非靶向代谢组结合转录组解析‘秋分籽’油茶种仁异黄酮代谢机制

Non targeted metabolomics combined with transcriptome analysis of isoflavonoid metabolism mechanism in Camellia oleifera ‘Qiufen’ kernel

摘要

【目的】‘秋分籽’油茶是我国特有的早熟油茶资源，本研究旨在揭示其种仁代谢物构成，阐明重要活性物质异黄酮类代谢物积累的动态规律，解析其代谢通路的关键核心基因及分子调控机制，以期为分子设计育种定向改良高异黄酮油茶优良种质提供基因资源和科学依据。【方法】取‘秋分籽’油茶果实生长期（QF0722）、油脂转化期（QF0821）和果实成熟期（QF0920）3 个关键发育时期的果实，经表型观察后对其种仁进行非靶向代谢组和转录组分析；通过对差异代谢物和差异表达基因的 KEGG 分析、关联分析和相关性分析，揭示调控‘秋分籽’油茶种仁发育，特别是异黄酮代谢关键基因和潜在分子机制。【结果】‘秋分籽’油茶果皮青色不变，但其果皮表面绒毛逐渐减少，种皮颜色不断加深并脱水为棕褐色。代谢组和转录组分别鉴定到 4 698 个代谢物和 27 211 个差异表达基因，通过 KEGG 分析和关联分析发现，苯丙烷类生物合成、异黄酮类生物合成、多种植物次生代谢产物的生物合成、萜类骨架生物合成、糖酵解/葡萄糖生成、丙酮酸代谢、α-亚麻酸代谢、抗坏血酸和醛酸代谢、甘油磷脂代谢/甘油酯代谢等通路在 3 个时期持续富集，嘌呤代谢、嘧啶代谢、赖氨酸降解、丁酸代谢、戊糖和葡萄糖醛酸相互转化等代谢通路在 QF0821 vs QF0722 中高度富集；而脂肪酸降解、核黄素代谢、硫代葡萄糖苷生物合成、精氨酸生物合成等代谢通路在 QF0920 vs QF0821 中高度富集。此外，通过整合代谢物−基因共表达网络，筛选出与 14 种异黄酮代谢物动态高度相关的 YCChr15 a_016660、 YCChr2 a_017080 和 novel.31123 等结构基因及 novel.22106、 novel.68240、 novel.5631 和 novel.34569 等 MYB 转录因子，为解析油茶异黄酮生物合成的转录调控机制提供了候选靶点。【结论】本研究基于‘秋分籽’油茶 3 个关键发育时期种仁的多组学分析，揭示了其早期氨基酸积累、中期次生代谢富集、晚期油脂合成的代谢变化特征，油脂转化期碳代谢支撑脂肪酸组装与细胞膜构建，成熟期脂肪酸修饰与异黄酮等防御物质协同积累的转录表达特性，阐明了‘秋分籽’油茶种仁发育过程中初生代谢与苯丙烷-异黄酮通路在转录代谢水平上的系统性协同机制，并筛选出与异黄酮代谢高度相关的 YCChr15 a_016660、 YCChr2 a_017080、 novel.31123、 novel.22106、 novel.68240、 novel.5631 和 novel.34569 等核心基因，为高异黄酮油茶分子育种提供了基因资源与科学依据。

Abstract

[Objective] Camellia oleifera ‘Qiufen’ is a unique early-maturing germplasm resource in China. This study aims to elucidate the metabolic profile of its kernels, clarify the dynamic accumulation patterns of important bioactive isoflavonoids, and decipher the key regulatory genes and molecular mechanisms underlying their metabolic pathways. The findings are expected to provide genetic resources and a scientific basis for the molecular design breeding of elite oil-tea varieties with high isoflavonoid content. [Method] Fruits of C. oleifera ‘Qiufen’ were collected at three critical developmental stages: fruit growth period (QF0722), oil conversion period (QF0821), and fruit maturity period (QF0920). Following phenotypic observation, their kernels were subjected to untargeted metabolomic and transcriptomic analyses. Through integrated KEGG analysis, association analysis, K-means clustering, and correlation analysis of differentially accumulated metabolites and differentially expressed genes, this study aims to uncover the key genes and potential molecular mechanisms regulating kernel development with a particular focus on isoflavonoid metabolism in C. oleifera ‘Qiufen’. [Result] While the fruit peel of C. oleifera ‘Qiufen’ remained green, the epidermal pubescence gradually diminished, and the seed coat color deepened progressively, eventually dehydrating to a brownish hue. Metabolomic and transcriptomic analyses identified 4 698 metabolites and 27 211 differentially expressed genes, respectively. KEGG enrichment and association analyses revealed that pathways such as phenylpropanoid biosynthesis, isoflavone biosynthesis, biosynthesis of various plant secondary metabolites, terpenoid skeleton biosynthesis, glycolysis/glucose production, pyruvate metabolism, alpha linolenic acid metabolism, ascorbic acid and aldehyde metabolism, glycerophospholipid metabolism/glycerolipid metabolism continued to accumulate in three periods. Metabolic pathways such as purine metabolism, pyrimidine metabolism, lysine degradation, butyric acid metabolism, and interconversion of pentose and glucuronic acid were highly enriched in QF0821 vs QF0722; and metabolic pathways such as fatty acid degradation, riboflavin metabolism, glucosinolate biosynthesis, and arginine biosynthesis were highly enriched in QF0920 vs QF0821. These findings indicate that the aforementioned metabolic pathways collectively regulate kernel development in C. oleifera ‘Qiufen’. In addition, by integrating the metabolite gene co expression network, we screened out structural genes such as YCChr15 a_016660, YCChr2 a_017080 and novel.31123 that are highly correlated with the dynamic changes of 14 isoflavone metabolites, as well as MYB transcription factors including novel.22106, novel.68240, novel.5631 and novel.34569, providing candidate targets for elucidating the transcriptional regulation mechanism of oil tea isoflavone biosynthesis. [Conclusion] This study conducted a multi-omics analysis of the seed kernels of C. oleifera ‘Qiufen’ during three key developmental stages, revealing the metabolic changes characteristics of amino acid accumulation in the early stage, secondary metabolism enrichment in the middle stage, and lipid synthesis in the late stage. It also elucidated the transcriptional expression characteristics of carbon metabolism supporting fatty acid assembly and cell membrane construction during the lipid transformation period, as well as the coordinated accumulation of fatty acid modifications and defense substances such as isoflavones during the mature stage. It clarified the systematic coordination of primary metabolism and the phenylpropanoid-isoflavone pathway at the transcriptional and metabolic levels during the development of the C. oleifera ‘Qiufen’ kernels. Moreover, it screened out core genes highly related to isoflavone metabolism, including YCChr15a_016660, YCChr2a_017080, novel.31123, novel.22106, novel.68240, novel.5631 and novel.34569, providing valuable genetic resources and scientific basis for molecular breeding of high-isoflavone Camellia.

关键词

‘秋分籽’油茶 / 非靶向代谢组 / 转录组 / 异黄酮代谢 / 定向改良 / 分子设计育种

Key words

Camellia oleifera ‘Qiufen’ / non targeted metabolomics / transcriptome / isoflavonoid metabolism / targeted improvement / molecular design breeding

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基金资助

湖北省重点研发计划项目(2025BBB010)

湖北省自然科学基金联合基金项目(2025AFD333)

湖北省中央引导地方科技发展专项(2024CSA071)

湖北省高等学校优秀中青年科技创新团队(T2023025)