目的 利用RNA-Seq数据筛选天麻Gastrodia elata稳定表达的通用内参基因，并分析天麻素合成途径相关基因的表达模式，为天麻分子生物学研究提供技术支撑。方法 收集了天麻剑麻阶段块茎、茎、花的转录组数据集，以及接菌后4个不同发育时期的转录组数据集共2个RNA-Seq数据集。通过分别计算2个数据集的Transcripts Per Million（TPM）表达矩阵，并采用M值结尾均值（trimmed mean of M-values，TMM）方法进行标准化，获得最终的表达量文件genes.TMM.TPM.matrix。以变异系数（coefficient of variation，CV）均小于0.05为阈值，从2个表达量文件中共同筛选出稳定表达基因作为候选内参基因，并基于本地PrimerServer2软件包设计特异性引物。利用实时荧光定量PCR分别检测候选基因在天麻块茎不同发育时期及组织部位的表达水平，使用delta-CT、geNorm、NormFinder、BestKeeper及RefFinder软件进行稳定性综合评价。同时，以筛选获得的最优内参基因分析天麻素合成途径上的6个关键酶基因的表达模式。结果 Gelref1基因在不同组织部位及发育时期样品中表达最稳定，可作为本研究条件下天麻qRT-PCR分析的适宜内参基因。以该基因为内参，6个天麻素合成途径关键酶基因在不同发育时期组织部位的表达模式存在明显差异，其中多数基因在块茎发育中后期均高表达。结论 提出了一套基于RNA-Seq数据的天麻内参基因系统筛选流程，为后续天麻素生物合成途径的分子机制研究及功能基因验证奠定了基础。

Objective To screen stable reference genes in Gastrodia elata and analyze the expression patterns of key enzyme genes in the gastrodin biosynthesis pathway, providing reliable normalization tools for molecular studies of G. elata. Methods Two transcriptome datasets, encompassing various tissues (tubers, stems, and flowers) at the jian-ma stage and four developmental stages post-inoculation, were integrated. Expression matrices were constructed using Transcripts Per Million (TPM) and normalized via the TMM method. Candidate reference genes were screened based on a coefficient of variation (CV) threshold of < 0.05, and specific primers were designed using the PrimerServer2 package. The expression levels of these candidates across different tissues and developmental stages were validated by qRT-PCR. Stability was comprehensively evaluated using delta-Ct, geNorm, NormFinder, BestKeeper, and RefFinder. Furthermore, the identified optimal reference gene was used to normalize the expression of six key enzyme genes in the gastrodin biosynthesis pathway. Results Gelref1 exhibited the highest stability across all samples, serving as a universal reference gene for various tissues and developmental stages of G. elata. Expression analysis revealed distinct patterns for the six key enzyme genes, with most showing high expression levels during the mid-to-late stages of tuber development. Conclusion This study establishes a systematic workflow for reference gene screening in G. elata based on RNA-Seq data, providing a reliable foundation for further research into the molecular mechanisms and functional validation of genes in gastrodin biosynthesis.

R282.12

湖北省中医药创新发展联合基金培育项目（2026AFC0923）；国家自然科学基金项目（32500333）；湖北省中医药管理局中医药创新团队项目（ZY2025J002）；中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”（2060302）