the coding sequence. Cis-acting element prediction showed that the promoter regions of BcWRKY genes were enriched in light-responsive elements and hormone-responsive elements associated with methyl jasmonate, salicylic acid, and abscisic acid. Chromosomal localization analysis demonstrated an uneven distribution of BcWRKY genes across chromosomes, with higher densities on ChrA01, ChrA02, and ChrA03. Collinearity analysis combined with selection pressure analysis suggested that the expansion of the BcWRKY gene family was primarily driven by whole-genome duplication and segmental duplication events, accompanied by purifying selection acting on some duplicated genes. RT-qPCR results indicated that the expression levels of BcWRKY22, BcWRKY42, and BcWRKY75 differed significantly between cultivated and wild accessions of B. chinense. Under stress treatments, BcWRKY10/42/75 showed remarkable upregulation in response to drought combined with high temperature. The interacting elements identified in the protein-protein interaction prediction results included those associated with hormone response, abiotic stress response, and photoperiodic signal transduction, providing predictive support for further investigation of BcWRKY10, BcWRKY42, and BcWRKY75 as core stress-responsive transcription factors. Conclusion This study preliminarily screened potential key WRKY genes that positively respond to variations in water and heat stress signals, offering potential targets for subsequent functional validation and molecular breeding, and laying a theoretical foundation for further understanding the molecular mechanisms underlying the quality formation of B. chinense.

R282.12

国家自然科学基金资助项目（82104329，32401226）；河南省青年学生科学基金项目（252300423976）；河南省重点研发专项（251111310500，231111312700，241111310200）；中国博士后科学基金特别资助项目（2014T170252）；河南省国际科技合作项目（252102520068）；河南省博士后科研专项（HN2025075）；河南省博士后经费资助