Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.
Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.
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DOI：
10.1186/1471-2229-10-252
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发表时间：
2010-11-18
影响因子：
5.3
通讯作者：
Facchini PJ
中科院分区：
文献类型：
--
作者：
Desgagné-Penix I;Khan MF;Schriemer DC;Cram D;Nowak J;Facchini PJ
罂粟是几种药用苄基异喹啉生物碱的来源，包括吗啡、可待因和血根碱。响应于用真菌诱导子处理，血根碱的生物合成和积累与罂粟细胞培养物中的其它植物防御反应一起被诱导沿着。培养细胞中生物碱代谢的转录诱导提供了一个机会，通过整合使用下一代技术生成的深度转录组和蛋白质组数据库来识别该过程的组成部分。用真菌诱导子处理罂粟细胞培养物10小时，制备cDNA文库。使用454 GS-FLX Titanium焦磷酸测序，产生427，369个表达序列标签（EST），平均长度为462 bp。这些序列的组装产生了93，723个unigenes，其中23，753个被分配了Gene Ontology注释。在EST数据库中鉴定了编码所有已知血根碱生物合成酶的转录本，其中5个转录本是50个最丰富的转录本中的代表。用真菌激发子处理50 h的细胞培养物的总蛋白提取物的液相色谱-串联质谱（LC-MS/MS）促进了1，004种蛋白质的鉴定。通过一维SDS-PAGE分级分离蛋白质，并用胰蛋白酶消化，然后进行LC-MS/MS分析。查询罂粟特异性EST数据库大大增强了肽的鉴定。10种已知血根碱生物合成酶和许多相关的主要代谢酶中的8种在肽数据库中表示。深度转录组和蛋白质组分析的整合提供了一个有效的平台，目录的次级代谢的组成部分，并确定编码未知酶的基因。在具有明确定义的代谢物谱的系统中建立由下一代技术生成的相应转录物和蛋白质数据库有助于改善基因、酶和途径组分之间的联系。蛋白质组数据库代表了最相关的类胡萝卜素生产酶，相比之下，更深，更完整的转录组库。转录本数据库包含编码大多数生物碱生物合成酶的全长mRNA，这是新型候选基因功能表征的关键要求。
Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.
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影响因子：
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