以代謝體學探討菸草毛狀根之尼古丁生合成機制

Abstract

毛狀根 (hairy root) 為植物受農桿菌 (Agrobacterium sp.) 感染後所生成之根狀組織，由於具有次級代謝物高累積量之特色，因此極具作為植物次級代謝物生產平台之潛力。本研究以菸草 (Nicotiana tabacum) 為模式植物探討其毛狀根之次級代謝物高累積量之機制。藉由對主要生物鹼與多胺類之標靶代謝體分析 (targeted metabolomics analysis) ，我們發現菸草毛狀根體內之尼古丁 (nicotine) 與腐胺 (putrescine) 含量呈負相關。為了進一步了解腐胺於毛狀根之尼古丁生合成路徑之調控，我們將腐胺添加至毛狀根培養基中，發現尼古丁含量較高之毛狀根，在外添加腐胺之培養條件下，其內生腐胺之濃度較低且生長狀態較佳，顯示毛狀根得以累積高含量之尼古丁可能與代謝腐胺之能力較強有關。本研究亦結合非標靶代謝體分析 (untargeted metabolomics analysis) 與機器學習之技術 (machine learning) ，挑選出許多生長快速且高尼古丁表現量之毛狀根之小分子生物標記 (putative biomarker)，並將進行分子結構鑑定。此外，本研究發現高尼古丁含量毛狀根特有之形態特徵，亦即可由形態特徵挑選具備高效率生產尼古丁能力之毛狀根品系。經由本研究得以更加了解高尼古丁含量菸草毛狀根之型態與生理機制，有利於以毛狀根作為植物次級代謝物生產平台之應用發展。

Hairy roots, which result from Agrobacterium infection, usually accumulate more secondary metabolites than other plant tissues. Therefore, hairy roots have great potential for producing secondary metabolites. In this work, Nicotiana tabacum hairy root was established as a model organism to study the secondary metabolism of hairy roots. First, by the application of targeted metabolomic analysis to major alkaloids and polyamines, we observed a negative correlation between the contents of nicotine and putrescine (the biosynthetic intermediate of nicotine). To further understand the role of putrescine in nicotine biosynthesis, putrescine was added to culture media, and clones with higher nicotine content were found to accumulate less putrescine and to grow better. These results indicated that high nicotine content in hairy roots may result from the high efficiency of their putrescine metabolism. Second, using untargeted metabolomic analysis associated with machine learning techniques, we found that several putative biomarkers were expressed differentially in hairy roots with both higher nicotine content and higher growth rate. These putative biomarkers will be further confirmed. In addition, we also found unique morphological traits of high nicotine content clones, which could help to increase the efficiency of the selection of high nicotine production clones. In summary, this study revealed the nicotine production machinery in Nicotiana tabacum hairy roots. We hope that these results facilitate the development of industrial-scale plant secondary metabolite production.