干旱胁迫下金银花绿原酸与木犀草苷积累降低的转录调控机理

Transcriptional regulation mechanism of reduced accumulation of chlorogenic acid and luteoloside in Lonicera japonica under drought stress

摘要:

为探究干旱胁迫对金银花主要次生代谢产物绿原酸和木犀草苷合成的调控机制，本研究通过设置5个干旱梯度(土壤含水量30%、24%、17%、14%、10%)，运用转录组高通量测序(RNA sequencing, RNA-seq)技术结合逆转录荧光定量聚合酶链式反应(reverse transcription quantitative polymerase chain reaction, RT-qPCR)，对差异表达基因(differentially expressed genes, DEGs)进行筛选与验证，并借助高效液相色谱分析(high-performance liquid chromatography, HPLC)技术，系统测定绿原酸与木犀草苷的含量动态变化。结果表明，干旱显著降低了次生代谢产物积累，且干旱程度越高下降越明显，其中特大干旱(土壤含水量10%)条件下二者含量分别显著下降至25.73 mg/g和11.33 mg/g (降幅分别为37.85%和9.58%)。转录组分析共鉴定到77 454条基因，其中特大干旱处理下DEGs数量达1 128个。基因本体论(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)富集分析显示，DEGs主要参与类黄酮合成、次生代谢物生物合成、植物激素信号转导及植物-病原互作通路，且调控绿原酸和木犀草苷合成的关键基因的表达显著下调。RT-qPCR验证了RNA-seq数据的准确性。本研究揭示了干旱胁迫通过抑制次生代谢通路关键基因表达降低主要次生代谢产物绿原酸和木犀草苷的含量，为金银花抗旱分子育种提供了候选基因资源。

Abstract:

To explore the regulatory mechanism of drought stress on the synthesis of chlorogenic acid and luteoloside in Lonicera japonica, we designed five drought gradients (soil water contents of 30%, 24%, 17%, 14%, and 10%) and screened and verified the differentially expressed genes (DEGs) by RNA sequencing (RNA-seq) and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Furthermore, we employed HPLC to systematically measure the content changes of chlorogenic acid and luteoloside. The results revealed that drought significantly reduced the accumulation of secondary metabolites, and severe drought led to more obvious reductions. Under extreme drought (soil water content of 10%), the content of chlorogenic acid and luteoloside decreased significantly to 25.73 mg/g and 11.33 mg/g (with the decrease rates of 37.85% and 9.58%, respectively). A total of 77 454 genes were identified via transcriptome analysis, among which the number of DEGs reached 1 128 under the extraordinary drought. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses revealed that the DEGs were mainly involved in flavonoid synthesis, secondary metabolite biosynthesis, plant hormone signal transduction and the plant-pathogen interaction pathways, and the expression of key genes regulating the synthesis of chlorogenic acid and luteoloside was significantly downregulated. RT-qPCR verified the accuracy of the RNA-seq data. This study revealed that drought stress reduced the content of chlorogenic acid and luteoloside, the main secondary metabolites, by inhibiting the expression of key genes in the secondary metabolism pathways. The findings provide candidate gene resources for molecular breeding of drought-tolerant Lonicera japonica.