Data from: Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants

Background: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. Results: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal (www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. Conclusions: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.背景：苄基异喹啉生物碱（Benzylisoquinoline Alkaloids, BIAs）是一类多样的植物特化代谢产物，其共同的生物合成起点均为酪氨酸。诸多BIAs具有强效药理活性，其富集植物在传统医学与文化实践中拥有悠久的应用历史。数十年来，学界以少数植物物种作为模式体系开展研究，使得包括吗啡、血根碱、小檗碱在内的主要BIA生物合成途径得以近乎完全阐明。但这一研究聚焦也造成了非模式物种相关研究的匮乏：这类物种同样可富集多种BIAs，但其生物合成途径至今仍完全未被探索。此外，这些非模式物种蕴含丰富的酶多样性资源，对植物生物化学家与新兴合成生物学研究具有重要价值。结果：为获取富集BIAs的非模式植物的遗传多样性资源，本研究选取了毛茛目4个科的20个物种。对每个物种提取的RNA分别采用1）罗氏GS-FLX Titanium测序平台与2）Illumina GA/HiSeq测序平台进行分析，最终共获得40个深度测序转录组文库。从头组装、注释以及后续的全长编码序列（Coding Sequence, CDS）预测结果显示，多数物种采用Illumina平台的组装效果更佳。各转录组的组装数据已上传至已有的基于网页的BLAST门户（www.phytometasyn.ca），以供公众访问。以BIA生物合成酶作为查询序列，通过同源性挖掘文库，共获得约850个可能参与生物碱生物合成的候选基因。采用库间FPKM标准化方法对这些候选基因进行表达分析，所得表达数据可为合理筛选候选基因提供依据，并可用于推测BIA代谢途径中潜在的代谢瓶颈。对15个不同的酶/蛋白家族分别进行系统发育分析，发现诸多新基因可能参与一类或多类生物碱的生物合成，包括吗啡烷类、阿朴啡类以及苯酞异喹啉类生物碱。利用本研究获得的转录组资源，对黄花海罂粟（Glaucium flavum）的候选N-甲基转移酶（N-methyltransferases, NMTs）开展了案例研究，验证了预测的酶活性并发现了新的酶活功能。结论：本研究为分离和发现1）BIA代谢途径中的功能同源蛋白以及2）全新的催化酶提供了关键资源。对黄花海罂粟NMTs的功能分析验证了该资源的实用性，并强调了通过实验验证推测酶功能的重要性。尽管本研究涉及的多数植物含有丰富的生物活性生物碱，且在传统医学中具有重要地位，但此前尚未有公开可访问、完成全面注释且支持BLAST检索的转录组数据。本研究所得结果可为BIA代谢途径的后续阐明提供关键序列信息，并为相关实验设计提供参考依据。