不同年份防风转录组学分析及有效成分生物合成关键基因挖掘

Transcriptome profiling of Saposhnikovia divaricata growing for different years and mining of key genes in active ingredient biosynthesis

防风为常用大宗药材,为探究防风活性成分色原酮和香豆素合成途径关键酶基因及其表达情况,该研究对一年生(S1)和二年生(S2)防风进行了转录组测序和生物信息学分析。利用IlluminaHiSeq平台进行测序,共得到40.8 Gb的数据。采用Trinity软件进行拼接后,得到110 732条transcripts和86 233条unigene。与NR、Swiss-Prot、GO、KEGG、PFAM各大数据库进行了比对和注释。注释结果显示防风与野胡萝卜同源的序列最多。KEGG代谢富集分析显示差异基因主要富集在植物激素信号传导、苯丙烷生物合成和黄酮类生物合成途径中。在防风有效成分合成相关通路中共鉴别出27条差异表达unigene,包含13种酶。二年生防风较一年生防风PAL、BGL、C4H、4CL、CYP98A、CSE、REF、CCoAOMT均上调表达,CHS、CAD、COMT均下调表达,HCT、POD既有上调表达的又有下调表达的unigene。其中PAL、C4H、4CL、BGL、CHS可作为防风有效成分合成的候选基因。对关键的4个酶基因进行RT-qPCR验证,基因表达结果与转录组测序一致。该研究丰富了防风的遗传数据信息,为鉴定参与次生代谢产物生物合成的候选基因提供了有力支持。

Saposhnikovia divaricata is a commonly used bulk medicinal plant. To explore the key enzyme genes and their expression in the biosynthesis of chromone and coumarin, the key active components, we carried out transcriptome sequencing(Illumina HiSeq) and bioinformatics analysis for the 1-year-old(S1) and 2-year-old(S2) plants of S. divaricata. A total of 40.8 Gb data was obtained. After the sequence assembly via Trinity, 110 732 transcripts and 86 233 unigenes were obtained, which were aligned and annotated with NR, Swiss-Prot, GO, KEGG, and PFAM. Daucus carota and S. divaricata had the highest sequence homology. KEGG pathway enrichment showed that the differentially expressed genes were mainly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis pathways. A total of 27 differentially expressed unigenes, including 13 enzyme genes, were identified in the pathways related to the synthesis of active ingredients in S. divaricata. Compared with S1 plant, S2 plant showed up-regulated expression of PAL, BGL, C4H, 4CL, CYP98A, CSE, REF, and CCoAOMT and down-regulated expression of CHS, CAD, and COMT. HCT and POD had both up-regulated and down-regulated unigenes. Among them, PAL, C4H, 4CL, BGL, and CHS can be used as candidate genes for the synthesis of the active ingredients in S. divaricata. The four key enzyme genes were verified by RT-qPCR, which showed the results consistent with transcriptome sequencing. This study enriches the genetic information of S. divaricata and provides support for the identification of candidate genes in the biosynthesis of secondary metabolites.