Stage and Cultivar Specific Regulation of Cannabinoid in Hemp: An Integrated Omics Perspective

we employed integrated transcriptomic and metabolomic analyses to elucidate the molecular basis of cannabinoid biosynthesis during early and late flowering stages in two hemp cultivars, CBG-Ambit and CBG-Limonene. A total of 338 differentially expressed genes (DEGs) were identified at the early stage (ES), while 342 at the late stage (LS) between CBG-Ambit (Amb) and CBG-Limonene (Lim) cultivars. Additionally, 1342 DEGs were identified between the early and late stages of Ambit, whereas 1152 DEGs among early and late stages in Limonene. Key candidate genes including cytochrome P450 78A9 (CYP78A9), 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGCR1), auxin transporter-like protein 2 (LAX2), myrcene synthase (MyrS), terpene synthase 29 (TS-29), geranylpyrophosphate: olivetolate geranyltransferase (GPP), ABSCISIC ACID-INSENSITIVE 5-like protein 7 isoform X1 (ABI5), MYB transcription factor 1, histone-lysine N-methyltransferase (SUVH6), TIFY 10A, tetraketide alpha-pyrone reductase 2 (TPKR2), ethylene-responsive transcription factor (ERF109), berberine bridge enzyme-like 8 (BBE8) and monoterpene synthase (MTS1), were implicated as central regulators of CBD biosynthesis and conformed by RT-qPCR. Functional enrichment (GO and KEGG) analysis demonstrated that these genes are concentrated in secondary metabolic processes, oxidoreductase activity, and transcriptional regulation, highlighting their central roles in cannabinoid and terpenoid biosynthesis. Along with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), metabolomic profiling further showed increased accumulation of CBDA and CBGA in late stage. Overall design: Identification of differentially expressed genes in early and late stage of CBG-Ambit and CBG-Lim for cannabinoid biosynthesis

本研究采用转录组学与代谢组学整合分析策略，解析两个大麻品种CBG-Ambit与CBG-Limonene在开花早、晚期的大麻素生物合成分子机制。
在CBG-Ambit（简写为Amb）与CBG-Limonene（简写为Lim）两个品种间，开花早期（ES）共鉴定得到338个差异表达基因（differentially expressed genes，DEGs），开花晚期（LS）则鉴定得到342个DEGs。
此外，Amb品种开花早、晚期间共鉴定得到1342个DEGs，Lim品种开花早、晚期间则鉴定得到1152个DEGs。
筛选得到的核心候选基因包括细胞色素P450 78A9（cytochrome P450 78A9，CYP78A9）、3-羟基-3-甲基戊二酰辅酶A还原酶1（3-hydroxy-3-methylglutaryl-coenzyme A reductase 1，HMGCR1）、生长素转运类蛋白2（auxin transporter-like protein 2，LAX2）、月桂烯合酶（myrcene synthase，MyrS）、萜烯合酶29（terpene synthase 29，TS-29）、香叶基焦磷酸：奥利韦托酸香叶基转移酶（geranylpyrophosphate: olivetolate geranyltransferase，GPP）、脱落酸不敏感5样蛋白7亚型X1（ABSCISIC ACID-INSENSITIVE 5-like protein 7 isoform X1，ABI5）、MYB转录因子1、组蛋白赖氨酸N-甲基转移酶（histone-lysine N-methyltransferase，SUVH6）、TIFY 10A、四酮α-吡喃酮还原酶2（tetraketide alpha-pyrone reductase 2，TPKR2）、乙烯响应转录因子（ethylene-responsive transcription factor，ERF109）、小檗碱桥酶样8（berberine bridge enzyme-like 8，BBE8）以及单萜合酶（monoterpene synthase，MTS1），上述基因被证实为大麻二酚（CBD）生物合成的核心调控因子，并经实时定量聚合酶链反应（RT-qPCR）验证。
基因本体（Gene Ontology，GO）与京都基因与基因组百科全书（KEGG）功能富集分析结果显示，上述基因显著富集于次级代谢过程、氧化还原酶活性以及转录调控通路，凸显其在大麻素与萜类化合物生物合成中的核心作用。
结合超高效液相色谱-质谱联用（UPLC-MS）的代谢组学分析进一步显示，晚期开花阶段CBDA与CBGA的积累量显著升高。
实验整体设计：针对大麻素生物合成相关基因，鉴定CBG-Ambit与CBG-Lim两个品种开花早、晚期的差异表达基因。