Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of, Medical Sciences, No.151, Malianwa North Road, Haidian District, Beijing, 100193, China.
Tianjin University of Traditional Chinese Medicine, No.10, Poyanghu Road, Jinghai District, Tianjin, 301617, China.
BMC Genomics. 2019 Aug 5;20(1):632. doi: 10.1186/s12864-019-6000-y.
Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis.
Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis.
We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.
绞股蓝皂苷是绞股蓝中的一组三萜皂苷，与人参属中的人参皂苷相同或非常相似。已经鉴定出几种参与人参皂苷生物合成的酶，这为阐明绞股蓝皂苷生物合成途径提供了重要线索。我们假设绞股蓝皂苷和人参皂苷可能具有相似的生物合成机制，并且两条途径中的相应酶在序列上可能具有相当大的相似性。为了进一步了解绞股蓝皂苷的生物合成，我们采用混合测序策略对绞股蓝转录组进行测序，然后通过构建系统发育树和基因表达分析来确定该途径中的候选基因。
按照 PacBio 标准分析流程，获得了 66046 个经过抛光的一致序列，而 Illumina 数据则使用 Trinity 软件组装成 140601 个 unigenes。然后，将这两条分析途径的输出序列合并。使用 CD-HIT 软件去除冗余数据后，共获得 140157 个最终的 unigenes。经过功能注释，选择了 5 个 2,3-氧化鲨烯环化酶基因、145 个细胞色素 P450 基因和 254 个 UDP-糖基转移酶基因，用于筛选绞股蓝皂苷生物合成相关基因。通过系统发育分析，将一些基因分为同一亚家族或与参与人参皂苷生物合成的特征酶密切相关的进化分支。使用实时 PCR 技术，在不同组织和茉莉酸甲酯诱导后的不同时间研究了它们的表达模式。由于同一生物合成途径中的基因通常共表达，我们推测 GpOSC1、GpCYP89 和 GpUGT35 是绞股蓝皂苷生物合成的主要候选基因。此外，六个 GpWRKY 和一个 GpbHLH 可能在调节绞股蓝皂苷生物合成中发挥作用。
我们开发了一种混合测序策略，以获得更长的转录组，提高准确性，这将极大地有助于下游基因的筛选和表征，从而提高我们阐明次生代谢物生物合成途径的能力。通过这种策略，我们发现了一些可能参与绞股蓝皂苷生物合成的候选基因，这为阐明该生物合成途径奠定了重要基础，从而极大地促进了该物种在代谢调控、合成生物学和分子育种方面的进一步研究。
