Specific oligonucleotides for qRT-PCR analysis.

Satureja is rich in phenolic monoterpenoids, mainly carvacrol, that is of interest due to diverse biological activities including antifungal and antibacterial. However, limited information is available regarding the molecular mechanisms underlying carvacrol biosynthesis and its regulation for this wonderful medicinal herb. To identify the putative genes involved in carvacrol and other monoterpene biosynthesis pathway, we generated a reference transcriptome in two endemic Satureja species of Iran, containing different yields (Satureja khuzistanica and Satureja rechingeri). Cross-species differential expression analysis was conducted between two species of Satureja. 210 and 186 transcripts related to terpenoid backbone biosynthesis were identified for S. khuzistanica and S. rechingeri, respectively. 29 differentially expressed genes (DEGs) involved in terpenoid biosynthesis were identified, and these DEGs were significantly enriched in monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, carotenoid biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways. Expression patterns of S. khuzistanica and S. rechingeri transcripts involved in the terpenoid biosynthetic pathway were evaluated. In addition, we identified 19 differentially expressed transcription factors (such as MYC4, bHLH, and ARF18) that may control terpenoid biosynthesis. We confirmed the altered expression levels of DEGs that encode carvacrol biosynthetic enzymes using quantitative real-time PCR (qRT-PCR). This study is the first report on de novo assembly and transcriptome data analysis in Satureja which could be useful for an understanding of the main constituents of Satureja essential oil and future research in this genus.香蓟属（Satureja）植物富含酚类单萜类化合物，其主要活性成分为香芹酚（carvacrol），该成分因兼具抗真菌、抗菌等多样生物活性而广受关注。然而，针对这一优良药用植物，有关香芹酚生物合成及其调控的分子机制的研究信息仍较为匮乏。为筛选参与香芹酚及其他单萜类生物合成途径的候选基因，本研究针对伊朗特有且香芹酚产量存在差异的两种香蓟属物种——胡齐斯坦香蓟（Satureja khuzistanica）与雷切尔香蓟（Satureja rechingeri）——构建了参考转录组。针对这两种香蓟属物种开展了跨物种差异表达分析。分别在胡齐斯坦香蓟和雷切尔香蓟中鉴定出210条和186条与萜类骨架生物合成（terpenoid backbone biosynthesis）相关的转录本。本研究共鉴定出29个参与萜类生物合成的差异表达基因（differentially expressed genes, DEGs），这些DEGs显著富集于单萜类生物合成、二萜类生物合成、倍半萜与三萜类生物合成、类胡萝卜素生物合成以及泛醌及其他萜类醌类生物合成等通路。研究人员对两种香蓟属植物中参与萜类生物合成途径的转录本表达模式进行了评估。此外，本研究还鉴定出19个可能调控萜类生物合成的差异表达转录因子，包括MYC4、bHLH及ARF18等。本研究通过实时荧光定量聚合酶链式反应（quantitative real-time PCR, qRT-PCR）验证了编码香芹酚生物合成酶的差异表达基因的表达水平变化。本研究首次报道了香蓟属植物的从头组装（de novo assembly）及转录组数据分析结果，可为解析香蓟属植物精油的主要成分及该属植物的后续相关研究提供重要参考。