De Novo Transcriptomes of Forsythia koreana Using a Novel Assembly Method: Insight into Tissue- and Species-Specific Expression of Lignan Biosynthesis-Related Gene.
De Novo Transcriptomes of Forsythia koreana Using a Novel Assembly Method: Insight into Tissue- and Species-Specific Expression of Lignan Biosynthesis-Related Gene.
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DOI：
10.1371/journal.pone.0164805
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Satake H
中科院分区：
文献类型：
--
作者：
Shiraishi A;Murata J;Matsumoto E;Matsubara S;Ono E;Satake H
连翘是多年生木本植物，由于其木脂素含量，是最广泛使用的中药和功能性膳食的药用来源之一。木脂素作为抗肿瘤药物和降低生活方式相关疾病风险的健康饮食的主要化合物受到广泛关注。然而，连翘的分子基础尚未建立。在这项研究中，我们使用Illumina HiSeq 1500平台重新验证了朝鲜连翘叶和愈伤组织的深层转录组。使用Trinity将总共8900万个读段组装成116，824个重叠群，其中1，576个重叠群显示出与负责植物特化代谢（包括木酚素生物合成）的酶的序列相似性。值得注意的是，基因本体论（GO）分析表明显着丰富的木脂素生物合成酶基因的愈伤组织转录组。然而，精确的注释和分子系统发育分析受到Trinity重叠群开放阅读框（ORF）部分序列的阻碍。为了获得更多的含有全长ORF的重叠群，我们开发了一种新的重叠布局共识为基础的程序，基于虚拟引物的序列重组（VP-seq）。VP-seq共解析了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比较叶和愈伤组织的表达谱揭示愈伤组织中开环异落叶松树脂醇脱氢酶（SIRD）的50倍上调。表达和系统发育聚类分析预测的候选人matairesinol-葡萄糖基化酶。我们还对其他富含木脂素的植物黄亚麻、亚麻和六蕊鬼耳草的转录组数据中的木脂素生物合成酶基因进行了VP-测序分析。通过比较分析发现，这两个基因簇既有参与matairesinol生物合成上游的共同基因簇，如SIRD，也有植物谱系特异性基因簇，特别是负责鬼臼毒素生物合成途径的基因，其中，在六蕊牡丹中没有发现鬼臼毒素生物合成的关键酶基因CYP 71 BE 54。flavum，尽管两者都是P. hexandrum。和L.黄曲霉产生鬼臼毒素。总之，这些数据建立了富有成效的连翘的分子基础，并提供了深入了解的分子进化和多样性的木脂素生物合成途径。
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.
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