目的 在全基因组水平，鉴定党参Codonopsis pilosula WRKY转录因子家族成员，分析党参WRKY转录因子家族的特征，并探讨其在盐胁迫响应中的潜在作用，为研究党参WRKY转录因子功能奠定基础。方法 利用隐马尔可夫模型基于蛋白质保守结构域在全基因组范围内进行筛选潜在基因家族成员。利用MAFFT、IQ-TREE软件、MEME在线工具、Plant CARE和TBtools等完成蛋白序列比对、保守结构域、进化树分析、基因共线性情况和启动子区顺式作用元件分析。基于根、茎、叶和花不同组织的转录组数据分析WRKY基因的表达模式，并利用qRT-PCR进行WRKY基因相对表达量分析，另外结合党参在0、60、120 mmol/L NaCl处理的转录组数据分析WRKY基因在盐胁迫条件下的表达变化。结果 从党参中鉴定到50个WRKY转录因子成员，蛋白基序分析显示其均有典型的WRKY结构域WRKYGQK，与拟南芥和水稻的WRKY转录因子构建系统进化树，将党参50个WRKY转录因子分为3组，其中第2大组可分为5个亚群。党参WRKY成员编码的氨基酸长度在103～986氨基酸之间，等电点为4.87～9.99，相对分子质量为12 435.14～107 946.28。基因表达结果表明党参WRKY基因中CpWRKY26、CpWRKY48、CpWRKY2、CpWRKY49在党参根、茎、叶和花中的表达水平具有显著差异性。盐胁迫处理结果表明，多数CpWRKY基因在不同浓度NaCl处理下呈现差异表达趋势，其中部分成员与已报道的抗盐WRKY基因在系统发育上聚类，提示其可能参与党参盐胁迫响应调控。结论 首次从全基因组水平对党参WRKY基因家族进行系统鉴定和生物信息学分析，并结合组织表达与盐胁迫响应分析揭示了党参WRKY基因可能参与党参生长发育、次生代谢物质生物合成及胁迫响应等生理过程，为深入研究党参WRKY基因的功能及其在药用成分调控中的作用机制奠定了理论基础，并为党参分子辅助育种提供了基因资源和科学依据。

Objective To identify members of the WRKY transcription factor family in Codonopsis pilosula at the genome-wide level and analyze their characteristics, and explore their potential roles in response to salt stress, thereby laying a foundation for functional studies of C. pilosula WRKY transcription factors. Methods Potential gene family members were screened across the entire genome based on conserved protein domains using the Hidden Markov model (HMM). Protein sequence alignment, conserved domain analysis, phylogenetic tree construction, gene synteny analysis, and cis-acting element analysis in the promoter region were performed using software and online tools including MAFFT, IQ-TREE, MEME, Plant CARE and TBtools. Expression patterns of WRKY genes were analyzed based on transcriptome data from different tissues (roots, stems, leaves, and flowers) and qRT-PCR was used to determine their relative expression levels. In addition, the transcriptome data of C. pilosula treated with 0, 60, and 120 mmol/L NaCl were analyzed to examine the expression changes of WRKY genes under salt stress. Results A total of 50 WRKY transcription factor members were identified in C. pilosula. Motif analysis showed that all members contained the typical WRKY domain with the conserved sequence WRKYGQK. Phylogenetic analysis with WRKY transcription factors from Arabidopsis thaliana and Oryza sativa classified the 50 C. pilosula WRKY transcription factors into three groups, with group II further divided into five subgroups. The amino acid lengths of the identified WRKY proteins ranged from 103 to 986 residues, with isoelectric points (pI) of 4.87−9.99 and molecular weights (MW) of 12 435.14−107 946.28. Gene expression analysis revealed significant differences in the expression levels of CpWRKY26, CpWRKY48, CpWRKY2, and CpWRKY49 among roots, stems, leaves, and flowers of C. pilosula. The results of salt stress treatment indicated that most CpWRKY genes showed differential expression trends under different concentrations of NaCl, and some members cluster phylogenetically with previously reported salt-tolerant WRKY genes, suggesting that they may be involved in the regulation of C. pilosula salt stress responses. Conclusion This study represents the first systematic identification and bioinformatics analysis of the WRKY gene family in C. pilosula at the genome-wide level. By combining tissue expression and salt stress response analyses, the study reveales that the WRKY genes in C. pilosula may be involved in physiological processes such as growth and development, secondary metabolite biosynthesis, and stress responses. This lays a theoretical foundation for further research on the functions of WRKY genes and their roles in regulating medicinal components, and provides gene resources and scientific evidence for molecular-assisted breeding of C. pilosula.

R282.12

国家重点研发计划项目（2022YFC3501703）；云南特色植物提取实验室开放研究项目基金资助（YKKF2024020）