目的 旨在系统鉴定人参FAD基因（Panax ginseng FAD，PgFAD）家族并验证PgFAD2-56的功能，以期揭示PgFAD基因的功能和胁迫响应机制。方法 利用生物信息学手段从人参T2T基因组中鉴定PgFAD基因家族成员，分析其理化性质、系统进化、染色体定位、基因结构、保守基序、顺式作用元件及共线性特征，以及胁迫条件下的表达模式；通过分子对接预测高表达FAD2与油酸的结合能力；以酿酒酵母作为异源表达底盘验证PgFAD2-56的功能。结果 从人参T2T基因组中共鉴定到98个PgFAD基因，根据氨基酸序列相似性可分为6个亚家族（ADS、SLD、FAD3/FAD7/FAD8、FAD6、FAD2和FAB），这种分类得到基因结构和保守基序的支持。启动子区鉴定出大量激素和应激响应顺式作用元件及转录因子结合位点（TFBS），表明PgFADs的表达调控受到多种因素影响。共线性分析发现FAD2亚族在人参中具有显著扩张。基因表达分析发现部分PgFADs在胁迫响应中呈显著差异表达。分子对接结果显示FAD2-56与油酸的结合能最低（−6.5 kcal/mol），底物偏好性优于其他高表达FAD2基因。酵母异源表达实验证实PgFAD2-56可改变脂肪酸组分，使转基因酵母产生亚油酸，并增强其对寒冷胁迫的抗性。结论 PgFAD基因家族具有强烈的亚族特异性，在人参抗逆中发挥着重要作用，FAD2亚族的扩张及PgFAD2-56的功能验证为人参抗逆分子育种提供了潜在靶点。

Objective FAD enzymes introduce double bonds at specific positions in fatty acids, converting saturated fatty acids into unsaturated fatty acids, playing crucial roles in plant growth, development, and stress responses. This study aimed to systematically identify the FAD gene family in Panax ginseng and verify the function of PgFAD2-56, so as to elucidate the functions and stress response mechanisms of ginseng FAD genes. Methods Using bioinformatics approaches, 98 PgFAD genes were identified from the P. ginseng T2T genome, and their physicochemical properties, phylogenetic relationships, chromosomal localization, gene structures, conserved motifs, cis-acting regulatory elements, collinearity, and expression patterns under stress conditions were analyzed. Molecular docking was performed to predict the binding affinity of highly expressed FAD2 proteins with oleic acid, and the function of PgFAD2-56 was verified using a yeast heterologous expression system. Results The results showed that the 98 PgFAD genes could be classified into six subfamilies (ADS, SLD, FAD3/FAD7/FAD8, FAD6, FAD2, and FAB) based on amino acid sequence similarity, which was consistently supported by their gene structures and conserved motifs. Numerous hormone- and stress-responsive cis-acting elements and transcription factor binding sites (TFBSs) were identified in the promoters of PgFAD genes. Collinearity analysis revealed significant expansion of the FAD2 subfamily in P. ginseng. Gene expression analysis demonstrated that some PgFADs displayed significant differential expression under stress conditions. Molecular docking indicated that FAD2-56 exhibited the lowest binding energy with oleic acid (−6.5 kcal/mol) among the highly expressed FAD2 proteins. Heterologous expression in yeast confirmed that PgFAD2-56 altered fatty acid composition by producing linoleic acid and enhanced cold stress resistance. Conclusion This study demonstrates that the ginseng FAD gene family exhibits strong subfamily specificity and plays a vital role in stress resistance, with the expansion of the FAD2 subfamily and functional characterization of PgFAD2-56 providing potential targets for stress-resistant molecular breeding in P. ginseng.

R282.12

中国农业产业发展基金（1260233）