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BACKGROUND

Persicaria minor (kesum) is an herbaceous plant with a high level of secondary metabolite compounds, particularly terpenoids. These terpenoid compounds have well-established roles in the pharmaceutical and food industries. Although the terpenoids of P. minor have been studied thoroughly, the involvement of microRNA (miRNA) in terpenoid regulation remains poorly understood and needs to be explored. In this study, P. minor plants were inoculated with the pathogenic fungus Fusarium oxysporum for terpenoid induction.

RESULT

SPME GC-MS analysis showed the highest terpenoid accumulation on the 6th day post-inoculation (dpi) compared to the other treatment time points (0 dpi, 3 dpi, and 9 dpi). Among the increased terpenoid compounds, α-cedrene, valencene and β-bisabolene were prominent. P. minor inoculated for 6 days was selected for miRNA library construction using next generation sequencing. Differential gene expression analysis showed that 58 miRNAs belonging to 30 families had significantly altered regulation. Among these 58 differentially expressed genes (DEGs), 27 [corrected] miRNAs were upregulated, whereas 31 [corrected] miRNAs were downregulated. Two putative novel pre-miRNAs were identified and validated through reverse transcriptase PCR. Prediction of target transcripts potentially involved in the mevalonate pathway (MVA) was carried out by psRobot software, resulting in four miRNAs: pmi-miR530, pmi-miR6173, pmi-miR6300 and a novel miRNA, pmi-Nov_13. In addition, two miRNAs, miR396a and miR398f/g, were predicted to have their target transcripts in the non-mevalonate pathway (MEP). In addition, a novel miRNA, pmi-Nov_12, was identified to have a target gene involved in green leaf volatile (GLV) biosynthesis. RT-qPCR analysis showed that pmi-miR6173, pmi-miR6300 and pmi-nov_13 were downregulated, while miR396a and miR398f/g were upregulated. Pmi-miR530 showed upregulation at 9 dpi, and dynamic expression was observed for pmi-nov_12. Pmi-6300 and pmi-miR396a cleavage sites were detected through degradome sequence analysis. Furthermore, the relationship between miRNA metabolites and mRNA metabolites was validated using correlation analysis.

CONCLUSION

Our findings suggest that six studied miRNAs post-transcriptionally regulate terpenoid biosynthesis in P. minor. This regulatory behaviour of miRNAs has potential as a genetic tool to regulate terpenoid biosynthesis in P. minor.

BACKGROUND

RESULT

CONCLUSION

背景

小酸模（kesum）是一种草本植物，具有高水平的次生代谢产物化合物，特别是萜类化合物。这些萜类化合物在制药和食品工业中具有很好的作用。尽管已经对小酸模的萜类化合物进行了深入研究，但 miRNA（microRNA）在萜类化合物调节中的作用仍知之甚少，需要进一步探索。在这项研究中，用致病真菌尖孢镰刀菌接种小酸模植物以诱导萜类化合物的产生。

结果

SPME GC-MS 分析显示，与其他处理时间点（0 dpi、3 dpi 和 9 dpi）相比，接种后第 6 天萜类化合物的积累最高。在增加的萜类化合物中，α-雪松烯、大根香叶烯和 β-倍半萜烯很突出。选择接种 6 天的小酸模植物用于下一代测序构建 miRNA 文库。差异基因表达分析显示，属于 30 个家族的 58 个 miRNA 有明显的调控变化。在这 58 个差异表达基因（DEGs）中，有 27 个 miRNA 上调，而 31 个 miRNA 下调。通过逆转录 PCR 鉴定并验证了两个假定的新前体 miRNA。通过 psRobot 软件预测可能参与甲羟戊酸途径（MVA）的靶转录本，得到 4 个 miRNA：pmi-miR530、pmi-miR6173、pmi-miR6300 和一个新的 miRNA，pmi-Nov_13。此外，预测到两个 miRNA，miR396a 和 miR398f/g，在非甲羟戊酸途径（MEP）中有其靶转录本。此外，鉴定出一个新的 miRNA，pmi-Nov_12，其靶基因参与绿叶挥发物（GLV）的生物合成。RT-qPCR 分析显示，pmi-miR6173、pmi-miR6300 和 pmi-nov_13 下调，而 miR396a 和 miR398f/g 上调。pmi-miR530 在 9 dpi 时上调，pmi-nov_12 则表现出动态表达。通过降解组序列分析检测到 pmi-miR6300 和 pmi-miR396a 的切割位点。此外，还通过相关性分析验证了 miRNA 代谢物和 mRNA 代谢物之间的关系。