School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, 610031, China.
Phytochemistry. 2023 Oct;214:113831. doi: 10.1016/j.phytochem.2023.113831. Epub 2023 Aug 19.
Fritillaria unibracteata is an endangered medicinal plant whose bulb has been used as a Chinese herb to suppress cough, asthma and excessive phlegm for centuries. Steroidal alkaloids, which are synthesized via the steroid synthesis pathways, are their significant bioactive constituents. However, few studies on genes involved in steroidal alkaloid biosynthesis in F. unibracteata have been reported, mainly due to the lack of the F. unibracteata genome. In this paper, comparative transcriptomic and metabolomic analyses of four different tissues of F. unibracteata (leaves, flowers, stems, and bulbs) were performed. Imperialine, peiminine, and peimisine were among the significant bioactive compounds that were considerably abundant in bulb tissue, according to the metabolomic findings. Then, 83.60 Gb transcriptome sequencing of four different tissues was performed, of which one gene encoding phosphomevalonate kinase was directly functionally characterized to verify the accuracy of sequences obtained from the transcriptome. A total of 9217 differentially expressed unigenes (DEGs) were identified in four different tissues of F. unibracteata. GO and KEGG enrichments revealed that phenylpropanoid biosynthesis, MVA-mediated terpenoid backbone biosynthesis, and steroid biosynthesis were enriched in bulb tissue, whereas enrichment of MEP-mediated terpenoid backbone biosynthesis, photosynthesis, photosynthesis-antenna protein and carotenoid biosynthesis was observed in aerial tissues. Moreover, clustering analysis indicated that the downstream steroid biosynthesis pathway was more important in steroidal alkaloid biosynthesis compared to the upstream terpenoid backbone biosynthesis pathway. Hence, MVA-mediated biosynthesis of steroidal alkaloids was proposed, in which 15 bulb-clustered DEGs were positively correlated with a high accumulation of bioactive steroid alkaloids, further validating our proposal. In addition, 36 CYP450s showing a positive correlation with bioactive steroidal alkaloids provided candidate enzymes to catalyze the subsequent steps of steroidal alkaloid biosynthesis. In addition, the transcription factors and ABC transporters clustered in bulb tissue might be responsible for the regulation and transportation of steroidal alkaloid biosynthesis. Protein-protein interaction analysis implied a highly complex steroid alkaloid biosynthesis network in which delta (24)-sterol reductase might be one of the central catalysts. Based on the integrated transcriptome and metabolome, this current study is a first step in understanding the tissue-specific biosynthesis of steroidal alkaloids in F. unibracteata. Furthermore, key genes and regulators identified herein could facilitate metabolic engineering to improve steroidal alkaloids in F. unibracteata.
贝母兰是一种濒危药用植物，其鳞茎被用作中药已有数百年历史，用于镇咳、哮喘和化痰。甾体生物碱是通过甾体合成途径合成的，是其重要的生物活性成分。然而，由于缺乏贝母兰基因组，对其甾体生物碱生物合成相关基因的研究较少。本文对贝母兰的四种不同组织（叶、花、茎和鳞茎）进行了比较转录组和代谢组分析。代谢组学研究发现，在鳞茎组织中，浙贝甲素、平贝甲素和贝母辛等重要生物活性化合物含量丰富。然后，对四种不同组织进行了 83.60 Gb 的转录组测序，其中一个编码磷酸甲羟戊酸激酶的基因被直接功能表征，以验证转录组获得序列的准确性。在贝母兰的四种不同组织中共鉴定出 9217 个差异表达的基因。GO 和 KEGG 富集分析表明，在鳞茎组织中，苯丙素生物合成、MVA 介导的萜类骨架生物合成和甾体生物合成被富集，而在地上组织中，MEP 介导的萜类骨架生物合成、光合作用、天线蛋白和类胡萝卜素生物合成被富集。此外，聚类分析表明，甾体生物碱生物合成的下游甾体生物合成途径比萜类骨架生物合成途径更为重要。因此，提出了 MVA 介导的甾体生物碱生物合成途径，其中 15 个与生物活性甾体生物碱高积累相关的鳞茎聚类 DEGs 进一步验证了我们的假设。此外，36 个与生物活性甾体生物碱呈正相关的 CYP450 为甾体生物碱生物合成的后续步骤提供了候选酶。此外，在鳞茎组织中聚类的转录因子和 ABC 转运蛋白可能负责甾体生物碱生物合成的调节和运输。蛋白-蛋白相互作用分析表明，甾体生物碱生物合成网络非常复杂，其中 δ（24）-固醇还原酶可能是其中的一个中心催化剂。基于转录组和代谢组的综合分析，本研究是了解贝母兰甾体生物碱组织特异性生物合成的第一步。此外，本文鉴定的关键基因和调控因子可促进代谢工程以提高贝母兰中的甾体生物碱含量。
