目的 探究不同生长时期覆盆子Rubus chingii果实中的黄酮类物质的变化规律，解析其苷类物质积累分子机制。方法 利用UPLC-MS/MS靶向代谢组学技术和高通量转录组测序（RNA sequencing，RNA-seq）分析了覆盆子果实5个不同生长时期S1（小青果）、S2（大青果）、S3（绿转黄果）、S4（黄转红果）、S5（红果）黄酮类化合物积累的变化，利用MetaboAnalyst结合KEGG、MWDB等数据库进行代谢物的功能注释，并利用主成分分析（principal component analysis，PCA）、正交偏最小二乘判别分析（orthogonal partial least squares-discriminant analysis，OPLS-DA）筛选差异代谢物，并将2组学进行联合分析。结果 利用UPLC-MS/MS从5个不同时期覆盆子果实共鉴定出87种代谢物，其中分别检测出81（S1）、77（S2）、78（S3）、76（S4）、77（S5）种差异积累代谢物，差异代谢物主要富集在黄酮生物合成、异黄酮生物合成、黄酮和黄酮醇生物合成、次级代谢物的生物合成等途径。利用高通量测序对5个不同时期覆盆子果实进行转录组文库构建，共获得114.7 Gb的Clean Data，每个样品均达到6.23 M clean reads以上，各样本Q₃₀碱基占92.3%及以上。将不同生长时期的转录组进行比对，差异基因表达分别为有2 354个（S1 vs S2）、1 027个（S1 vs S3）、3 370个（S1 vs S4）、10 249个（S1 vs S5）。结论 从代谢和转录的2个层面上对覆盆子不同生长时期中的黄酮代谢物变化和转录变化展开分析，共注释到了黄酮合成途径的15个黄酮代谢物和95个基因，将其进行相关性分析发现，查耳酮异构酶（chalcone isomerase，CHI）、对羟基肉桂酰转移酶（hydroxycinnamoyl transferase，HCT）、查耳酮合酶（chalcone synthase，CHS）、黄酮醇合酶（flavonol synthase，FLS）、黄烷酮-3-羟化酶（flavanone 3-hydroxylase，F3H）、黄酮3’-羟化酶（flavonoid 3'-hydroxylase，F3′H）等基因的表达水平与黄酮代谢物的积累显著相关，表明这些基因参与调控黄酮类化合物的生物合成。首次阐述了覆盆子的黄酮类成分的合成途径和相关基因，为利用生物工程技术生产其黄酮类化合物提供了依据，对于扩大药用资源具有重要意义。

Objective To investigate the variation patterns of flavonoid compounds in Rubus chingii at different developmental stages and to elucidate the molecular mechanisms underlying the accumulation of glycosides. Methods Targeted metabolomics based on UPLC-MS/MS and high-throughput transcriptome sequencing (RNA sequencing, RNA-seq) were used to analyze the changes in flavonoid compound accumulation during five developmental stages of R. chingii: S1 (little green fruit), S2 (large green fruit), S3 (green-to-yellow fruit), S4 (yellow-to-red fruit), S5 (red fruit). Functional annotation of metabolites was performed using MetaboAnalyst in conjunction with KEGG, MWDB, and other databases. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were employed to screen differential metabolites. An integrated analysis of metabolomics and transcriptomics was conducted. Results A total of 87 metabolites were identified across the five developmental stages using targeted metabolomics. Differentially accumulated metabolites were detected in the following numbers: 81 (S1), 77 (S2), 78 (S3), 76 (S4), and 77 (S5). These metabolites were mainly enriched in pathways including flavonoid biosynthesis, is oflavonoid biosynthesis, flavone and flavonol biosynthesis, and secondary metabolite biosynthesis. High-throughput sequencing generated a total of 114.7 Gb of clean data, with each sample producing over 6.23 M Clean reads and Q₃₀base percentages above 92.3%. Comparative transcriptome analysis across developmental stages revealed differentially expressed genes: 2 354 (S1 vs S2), 1 027 (S1 vs S3), 3 370 (S1 vs S4), and 10 249 (S1 vs S5); Conclusion This study analyzed the changes in flavonoid metabolites and transcriptome profiles of R. chingii at different developmental stages from both metabolic and transcriptional perspectives. A total of 15 flavonoid metabolites and 95 genes involved in the flavonoid biosynthesis pathway were annotated. Correlation analysis revealed that the expression levels of genes such as CHI, HCT, CHS, FLS, F3H, and F3’H were significantly associated with flavonoid accumulation, indicating their regulatory roles in flavonoid biosynthesis. This is the first study to elucidate the flavonoid biosynthesis pathway and related genes in R. chingii, providing a theoretical basis for the biotechnological production of flavonoid compounds and offering significant value for the development of medicinal resources.

R286.12

浙江省公益基金计划项目（LTGN24H280001）；浙江省自然科学基金重大项目（LD25H280001）