目的 以地黄Rehmannia glutinosa品种85-5为试验材料，借助Stereo-seq空间转录组技术解析地黄块根基因原位表达模式，揭示块根细胞的表达空间异质性。方法 通过荧光信号与灰度值分析筛选地黄块根的最优透化时间和透化条件，对比不同Bin分辨率的基因检出量与细胞聚类效果确定适配地黄块根分析的最佳Bin参数，利用主成分分析（principal component analysis，PCA）降维与均匀流形近似与投影（uniform manifold approximation and projection，UMAP）聚类方法完成块根细胞类群划分，利用Seurat软件筛选各细胞类群差异表达基因，以qRT-PCR验证空间转录组分析结果的可靠性。结果 18 min为地黄块根冷冻切片的最佳透化时间。Bin 200分辨率更适用于地黄块根的宏观细胞类群的解析。经PCA降维和UMAP聚类，将地黄块根划分为5个细胞类群，各细胞类群具有明显空间区位特异性，其中类群1和类群2主要分布于韧皮部，类群3主要分布于周皮，类群4则主要分布于形成层。qRT-PCR结果显示，DH2020-016897为周皮特异表达的基因，DH2020-009767和DH2020-009768在形成层中优势表达。结论 成功构建了地黄块根空间转录组图谱，揭示了不同细胞类群的空间分布格局与部分基因的表达特征，为深入解析地黄块根发育、次生代谢产物的合成及逆境应答的空间分子调控机制提供了重要参考。

Objective Using cultivar 85-5 as the experimental material, Stereo-seq spatial transcriptomics technology was employed to analyze the in situ gene expression patterns of tuberous roots, thereby revealing the spatial heterogeneity of gene expression in root cells. Methods The optimal permeabilization duration was determined by fluorescence intensity and grayscale quantification, and the best bin resolution was identified by comparing gene detection efficiency and cell clustering performance across multiple parameter groups. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) were used for cell clustering. Cluster-specific differentially expressed genes (DEGs) were identified using the Seurat package, and qRT-PCR was performed to validate the reliability of spatial transcriptomic data. Results The optimal permeabilization time for frozen sections of R. glutinosa tuberous roots is 18 min, and a bin size of 200 is more suitable for the analysis of macroscopic cell populations in R. glutinosa tuberous roots. Root cells of R. glutinosa were classified into five distinct populations with unique spatial localization via PCA and UMAP. Specifically, clusters 1 and 2 were predominantly distributed in the phloem, cluster 3 in the periderm, and cluster 4 in the cambium. The qRT-PCR verified that gene DH2020-016897 was specifically expressed in the periderm, whereas DH2020-009767 and DH2020-009768 exhibited predominant expression in the cambium. Conclusion In this study, we established a high-resolution spatial transcriptomic atlas of R. glutinosa tuberous roots, delineating spatial distribution patterns of individual cell clusters and the expression profiles of key genes. These findings provide a fundamental resource for exploring tuberous roots development, secondary metabolite biosynthesis, and spatially resolved molecular regulatory networks underlying stress responses in R. glutinosa.

R282.6

国家自然科学基金项目（82373985）；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）