摘要：

朱砂根是紫金牛科中药植物,主要活性成分为齐墩果烷型三萜类化合物朱砂根皂苷。为探索朱砂根三萜皂苷生物合成的分子基础,采用Illunima HiseqTM 4000对无菌水、2 mmol/L SA处理的3年生朱砂根植株进行转录组测序,基于Blast完成Unigene分类、功能注释、代谢通路分析、蛋白功能注释、差异表达基因分析、代谢途径关键基因挖掘等。共获得41.63 Gb数据,拼接组装获得102 491条Unigenes,平均长度831 bp,注释率50.21%。筛选出3 417个SA应答差异表达基因,其中2 725个基因上调,692个基因下调。差异表达基因显著富集于次生代谢物生物合成、代谢途径、氨基糖和核苷酸糖代谢、糖酵解/糖异生、碳代谢等通路。与朱砂根三萜皂苷相关的代谢通路,包括萜类骨架生物合成(ko00900)和倍半萜与三萜生物合成(ko00909),共筛选出8个关键酶、11条差异表达Unigenes。SA处理后萜类骨架生物合成MVA途径HMGR、HMGS、MVK、GGPS、SS、SQE基因差异表达显著,MEP途径DXS、DXR、MCT、CMK、MDS、HDR基因差异表达不显著。催化生成齐墩果烷型五环三萜前体的关键酶β-香树素合成酶基因(β-AS)表达量下调,后修饰基因CYP450s(CYP72A67)表达量也下调。荧光定量qPCR值与RNA-seq表达量FPKM值变化趋势一致。推测SA通过诱导MVA 途径的关键酶基因表达影响朱砂根三萜皂苷的积累,MEP途径中的酶基因可能参与其他支路次生代谢产物的合成。

关键词: 朱砂根, 三萜皂苷, 转录组, 水杨酸, 差异表达, qPCR

Abstract:

Ardisia crenata Sims is a traditional Chinese medicine plant belonging to Myrsinaceae family.The oleanane-type triterpenoid saponin is regarded as the main active ingredient accumulated in the root.In order to explore the molecular basis of the biosynthesis of triterpenoid saponins,the Illumina HiseqTM 4000 high throughput sequencing method was used to analyze the transcriptome of 3-year-old plants of A.crenata treated with sterile water and 2 mmol/L SA.The transcriptome data of roots were obtained,and GO classification,KOG functional annotation,KEGG metabolic pathway analysis,protein function annotation analysis,differential gene analysis and discovery of key genes in metabolic pathways were completed based on Blast.A total of 41.63 Gb Clean data were obtained,102 491 Unigenes were assembled by de novo splicing with an average length of 831 bp,and the annotation rate was 50.21%.Through differential analysis of genes responding to SA,a total of 3 417 differentially expressed genes(DEGs)were found,of which 2 725 were up-regulated and 692 were down-regulated.DEGs were significantly enriched in secondary metabolite biosynthesis,metabolic pathways,amino and nucleotide metabolism,Glycolysis/Gluconeogenesis,carbon metabolism pathways.Metabolic pathways related to triterpenoid saponin in A.crenata included the pathways of terpene skeleton biosynthesis(ko00900)and sesquiterpene and triterpene biosynthesis(ko00909).A total of 8 key enzymes and 11 differentially expressed Unigenes were screened.Under salicylic acid treatment, the differential expression of HMGR, HMGS, MVK, GGPS, SS, and SQE genes was significant in MVA pathway of terpene skeleton biosynthesis, while the differential expression of DXS, DXR, MCT, CMK, MDS, and HDR genes in MEP pathway was not significant. The gene expression of beta-amyrin synthase(β-AS),the key enzyme that catalyzes the formation of oleanolane pentacyclic triterpene precursors,was down-regulated,as was the expression of the post-modification genes CYP450s(CYP72A67).The change trend of qPCR value was consistent with that of FPKM value of RNA-seq.It is speculated that SA affected the accumulation of triterpenoid saponins by inducing the expression of key enzyme genes in the MVA pathway,and the enzyme genes in MEP pathway may participate in the synthesis of secondary metabolites in other pathways.

Key words: Ardisia crenata Sims, Triterpenoid saponins, Transcriptome, Salicylic acid, Differential expression, qPCR