Wu Fang, Zhang Jing, Yang Hongshan, Duan Huirong
Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
PLoS One. 2025 May 8;20(5):e0322981. doi: 10.1371/journal.pone.0322981. eCollection 2025.
Glucosinolate are one of the vital secondary metabolites in alfalfa (Medicago sativa L.), and primarily present as β-D-glucosinolate derivatives, improving the resistance in response to biotic and abiotic stresses of alfalfa. CYP79 (Cytochrome P450 monooxygenases) and CYP83 gene families play an important role in the core structure biosynthesis of glucosinolate. Nevertheless, a comprehensive exploration of CYP79 and CYP83 family members in alfalfa has thus far not been study. The types of glucosinolate in alfalfa were qualitative and quantitative analysis by UPLC-MS/MS. Then, we identified MsCYP79 and MsCYP83 gene families in alfalfa, and scrutinized the physicochemical attributes, gene architecture, collinearity, evolutionary trajectories, as well as expression patterns under mechanical damage. The findings revealed the glucosinolate metabolites of alfalfa divided into three classes, including 27 aliphatic glucosinolates, 9 aromatic glucosinolates, and 5 indole glucosinolates. In addition, 59 MsCYP79 family members and 56 MsCYP83 family members were identified in alfalfa, which were classified into eight main groups based on phylogenetic analysis. MsCYP79 and MsCYP83 were distributed unevenly on 26 chromosomes and had 2-6 exons. Then, employing MEME software unveiled 15 conserved motifs within the protein structures of MsCYP79 and MsCYP83. Real-time quantitative PCR was used to detect the expression level of MsCYP79 and MsCYP83 genes and demonstrated that the selected genes in alfalfa were tissue-specific and had different expression patterns in response to mechanical damage. This investigation laid a robust groundwork for substantiating the functions of MsCYP79 and MsCYP83 and facilitating the cultivation of alfalfa varieties enriched in glucosinolate content.
硫代葡萄糖苷是紫花苜蓿（Medicago sativa L.）中重要的次生代谢产物之一，主要以β-D-硫代葡萄糖苷衍生物的形式存在，可提高紫花苜蓿对生物和非生物胁迫的抗性。细胞色素P450单加氧酶（CYP79）和CYP83基因家族在硫代葡萄糖苷的核心结构生物合成中起重要作用。然而，迄今为止尚未对紫花苜蓿中的CYP79和CYP83家族成员进行全面探索。采用超高效液相色谱-串联质谱法（UPLC-MS/MS）对紫花苜蓿中硫代葡萄糖苷的种类进行了定性和定量分析。然后，我们在紫花苜蓿中鉴定了MsCYP79和MsCYP83基因家族，并详细研究了它们的理化性质、基因结构、共线性、进化轨迹以及机械损伤后的表达模式。研究结果表明，紫花苜蓿中的硫代葡萄糖苷代谢产物分为三类，包括27种脂肪族硫代葡萄糖苷、9种芳香族硫代葡萄糖苷和5种吲哚族硫代葡萄糖苷。此外，在紫花苜蓿中鉴定出59个MsCYP79家族成员和56个MsCYP83家族成员，根据系统发育分析将它们分为八个主要组。MsCYP79和MsCYP83不均匀地分布在26条染色体上，有2至6个外显子。然后，使用MEME软件揭示了MsCYP79和MsCYP83蛋白质结构中的15个保守基序。采用实时定量PCR检测MsCYP79和MsCYP83基因的表达水平，结果表明紫花苜蓿中所选基因具有组织特异性，并且在机械损伤后具有不同的表达模式。本研究为阐明MsCYP79和MsCYP83的功能以及培育硫代葡萄糖苷含量丰富的紫花苜蓿品种奠定了坚实的基础。
