外源水杨酸影响苦荞类黄酮生物合成及相关基因表达模式

Effects of exogenous salicylic acid on flavonoid biosynthesis and expression patterns of related genes in tartary buckwheat

水杨酸(salicylic acid,SA)作为一个重要的信号分子,具有调节植物次生代谢产物的功能。前期研究表明外源SA可促进苦荞幼苗总黄酮积累并影响相关基因表达,然而对于其影响苦荞籽粒中类黄酮合成还鲜有报道。本研究对‘黑丰1号’苦荞籽粒于绿熟期外源喷施0.5 mmol·L^(-1)SA,发现总黄酮含量较对照提高1.21倍;比较转录组分析共鉴定出2317个差异表达基因,包括988个上调和1329个下调基因;KEGG富集分析揭示差异表达基因主要集中在类黄酮生物合成途径上,在该途径中鉴定到26个表达差异显著的关键酶基因,其中苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)、反式肉桂酸4-单加氧酶(trans-cinnamate4-monooxygenase,C4H)基因家族成员既有上调又有下调,而4-香豆酸酯-CoA连接酶(4-coumarate-CoA ligase,4CL)、查尔酮合酶(chalcone synthase,CHS)和黄烷酮3-羟化酶(flavanone 3-hydroxylase,F3H)上调1.32~4.33倍。qRT-PCR结果表明,其表达趋势与转录组分析结果一致。对类黄酮代谢通路的26个基因进行启动子分析发现有12个基因均含有SA响应元件,表明该途径关键酶基因具有参与响应SA信号的功能。基于加权共表达网络分析结合拟南芥同源基因功能注释共筛选到3个SA介导下调控类黄酮合成相关基因的转录因子FtWRKY7(FtPinG0002142900)、FtWRKY2(FtPinG0002177700)及FtWRKY65(FtPinG0002493300)。以上结果暗示SA可调控类黄酮合成通路相关基因的表达水平,从而提升苦荞籽粒总黄酮积累。本研究初步揭示了SA调控苦荞籽粒类黄酮合成的分子机制,同时为外源喷施SA实现苦荞籽粒黄酮类物质生物强化,提升其营养价值提供一定的理论参考。

Salicylic acid(SA)is an important signaling molecule that regulates plant secondary metabolites.Previous studies have shown that exogenous salicylic acid can promote total flavonoid accumulation and related gene expression in Tartary buckwheat seedlings.However,the effects of flavonoid metabolism in Tartary buckwheat grains are rarely reported.In this study,at the green ripening stage of Tartary buckwheat grains,the total flavonoid content of‘Heifeng 1’was increased by 1.21 times compared with the control when 0.5 mmol·L^(-1) SA was applied externally.A total of 2317 differentially expressed genes,including 988 up-regulated and 1329 down-regulated genes,were identified by comparative transcriptome analysis.KEGG enrichment analysis revealed that the differentially expressed genes were mainly concentrated in the flavonoid biosynthesis pathway,and 26 key enzyme genes with significant differences in expression were identified in this pathway,phenylalanine ammonia-lyase(PAL)and trans-cinnamate 4-monooxygenase(C4H),were both up-regulated and down-regulated,while 4-coumarate-CoA ligase(4CL),chalcone synthase(CHS)and flavanone 3-hydroxylase(F3H)were up-regulated by 1.32—4.33 times.qRTPCR showed that the expression trend was consistent with that of transcriptome analysis.The promoter analysis of 26 genes of flavonoid metabolism pathway found that 12 genes contained SA response elements,indicating that the key enzyme genes of this pathway were involved in the function of responding to SA signal.Based on weighted coexpression network analysis and homologous gene analysis,3 transcription factors were identified that regulate flavonoid synthesis mediated by exogenous salicylic acid Ft-WRKY7(FtPinG0002142900),FtWRKY2(FtPinG0002177700)and FtWRKY65(FtPinG0002493300).These results suggested that exogenous salicylic acid in-directly regulated the expression level of genes related to flavonoid metabolic pathway,thereby increasing the total flavonoid accumulation in Tartary buckwheat grains.This study preliminarily revealed the molecular mechanism of salicylic acid regulating flavonoid synthesis in Tartary buckwheat grains.It provided some theoretical reference for exogenous SA biospraying to enhance the flavonoid in Tartary buckwheat grains and improve its nutritional value.