转录组测序分析膜荚黄芪紫茎花青素生物合成的关键基因

Analysis of the Key Genes on the Anthocyanin Biosynthesis in the Purple Stems of Astragalus membranaceus based on Transcriptome Sequencing

目的:筛选与膜荚黄芪紫茎花青素生物合成相关的关键基因。方法:以膜荚黄芪紫茎和绿茎为材料,利用超高液相色谱法(UPLC)、转录组测序(RNA-Seq)和实时荧光定量PCR(qRT-PCR)等方法,分析了紫茎和绿茎中花青素含量,花青素生物合成相关基因的表达水平。结果:紫茎中飞燕草素和矢车菊素含量分别为118.23μg/g、80.62μg/g,而绿茎中不含这两种花青素。转录组测序共获得128932个Unigene,有72470个Unigene在公共数据库获得注释。紫茎和绿茎共有6466个基因差异表达,在花青素生物合成途径有33个差异表达的结构基因,其中9个在紫茎中上调表达,另外还筛选出在紫茎中高表达的2个MYB和1个bHLH转录因子。对12个与花青素生物合成相关的基因经转录组测序结果验证和田间材料2次qRT-PCR分析,从基因表达模式推测F3′5′H、DFR、ANS、bHLH基因可能参与膜荚黄芪紫茎花青素生物合成途径。结论:通过膜荚黄芪紫茎和绿茎花青素含量和转录组测序结果的比较,所筛选出的花青素代谢途径中3个结构基因(F3′5′H、DFR、ANS)与1个bHLH转录因子在调控膜荚黄芪紫茎花青素生物合成过程中具有重要作用。

Objective:To explore the key genes related to the anthocyanin biosynthesis in purple stems of Astragalus membranaceus.Methods:The purple stems and green stems of Astragalus membranaceus were used as materials,the content of anthocyanin and the expression levels of anthocyanin biosynthesis related genes in purple stems and green stems were analyzed by ultra-high liquid chromatography(UPLC),transcriptomic sequencing(RNA-Seq)and real-time fluorescence quantitative PCR(qRT-PCR).Results:The contents of delphindin and cyanidin in purple stems were 118.23 and 80.62μg/g,respectively,but the green stems did not contain these two anthocyanins.A total of 128932 Unigenes were obtained by transcriptome sequencing and 72470 Unigenes were annotated in the public database.There were 6466 differentially expressed genes in purple stems and green stems,33 differentially expressed structural genes in anthocyanin biosynthesis pathway,nine of which were up-regulated in purple stems,and two MYB and one bHLH transcription factors that were highly expressed in purple stems were also screened out.Twelve genes related to anthocyanin biosynthesis were verified by transcriptome sequencing results and qRT-PCR analysis of field materials for two times,it was speculated that F3′5′H,DFR,ANS and bHLH genes might be involved in anthocyanin biosynthesis pathway of Astragalus membranaceus purple stems from the gene expression pattern.Conclusion:The comparisons of anthocyanin content in purple stems and green stems of Astragalus membranaceus and transcriptome sequencing results show that three structural genes(F3′5′H,DFR,ANS)and one bHLH transcription factor in the metabolic pathway of anthocyanin play an important role in the anthocyanin biosynthesis in purple stems of Astragalus membranaceus.