基于多组学分析不同生长阶段金线兰内源激素对类黄酮的潜在调控差异

doi:10.13560/j.cnki.biotech.bull.1985.2025-0861

摘要/Abstract

摘要：

目的探究不同生长阶段的金线兰内源激素对类黄酮代谢的影响，揭示其类黄酮积累差异的分子基础。方法以金线兰为实验材料，分别选取不同生长阶段（S3：3个月，S6：6个月，S10：10个月）的叶片，采用高通量RNA测序技术（RNA-Seq）和液相色谱-质谱联用技术（LC-MS/MS）获得金线兰的转录组学与代谢组学数据。通过生物信息学分析，预测与类黄酮有关的差异酶基因、代谢物与差异激素代谢物，并探讨它们之间的关系。结果共预测出与类黄酮有关差异酶基因17个、差异代谢物16种，与激素有关的差异代谢物19种。F3'H和FLS是影响金线兰叶片类黄酮代谢的关键基因，而矢车菊素5-O-β-D-葡萄糖苷3-O-β-D-樱草糖苷和矢车菊素3-O-β-D-樱草糖苷可能是金线兰叶片颜色呈现差异的关键代谢物。相关性分析表明，共有11个激素差异代谢物和CHS、PGT1、F3H等13个类黄酮差异酶基因显著相关；同时预测到19个转录因子参与调控F3H、F3'H、FLS、CHS、PGT1、CCoAOMT等8个差异酶基因，并且16个转录因子含有响应植物激素（水杨酸、脱落酸、甲基茉莉酸、赤霉素、乙烯和生长素）的顺式元件；并通过RT-qPCR验证了6个基因（3个转录因子和3个酶基因）的表达量，结果与转录组分析一致。结论金线兰不同生长阶段共有16个转录因子响应5类激素元件，进而调控类黄酮代谢相关酶基因F3'H、FLS的表达，最终影响类黄酮类代谢产物飞燕草素5-O-β-D-葡糖苷3-O-β-D-桑布糖苷和矢车菊素3-O-β-D-桑布双糖苷的积累。

关键词: 金线兰, 类黄酮, 植物激素, 转录组, 代谢组

Abstract:

Objective To investigate the influences of endogenous hormones at different growth stages of Anoectochilus roxburghii on flavonoid metabolism and to reveal the molecular basis for the differences in flavonoid accumulation. Method Using A. roxburghii as the experimental material, leaves from different growth stages (S3: 3 months, S6: 6 months, S10: 10 months) were selected. High-throughput RNA sequencing (RNA-Seq) and liquid chromatography-mass spectrometry (LC-MS/MS) were employed to obtain the transcriptomic and metabolomic data of A. roxburghii. Bioinformatics analysis was used to predict the differential enzyme genes related to flavonoids, metabolites, and differential hormone metabolites, and to explore the relationships among them. Result A total of 17 differential enzyme genes related to flavonoids, 16 differential metabolites, and 19 differential hormone metabolites were predicted. F3'H and FLS were identified as the key genes affecting flavonoid metabolism in the leaves of A. roxburghii, while cyanidin 5-O-β-D-glucoside 3-O-β-D-primeveroside and cyanidin 3-O-β-D-primeveroside might be the key metabolites causing the color differences in the leaves. Correlation analysis showed that 11 differential hormone metabolites were significantly related to 13 differential enzyme genes related to flavonoids, such as CHS, PGT1 and F3H. Moreover, 19 transcription factors were predicted to regulate eight differential enzyme genes, including F3H, F3'H, FLS, CHS, PGT1, and CCoAOMT. Among these transcription factors, 16 contained cis-elements responding to plant hormones (salicylic acid, abscisic acid, methyl jasmonate, gibberellin, ethylene, and auxin). The expressions of six genes (three transcription factors and three enzyme genes) were verified by qRT-PCR, and the results were consistent with the transcriptomic analysis. Conclusion At different growth stages, 16 transcription factors in A. roxburghii respond to five types of hormone elements, thereby regulating the expressions of flavonoid metabolism-related enzyme genes F3'H and FLS, and ultimately affecting the accumulation of flavonoid metabolites, delphinidin 5-O-β-D-glucoside 3-O-β-D-sambubioside and cyanidin 3-O-β-D-sambubioside.

Key words: Anoectochilus roxburghii, flavonoids, plant hormones, transcriptome, metabolome

曾菁菁, 罗盼兰, 闫淑君, 郑涛, 杨俊杰, 蔡坤秀, 曹佳玉, 张天翔, 李銮, 陈莹. 基于多组学分析不同生长阶段金线兰内源激素对类黄酮的潜在调控差异[J]. 生物技术通报, 2025, 41(12): 190-200.

ZENG Jing-jing, LUO Pan-lan, YAN Shu-jun, ZHENG Tao, YANG Jun-jie, CAI Kun-xiu, CAO Jia-yu, ZHANG Tian-xiang, LI Luan, CHEN Ying. Potential Regulatory Differences of Endogenous Hormones on Flavonoids in Anoectochilus roxburghii at Different Growth Stages Based on Multi-omics Analysis[J]. Biotechnology Bulletin, 2025, 41(12): 190-200.

图/表 8

图1 不同生长阶段的金线兰从左到右依次为三月龄、六月龄和十月龄。下同

Fig. 1 Anoectochilus roxburghii at different growth stagesFrom left to right, they are three-month-old, six-month-old, and ten-month-old, respectively

图2 不同生长阶段金线兰叶片中差异类黄酮代谢物与植物激素代谢物聚类热图A：类黄酮代谢物；B：植物激素代谢物

Fig. 2 Clustered heatmap of differential flavonoid and plant hormone metabolites in the leaves of A. roxburghii at various growth stagesA: Flavonoid metabolites; B: Plant hormone metabolites

图3 不同生长阶段的金线兰叶片中类黄酮合成相关差异基因聚类热图

Fig. 3 Hierarchical-clustering heatmap of flavonoid-synthesis-related differentially expressed genes in A. roxburghii leaves at different growth stages

图4 不同生长阶段的金线兰叶片类黄酮转录代谢联合分析图

Fig. 4 Integrated transcriptome-metabolome analysis of flavonoids in A. roxburghii leaves at different growth stages

图5 不同生长阶段的金线兰叶片中“植物激素-类黄酮基因”相关性网络植物激素代谢物用KEGG注释号来表示，实线表示正向调控，虚线表示负向调控

Fig. 5 Correlation network between phytohormones and flavonoid-related genes in A. roxburghii leaves at different growth stagesPlant hormone metabolites are denoted by KEGG annotation numbers. Solid lines indicate positive regulation, and dashed lines indicate negative regulation

图6 不同生长阶段金线兰叶片“转录因子-类黄酮合成关键酶基因表达”调控网络图

Fig. 6 Regulatory network between transcription factors and key enzyme genes of flavonoid synthesis in A. roxburghii leaves at different growth stages

图7 不同生长阶段金线兰叶片中类黄酮相关转录因子的启动子顺式元件分析

Fig. 7 Cis-regulatory element profiling of flavonoid-related transcription factors in A. roxburghii leaves at different growth stages

图8 不同生长阶段的金线兰叶片中差异酶和转录因子RT-qPCR分析

Fig. 8 RT-qPCR analysis of differential enzymes and transcription factors in A. roxburghii leaves at different growth stages

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