Transcriptome Profiling of Khat (<i>Catha edulis</i>) and <i>Ephedra sinica</i> Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis

Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.苯丙胺类类似物(Amphetamine analogues)由麻黄属(Ephedra)植物以及巧茶(khat, Catha edulis)合成，其中包括广泛应用的减充血剂与食欲抑制剂(1S,2S)-伪麻黄碱和(1R,2S)-麻黄碱。这类源自L-苯丙氨酸的代谢产物的生物合成涉及多步途径，该途径的部分生化机制已通过其他植物中已阐明的苯甲酸代谢知识，以及以巧茶和麻黄属物种为模型系统获得的直接证据得以解析。尽管苯丙胺类生物碱具有重要的商业价值，但其生物合成途径中仅单一步骤在分子层面得到了阐明。本研究采用Illumina二代测序(Illumina next-generation sequencing)技术，结合Trinity与Velvet-Oases组装平台，为草麻黄(Ephedra sinica)和巧茶建立了数据挖掘框架。涵盖总计20万个单基因簇(unigene)的序列文库，通过针对公共数据库的直接检索流程完成了注释。注释内容包括分配基因本体(Gene Ontology, GO)术语，以将单基因簇归类至不同功能类别。作为本研究旨在发现新基因的功能基因组学计划的一部分，我们对数据库进行了挖掘，以筛选出可能参与生物碱生物合成的候选酶。本次挖掘所用的查询序列包括在苯甲酸代谢中已明确功能的酶，以及催化与苯丙胺类生物碱代谢预测反应相似的酶。候选基因基于系统发育关系、基于FPKM的表达数据以及机制合理性进行了评估。构建大规模序列资源是解析该途径的关键一步，这一目标兼具学术与工业应用价值。