Ghanbari Moheb Seraj Rahele, Ahmadikhah Asadollah, Esmaeilzadeh-Salestani Keyvan, Shariati Vahid, Behnamian Mahdi, Tariverdizadeh Neda, Emadi Ali, Dezhsetan Sara
Department of Horticultural Sciences, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran.
Department of Cell & Molecular Biology, Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran.
BMC Plant Biol. 2025 Aug 28;25(1):1150. doi: 10.1186/s12870-025-07272-5.
To evaluate the milk thistle transcriptome under drought stress in field conditions, irrigation was applied using a weighted method at three levels: 100% F.C every 2 days, 70% F.C every 4 days, and 40% F.C every 8 days. Sampling was performed after 8 days at the flowering stage. Plant leaves were collected for RNA-seq analysis, seeds for oily and methanolic extracts, and downstream analyses were performed. Since there was no annotated reference genome for this plant, the De novo Assembly method was implemented to assemble the transcriptome. Contigs were blasted against five databases: NT, NR, Uniprot, and protein databases of Arabidopsis thaliana and Helianthus annuus. A total of 9,517 genes (~ 73% of Uniprot genes) were common across all databases and selected for further analysis due to their comprehensive annotation. Then, DEGs were identified and functionally annotated using Gene Ontology (GO) analysis with the ShinyGO platform, biological pathway analysis through KEGG, and transcription factor identification via PlantTFDB. Next, silybinin content was measured using HPLC. Generally, the most repeated pathways in all treatments include the Biosynthesis of secondary metabolites and the MAPK signaling pathway. Also, most biological processes are related to the oxidation-reduction process, and response to stress, and most molecular functions are protein and mRNA binding. Our results indicate the active role of transcription factors ERF, C3H, and bHLH in drought stress tolerance. Silybin a and b showed that severe drought stress enhanced the accumulation of silybinin compared with seeds from the control. Eight differentially expressed genes (CYP86A1, CYP710A1, FATA2, LACS3, LOX2, PAL, PLA2-ALPHA, and PXG3) were used to validate the RNA-Seq data. qRT-PCR results confirmed strong consistency with the RNA-Seq findings. Finally, the genes involved in the silymarin pathway were identified, and their expression was determined through RNA-Seq data and compared with the silymarin contents.
为了评估田间干旱胁迫下水飞蓟的转录组，采用加权法在三个水平上进行灌溉：每2天100%田间持水量、每4天70%田间持水量、每8天40%田间持水量。在开花期8天后进行采样。采集植物叶片用于RNA测序分析，采集种子用于油性和甲醇提取物，并进行下游分析。由于该植物没有注释的参考基因组，因此采用从头组装方法来组装转录组。将重叠群与五个数据库进行比对：NT、NR、Uniprot以及拟南芥和向日葵的蛋白质数据库。共有9517个基因（约占Uniprot基因的73%）在所有数据库中都有，因其注释全面而被选择进行进一步分析。然后，使用ShinyGO平台通过基因本体（GO）分析鉴定差异表达基因（DEG）并进行功能注释，通过KEGG进行生物途径分析，通过PlantTFDB鉴定转录因子。接下来，使用高效液相色谱法测量水飞蓟宾含量。一般来说，所有处理中最常出现的途径包括次生代谢物的生物合成和丝裂原活化蛋白激酶（MAPK）信号通路。此外，大多数生物过程与氧化还原过程和对胁迫的反应有关，大多数分子功能是蛋白质和mRNA结合。我们的结果表明转录因子ERF、C3H和bHLH在干旱胁迫耐受性中发挥积极作用。水飞蓟宾a和b表明，与对照种子相比，严重干旱胁迫增强了水飞蓟宾的积累。使用八个差异表达基因（CYP86A1、CYP710A1、FATA2、LACS3、LOX2、PAL、PLA2 - ALPHA和PXG3）来验证RNA测序数据。定量逆转录聚合酶链反应（qRT - PCR）结果证实与RNA测序结果高度一致。最后，鉴定了参与水飞蓟素途径的基因，并通过RNA测序数据确定其表达，并与水飞蓟素含量进行比较。
