Transcriptome Sequencing and Expression Analysis of Terpenoid Biosynthesis Genes in Litsea cubeba

BackgroundAromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. ResultsUsing Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. Denovo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. ConclusionRNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of essential oils in L. cubeba fruits.背景：从山鸡椒（Litsea cubeba (Lour.) Pers.）新鲜果实中提取的芳香精油具有多样的医疗与经济价值。该类精油的优势成分为单萜与倍半萜。解析萜类生物合成的分子机制，对提升萜类物质的产量与品质至关重要。然而，公共数据库中现存的山鸡椒核苷酸序列仅40条，远不足以支撑相关分子机制的研究。因此，为获取大规模转录组序列以用于基因挖掘（尤其是萜类生物合成相关基因），对山鸡椒开展高通量转录组测序实属必要。 研究结果：采用Illumina双端测序技术，共获得约2350万条高质量reads。通过从头组装（de novo assembly）得到68648条单基因簇（unigene），平均长度为834 bp。总计38439条（占比56%）单基因簇获得功能注释，其中35732条、25806条单基因簇可分别比对至基因本体（Gene Ontology，GO）数据库与直系同源群（Cluster of Orthologous Groups，COG）数据库。通过比对京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路数据库，16130条单基因簇被归类至297条KEGG通路中，其中61条单基因簇与萜类骨架生物合成相关，涵盖甲羟戊酸途径与2-甲基-D-赤藓糖醇-4-磷酸途径。借助PlantCyc数据库进行注释，12963条单基因簇中有285条被归入萜类代谢通路。此外，本研究从该转录组中鉴定出14个萜合酶基因。采用实时定量聚合酶链反应（reverse transcription quantitative PCR，RT-qPCR）分析了16个萜类生物合成相关基因的表达模式，以探究其潜在功能。 结论：转录组测序可有效获取大量序列信息。据我们所知，本研究是首次对山鸡椒转录组进行系统性解析，所获得的大规模转录组数据将加速解析精油生物合成的分子机制。本研究结果可为后续开展山鸡椒果实精油化学组成背后的分子遗传与基因组学研究提供支撑。