Shiraishi Akira, Murata Jun, Matsumoto Erika, Matsubara Shin, Ono Eiichiro, Satake Honoo
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Kyoto, 619-0284, Japan.
Research Institute, Suntory Global Innovation Center (SIC) Ltd., Kyoto, 619-0284, Japan.
PLoS One. 2016 Oct 21;11(10):e0164805. doi: 10.1371/journal.pone.0164805. eCollection 2016.
Forsythia spp. are perennial woody plants which are one of the most extensively used medicinal sources of Chinese medicines and functional diets owing to their lignan contents. Lignans have received widespread attention as leading compounds in the development of antitumor drugs and healthy diets for reducing the risks of lifestyle-related diseases. However, the molecular basis of Forsythia has yet to be established. In this study, we have verified de novo deep transcriptome of Forsythia koreana leaf and callus using the Illumina HiSeq 1500 platform. A total of 89 million reads were assembled into 116,824 contigs using Trinity, and 1,576 of the contigs displayed the sequence similarity to the enzymes responsible for plant specialized metabolism including lignan biosynthesis. Notably, gene ontology (GO) analysis indicated the remarkable enrichment of lignan-biosynthetic enzyme genes in the callus transcriptome. Nevertheless, precise annotation and molecular phylogenetic analyses were hindered by partial sequences of open reading frames (ORFs) of the Trinity-based contigs. To obtain more numerous contigs harboring a full-length ORF, we developed a novel overlapping layout consensus-based procedure, virtual primer-based sequence reassembly (VP-seq). VP-seq elucidated 709 full-length ORFs, whereas only 146 full-length ORFs were assembled by Trinity. The comparison of expression profiles of leaf and callus using VP-seq-based full-length ORFs revealed 50-fold upregulation of secoisolariciresinol dehydrogenase (SIRD) in callus. Expression and phylogenetic cluster analyses predicted candidates for matairesinol-glucosylating enzymes. We also performed VP-seq analysis of lignan-biosynthetic enzyme genes in the transcriptome data of other lignan-rich plants, Linum flavum, Linum usitatissimum and Podophyllum hexandrum. The comparative analysis indicated both common gene clusters involved in biosynthesis upstream of matairesinol such as SIRD and plant lineage-specific gene clusters, in particular, genes responsible for biosynthetic pathways for production of podophyllotoxin; CYP71BE54, a key enzyme gene for podophyllotoxin biosynthesis in P. hexandrum, was not found in L. flavum, although both P. hexandrum. and L. flavum yield podophyllotoxin. Altogether, these data have established the fruitful molecular basis of Forsythia and provided insight into the molecular evolution and diversity of lignan biosynthetic pathways.
连翘属植物是多年生木本植物，由于其木脂素含量，是中药和功能性食品中使用最广泛的药用来源之一。木脂素作为抗肿瘤药物开发和降低生活方式相关疾病风险的健康饮食中的主要化合物，受到了广泛关注。然而，连翘的分子基础尚未建立。在本研究中，我们使用Illumina HiSeq 1500平台验证了朝鲜连翘叶片和愈伤组织的从头深度转录组。使用Trinity将总共8900万个读段组装成116,824个重叠群，其中1576个重叠群与负责包括木脂素生物合成在内的植物特殊代谢的酶具有序列相似性。值得注意的是，基因本体（GO）分析表明愈伤组织转录组中木脂素生物合成酶基因显著富集。然而，基于Trinity的重叠群的开放阅读框（ORF）部分序列阻碍了精确注释和分子系统发育分析。为了获得更多含有全长ORF的重叠群，我们开发了一种基于重叠布局共识的新方法，即基于虚拟引物的序列重新组装（VP-seq）。VP-seq阐明了709个全长ORF，而Trinity仅组装了146个全长ORF。使用基于VP-seq的全长ORF对叶片和愈伤组织的表达谱进行比较，发现愈伤组织中开环异落叶松脂醇脱氢酶（SIRD）上调了50倍。表达和系统发育聚类分析预测了罗汉松脂醇糖基化酶的候选基因。我们还对其他富含木脂素的植物亚麻、亚麻和六角莲转录组数据中的木脂素生物合成酶基因进行了VP-seq分析。比较分析表明，在罗汉松脂醇生物合成上游涉及的常见基因簇，如SIRD和植物谱系特异性基因簇，特别是负责鬼臼毒素生产生物合成途径的基因；尽管六角莲和亚麻都能产生鬼臼毒素，但在亚麻中未发现六角莲中鬼臼毒素生物合成的关键酶基因CYP71BE54。总之，这些数据建立了连翘丰富的分子基础，并为木脂素生物合成途径的分子进化和多样性提供了见解。
