SummaryAn integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant‐specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid‐producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI‐FTICR‐MS and ESI‐MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N‐methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N‐methyltransferase (TfPavNMT), which showed a unique preference for (±)‐pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species‐dependent specialized metabolite profiles.
摘要 为了发现参与植物特化代谢的新
基因并确定其功能特征,我们开发了一种综合方法,利用目标代谢物图谱和适度的EST文库(每个文库包含约3500个单体
基因)。已经建立了苄基
异喹啉生物碱生产
细胞培养物的 EST 数据库,这些
细胞培养物包括 Eschscholzia californica、Papaver bracteatum 和 Thalictrum flavum,它们是
生物碱生物合成
基因的丰富宝库。ESI-FTICR-MS 和 ESI-MS/MS 分析有助于对每个系统中的
生物碱进行明确鉴定和相对定量。将每个 EST 文库中的已知和候选
生物合成
基因与根据经验代谢物图谱组装的苄基
异喹啉生物碱生物合成网络进行人工整合,可以鉴定出 4 个 N-甲基转移酶(N
MT)并确定其功能特征。其中一个来自 T. flavum 的 cDNA 编码了帕维因 N-甲基转移酶(TfPavN
MT),该酶对 (±)- 帕维因有独特的偏好,代表了参与帕维因
生物碱分支途径的第一个分离酶。特定
生物碱的出现、编码已知苄基
异喹啉生物碱生物合成
基因的ESTs的补充以及特征N
MTs的不同底物范围之间的相关性证明了双边预测酶功能和依赖物种的特殊代谢物特征的可行性。