转录组和代谢组联合分析蒙古黄芪茎表皮毛形成及其对药效成分积累的影响

doi:10.13560/j.cnki.biotech.bull.1985.2026-0172

摘要/Abstract

摘要：

目的筛选蒙古黄芪茎表皮毛形成关键基因，探究茎表皮毛对黄酮和皂苷类药效成分的影响。方法通过扫描电子显微镜（SEM）观察蒙古黄芪多毛茎与无毛茎表型特征，转录组（RNA-seq）和非靶向代谢组（UPLC-MS/MS）技术分析茎表皮毛形成机制及代谢物积累变化；高效液相色谱（HPLC）探究2种表型对茎和根部主要药效成分积累的影响。结果蒙古黄芪茎表皮毛为非腺毛，转录组共筛选出15 917个差异表达基因（DEGs），这些DEGs涉及表皮毛发育、植物激素信号转导及类黄酮生物合成等通路，其中与表皮毛发育相关的基因GIS、GIS3-like、GIS3、HAT1和ETC1可能较高程度参与非腺毛的形成。代谢组学共检测到670个差异积累代谢物（DAMs）。与多毛茎相比，无毛茎中443个代谢物上调、227个下调，DAMs主要富集于苯丙氨酸、酪氨酸和色氨酸生物合成、核苷酸代谢通路等。HPLC分析表明，无毛茎蒙古黄芪茎部毛蕊异黄酮苷含量极显著低于多毛茎茎部（P<0.01），芒柄花素含量显著高于多毛茎茎部（P<0.05）；无毛茎根部毛蕊异黄酮含量（P<0.05）、4种皂苷类成分总含量（P<0.05）均显著低于多毛茎根部。结论筛选出多个调控蒙古黄芪茎表皮毛的候选基因。蒙古黄芪茎表皮毛与异黄酮类和皂苷类药效成分积累存在一定关联，多毛茎较无毛茎表型更优。

关键词: 蒙古黄芪, 代谢组, 转录组, 非腺毛, 黄酮类, 皂苷类

Abstract:

Objective This study aimed to identify key genes regulating stem trichome formation in Astragalus mongholicus and to investigate how pilose and glabrous stems affect the accumulation of flavonoids and saponins. Method Phenotypic characteristics of glabrous and pilose stems were examined using scanning electron microscopy (SEM). An integrated approach combining transcriptome sequencing (RNA-seq) and untargeted metabolomics (UPLC-MS/MS) was employed to elucidate the regulatory mechanism of trichome formation and associated metabolic changes. Furthermore, high-performance liquid chromatography (HPLC) was employed to analyze the effects of these two phenotypes on the accumulation of major bioactive compounds in both stems and roots. Result SEM revealed that the stem epidermal trichomes of A. mongholicus are non-glandular trichomes. RNA-seq analysis identified 15 917 differentially expressed genes (DEGs), which were significantly enriched in the pathways related to trichome development, plant hormone signal transduction, and flavonoid biosynthesis. Among them, the trichome development-related genes GIS, GIS3-like, GIS3, HAT1 and ETC1 may be highly involved in the formation of non-glandular trichomes. Metabolomics profiling revealed 670 differentially accumulated metabolites (DAMs) in the epidermis of glabrous stems using the pilose stem epidermis as a control, among which 443 metabolites were up-regulated and 227 were down-regulated. The DAMs are mainly enriched in the biosynthetic pathways of phenylalanine, tyrosine and tryptophan, as well as nucleotide metabolism pathways. HPLC quantification confirmed a significant decrease (P<0.01) in the content of calycosin-7-O-β-D-glucoside in glabrous stems. In the roots of glabrous plants, the contents of calycosin and total content of the four saponin components were significantly reduced (P<0.05). Conclusions Several candidate genes regulating stem trichome formation in A. mongholicus were screened out. The stem trichomes of A. mongholicus are closely associated with the accumulation of bioactive ingredients including isoflavonoids and saponins. The pilose stem phenotype exhibits better agronomic performance than the glabrous stem phenotype.

Key words: Astragalus mongholicus, metabolomics, transcriptomics, non-glandular trichomes, flavonoids, saponins

雷子莹, 武延慧, 赵生婷, 张瑜, 王嘉蕾, 贺润丽, 刘建霞, 王瑞军. 转录组和代谢组联合分析蒙古黄芪茎表皮毛形成及其对药效成分积累的影响[J]. 生物技术通报, 2026, 42(11): 210-221.

LEI Zi-ying, WU Yan-hui, ZHAO Sheng-ting, ZHANG Yu, WANG Jia-lei, HE Run-li, LIU Jian-xia, WANG Rui-jun. Integrated Transcriptomic and Metabolomic Analysis Reveals the Mechanism Underlying Trichome Formation on the Stem Epidermis of A. mongholicus and Its Impact on Bioactive Compound Accumulation[J]. Biotechnology Bulletin, 2026, 42(11): 210-221.

图/表 12

图1 PCA得分图（A）和DEGs火山图（B）

Fig. 1 PCA scoring plot (A) and volcano plot of DEGs (B)

图2 DEGs GO富集分析圈图第1圈表示富集条目与对应分类，分别为生物过程（蓝色），分子功能（灰色）和细胞成分（红色）3种类型；第2圈显示每个条目中背景基因的数目及该分类的统计显著性值-log10 （q value），条形图越长表示富集到该GO条目基因越多，颜色越深代表q值越小，富集越显著；第3圈表示前景基因总数，条形图越长表示基因数目越多；第4圈不同高低条形图表示富集因子大小，越高代表富集因子越大

Fig. 2 GO enrichment analysis plot of DEGsThe first circle represents the enriched terms and their corresponding categories, namely biological process (blue), molecular function (gray), and cellular component (red). The second circle shows the number of background genes in each term and the statistical significance value -log10 (q value) for the category; a longer bar indicates more genes enriched in the GO term, and a darker color denotes a smaller q value, meaning more significant enrichment. The third circle indicates the total number of foreground genes, with a longer bar standing for a larger number of genes. The fourth circle with bars of different heights represents the enrichment factor, where a higher bar corresponds to a larger enrichment factor

图3 差异表达基因KEGG富集分析图

Fig. 3 KEGG enrichment analysis plot of DEGs

表1 蒙古黄芪黄酮和皂苷类线性回归方程

Table 1 Linear regression equations for flavonoids and saponins in A. mongholicus

化合物 Compound	线性方程 Linear regression equation	R²	线性范围 Linear range （μg）
毛蕊异黄酮苷	Y=39 109 157 C+180 233	0.999 5	0.050-4.970
芒柄花苷	Y=27 793 749 C-48 296	0.999 8	0.128-7.680
毛蕊异黄酮	Y=6 355 527 C-7 323	0.999 9	0.038-2.280
芒柄花素	Y=5 436 799 C-20 586	0.999 8	0.031-1.860
黄芪甲苷	Y=1.660 2 C+5.335 3	0.999 2	0.180-3.300
黄芪皂苷Ⅰ	Y=1.543 2 C+5.947 6	0.999 9	0.588-9.420
黄芪皂苷Ⅱ	Y=1.585 7 C+5.442	0.999 5	0.300-4.800
黄芪皂苷Ⅲ	Y=1.323 2 C+6.408	0.999 8	0.242-3.880

图4 激素相关差异表达基因（A）和差异表达转录因子热图（B）热图数据为相对表达量（TPM）值按行标准化处理，红色代表每行内基因相对表达量上调，蓝色代表下调，下同

Fig. 4 Heat map of hormone-related DEGs (A) and differentially expressed transcription factors (B)The data in the heatmap are row-normalized relative expression (TPM) values. Red indicates upregulated relative expression within each row and blue indicates downregulation. The same below

表2 蒙古黄芪黄酮和皂苷类含量测定结果

Table 2 Determination results of flavonoid and saponin contents in A. mongholicus (x¯±s, n=10)

组别（mg/g）	PHS	GHS	PHR	GHR
毛蕊异黄酮苷	0.11±0.04	0.04±0.01**	1.48±0.50	1.76±0.35
芒柄花苷	0.04±0.01	0.04±0.01	0.53±0.37	0.44±0.17
毛蕊异黄酮	0.15±0.08	0.18±0.06	0.25±0.12	0.13±0.07^#
芒柄花素	0.01±0.01	0.02±0.01*	0.04±0.03	0.02±0.03
4种异黄酮总和	0.32±0.08	0.28±0.05	2.30±0.52	2.35±0.50
黄芪甲苷	-	-	0.25±0.07	0.26±0.08
黄芪皂苷Ⅰ	-	-	1.05±0.24	0.78±0.35
黄芪皂苷Ⅱ	-	-	0.61±0.18	0.48±0.16
黄芪皂苷Ⅲ	-	-	0.08±0.01	0.13±0.07
4种皂苷成分总和	-		1.98±0.36	1.65±0.36^#

图5 黄酮相关差异表达基因热图

Fig. 5 Heat map of flavonoid-related DEGs

图6 RT-qPCR验证结果图柱状图为RT-qPCR测定的各靶基因相对表达水平（2-ΔΔCt），红色线条表示对应RNA-seq（TPM值），x¯±s，n=3

Fig. 6 Validation results of RT-qPCRThe histograms represent the relative expression levels of each target gene measured by RT-qPCR (2-ΔΔCt), and the red lines indicate the corresponding RNA-seq (TPM values), x¯±s, n=3

图7 代谢物PCA（A）和OPLS-DA（B）图PC1表示第一主成分，PC2表示第二主成分，百分比表示该主成分对数据集的解释率；图中的每个点表示1个样品，同一个组的样品使用同一种颜色表示

Fig. 7 PCA (A) and OPLS-DA (B) score plots of metabolitesPC1 represents the first principal component and PC2 the second principal component, with the percentages indicating their respective explanation rates of the dataset; each dot in the figure denotes a sample, and samples from the same group are colored the same

图8 DAMs数据图A：DAMs火山图；B：DAMs分类图；C：黄酮差异积累代谢物热图

Fig. 8 Data plot of DAMsA: volcano plot of DAMs; B: classification chart of DAMs; C: heat map of differentially accumulated flavonoid metabolites

图9 DAMs KEGG富集分析图

Fig. 9 KEGG enrichment analysis plot of DAMs

图10 茎部类黄酮通路DEGs和DAMs图方框内标注的为代谢物：红色表示代谢物含量升高，绿色表示代谢物含量降低；箭头旁标注的为相关基因：红色标注的基因表示表达上调，绿色标注的基因表示表达下调，蓝色表示既有上调又有下调基因；图中不同颜色的矩形框对应不同的代谢分支：黄色代表苯丙烷代谢途径，粉色代表异黄酮代谢途径，蓝色代表黄酮和黄酮醇代谢途径

Fig. 10 Diagram of DEGs and DAMs in the flavonoid pathway of stemsMetabolites are marked in boxed labels: red indicates significantly increased metabolite content, while green indicates decreased metabolite content. Related genes are annotated beside the arrows: red represents upregulated genes, green represents downregulated genes, and blue indicates genes with both upregulated and downregulated expression patterns. Rectangular boxes of different colors in the diagram correspond to distinct metabolic branches: the yellow box denotes the phenylpropanoid pathway, the pink box represents the isoflavonoid pathway, and the blue box refers to the flavone and flavonol biosynthesis pathway

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