基于代谢组学和转录组学的不同生长年限下银杏萜类生物合成关键基因表达分析

Expression analysis of key genes in terpenoid biosynthesis of Ginkgo biloba under different growth years based on metabolomics and transcriptome

目的以不同发育阶段不同树龄的银杏叶(银杏幼树叶,多年生银杏树叶)为对象,分析次生代谢物变化规律,测定黄酮及萜内酯含量,分析萜类生物合成的通路和关键基因表达,为提高银杏萜内酯产量提供分子生药学数据。方法采用液相色谱-质谱联用(LC-MS)技术,并进行高通量转录组学测序(RNA-seq),从差异表达基因中寻找次生代谢物生物合成的关联酶基因。结果代谢数据表明,不同树龄,不同发育阶段的银杏叶次生代谢物明显不同,黄酮类成分,老树叶片与幼树叶4~7月含量均呈下降趋势;同发育阶段,幼树叶含量高于老树叶片含量;萜内酯类成分,老树叶片4~7月含量略微呈上升趋势,幼树叶略微呈缓降趋势;同发育阶段,幼树叶含量高于老树叶片含量。转录组分析出萌发期,老树叶片与幼树叶片生长状态相似,5~7月老树叶与幼树叶基因表达发生较大变化。筛选出49条与萜类合成有关的候选基因,分析后发现甲羟戊酸途径(mevalonate pathway,MVA)中老树叶表达高于幼树叶;甲基赤藻糖醇磷酸途径(methylerythritol 4-phosphate pathway,MEP)中幼树叶表达高于老树叶。结论幼树叶比老树叶药用活性成分含量更高,适合作为药材。银杏叶内萜类合成途径可能主要依赖于MEP途径,且相关基因表达量与代谢成分相互证明。

Objective Ginkgo biloba leaves(G.nursery leaves,perennial G.biloba leaves)at different development stages and ages were used as objects to determine the content of flavonoids and terpene lactones in their secondary metabolites,to analyze the pathways of terpene biosynthesis and the expression of key genes,and to provide molecular biopharmaceutical data for improving the yield of ginkgo terpenolactone.Methods Based on liquid chromatography-mass spectrometry(LC-MS)metabolomic technology,and high-throughput transcriptomics sequencing(RNA-seq)to find the related enzymes of secondary metabolite biosynthesis from differentially expressed genes.Results It was found that the secondary metabolites of G.biloba leaves at different ages and developmental stages were significantly different in metabolomics.Firstly,the content of flavonoids,both in the tree leaves and nursery leaves,was presented by a decreasing trend from April to July.While at the same developmental stage,the content in nursery leaves was higher than that in tree leaves.Secondly,the content of terpene lactones in tree leaves was presented by a slightly increasing trend from April to July,however,a slightly decreasing trend in nursery leaves.While at the same development stage,the content in nursery leaves was higher than that in tree leaves.Then analyzed in the transcriptome,the growth status of old leaves was similar with the nursery leaves in germination stage,and there was a relatively large change in the gene expressions of old leaves and nursery leaves in May,June and July.After screening out 49 candidate genes related to terpenoid synthesis and analyzing them,it was found that the expression of tree leaves in mevalonate(MVA)pathway pathway was higher than that in nursery leaves;The later was higher than the former in methylerythritol 4-phosphate(MEP)pathway.Conclusion The nursery leaves have a higher content of medicinal active ingredients than tree leaves,which are suitable as medicinal materials.The terpenoid synthesis pathway in G.biloba leaves may mainly depend on the MEP pathway,and the expression levels of related genes and metabolic components are mutually proven.