N⁶-甲基腺嘌呤（m⁶A） 是真核生物 mRNA 内部最丰富的一种表观化学修饰，能够被甲基化酶、去甲基化酶及结合蛋白所动态的添加、去除及特异性识别。其中，m⁶A结合蛋白通过特异性识别并结合m⁶A精密调控mRNA前体剪接、出核、稳定性、翻译效率等RNA代谢过程，在转录后水平调控基因表达，影响生物体内诸多生理过程。YTH（含 YT521B homology 保守结构域）是植物中发现的主要m⁶A 结合蛋白，在拟南、水稻、番茄中发挥重要生物学功能，但在药用植物中YTH蛋白的功能及其调控机制尚不清楚。本研究以药用模式植物丹参为研究材料，开展YTH家族基因的鉴定及代表性YTH基因功能的研究。主要研究结果如下：

（1）基于已发表的丹参高质量基因组，以拟南芥、水稻YTH蛋白序列为参照，通过同源序列比对，共鉴定出19个丹参YTH基因（SmYTH）。它们分布在5条染色体上，其中SmYTH1、SmYTH2和SmYTH3分别位于1、5和7号染色体，SmYTH4与SmYTH7分布于8号染色体，SmYTH5和SmYTH6分布于3号染色体，而SmYTH8-SmYTH19以串联基因簇的形式分布于8号染色体。

（2）进化分析显示，SmYTH3属于YTHDF-1家族，SmYTH1属于YTHDF-2家族，其余成员则属于YTHDF-3家族。利用MEGA11软件，对拟南芥、大豆、水稻、玉米以及丹参的76个YTH蛋白序列使用邻位连接法构建了系统发育树。发现除SmYTH3和SmYTH1外，其他YTH都属于YTHDF-3亚家族。丹参YTH家族中不存在YTHDC亚家族成员。

（3）SmYTH1-SmYTH4基因可能在丹参的生长发育以及次生代谢中发挥重要作用。应用生物信息学软件，对SmYTH基因结构、蛋白保守结构域及二、三级结构、相分离等进行预测和分析，发现SmYTH1-SmYTH4的基因结构相较于其他成员更复杂，推断的蛋白序列含有与m⁶A结合的、由三个色氨酸残基（WWW）构成的芳香笼结构，且N端存在能够促进相分离发生的无序化PrLDs结构域。SmYTH5-SmYTH19的基因结构相对简单，推断的蛋白没有促进相分离发生的PrLDs结构域，却含有一个F-box蛋白相互作用结构域（F_box_assoc_1）。

（4）SmYTH1-SmYTH4基因在根和叶中高度表达，其中SmYTH3在丹参药用部位表达最强烈。这些基因都对外源的激素茉莉酸甲酯（MeJA）和水杨酸（SA）响应显著，。SmYTH家族基因的组织特异性表达模式分析显示，SmYTH1-SmYTH4基因在根和叶中高度表达，而SmYTH8-SmYTH19主要在叶中表达,这与RNA-seq结果一致，暗示了SmYTH蛋白成员功能上的差异。另外，SmYTH基因响应植物生长调节剂MeJA和SA，并影响了活性成分丹酚酸B和迷迭香酸的含量，这表明SmYTH家族可能参与调控了丹参次生代谢过程。

（5）SmYTH3能够调控丹酚酸的两个关键酶基因表达，影响丹酚酸的含量。我们以SmYTH3为研究目标进一步探索其在丹参中的生物功能及调控机制。通过构建SmYTH3过表达和敲除载体，诱导转基因毛状根，获得了3个过表达毛状根株系（OE-SmYTH3-line2,8,10）以及一个纯合突变体株系（smyth3-line10）。表型分析显示，相较于野生型毛状根，突变体侧枝分叉增多，悬浮培养时生长较快，而过表达株系侧枝减少，悬浮培养时生长较慢。高效液相色谱HPLC结果显示，突变体中丹酚酸B和迷迭香酸含量显著升高，而过表达株系中丹酚酸B和迷迭香酸含量显著下降。通过进一步qPCR实验发现丹酚酸合成途径两个关键酶基因SmRAS1和SmCYP98A14在突变体中表达量上升而过表达株系中表达量下降，由此推断，SmYTH3可能以依赖m⁶A的方式抑制了SmRAS1和SmCYP98A14基因的表达。

综上所述，本研究在药用植物丹参中鉴定了m⁶A结合蛋白 YTH家族，研究了其进化关系和表达模式，同时对SmYTH中的代表蛋白SmYTH3进行了初步研究，发现SmYTH3蛋白能够调控丹参次生代谢，从转录后层面丰富了丹参次生代谢调控网络。

N⁶-methyladenosine (m⁶A), the most abundant internal epigenetic chemical modification in eukaryotic mRNA, is dynamically added, removed, and specifically recognized by methyltransferases, demethylases, and binding proteins. Among these, m⁶A-binding proteins precisely regulate RNA metabolism processes—including pre-mRNA splicing, nuclear export, stability, and translation efficiency—through the specific recognition and binding of m⁶A. This constitutes a crucial mechanism for post-transcriptional gene expression regulation, influencing numerous physiological processes within organisms. Currently, the YTH family (containing the conserved YT521B homology domain) has been identified as the primary m⁶A-binding proteins in plants, playing significant biological roles in Arabidopsis thaliana, Oryza sativa, and Solanum lycopersicum. However, the characterization and regulatory mechanisms of YTH family proteins in medicinal plants remain largely unexplored. Therefore, this study utilized the medicinal model plant Salvia miltiorrhiza to identify its YTH family proteins and investigate the functional mechanisms of representative YTH members. The main findings are summarized as follows:

(1) Based on the existing high-quality genome of Salvia miltiorrhiza, nineteen YTH family genes were identified through homologous sequence alignment using the reference sequences of the YTH protein from Arabidopsis thaliana and Oryza sativa. These genes are distributed across 5 chromosomes: SmYTH1, SmYTH2, and SmYTH3 are located on chromosomes 1, 5, and 7, respectively; SmYTH4 and SmYTH7 co-localize on chromosome 8; SmYTH5 and SmYTH6 reside on chromosome 3; while SmYTH8 to SmYTH19 form a tandem gene cluster on chromosome 8.

(2) Phylogenetic analysis revealed that SmYTH3 is classified into the YTHDF-1 family, SmYTH1 belongs to the YTHDF-2 family, while the remaining members fall within the YTHDF-3 family. A phylogenetic tree was constructed using the neighbor-joining method in MEGA11 software, incorporating 76 YTH protein sequences from Arabidopsis thaliana, Glycine max, Oryza sativa, Zea mays, and Salvia miltiorrhiza. This analysis confirmed that all SmYTH genes except SmYTH3 and SmYTH1 cluster within the YTHDF-3 subfamily. Furthermore, no members of the YTHDC subfamily were identified within the SmYTH family.

(3) SmYTH1-SmYTH4 genes are likely involved in regulating growth, development, and secondary metabolism in Salvia miltiorrhiza. Bioinformatic analyses were performed to characterize the gene structure, conserved protein domains, secondary and tertiary structures, and phase separation potential of the SmYTH gene family. SmYTH1-SmYTH4 genes exhibited more complex gene structures compared to other family members. These genes were predicted to contain an aromatic cage structure composed of three tryptophan residues (WWW) for m⁶A binding. Additionally, predictive analysis suggested the presence of intrinsically disordered Prion-Like Domains (PrLDs) at their N-termini, which are known to promote liquid-liquid phase separation (LLPS). In contrast, SmYTH5-SmYTH19 genes possessed relatively simple gene structures. Predictive analysis indicated that these genes lack PrLDs capable of promoting LLPS but contain an F-box protein interaction domain (F_box_assoc_1).

(4) SmYTH1-SmYTH4 genes are highly expressed in roots and leaves of Salvia miltiorrhiza. Analysis of the tissue-specific expression patterns of SmYTH family genes revealed that SmYTH1-SmYTH4 genes are highly expressed in roots and leaves, while SmYTH8-SmYTH19 are predominantly expressed in leaves. This expression pattern is consistent with RNA-seq data and suggests functional divergence among SmYTH protein members. Furthermore, SmYTH genes respond to the phytohormones methyl jasmonate (MeJA) and salicylic acid (SA) and modulate the accumulation of the bioactive compounds salvianolic acid B and rosmarinic acid. This indicates that the SmYTH family likely participates in regulating the secondary metabolic processes in Salvia miltiorrhiza.

(5) SmYTH3 was found to modulate salvianolic acid accumulation in Salvia miltiorrhiza by regulating the expression of genes encoding two key biosynthetic enzymes. Building upon the initial characterization, SmYTH3 was selected as a primary target to investigate its biological function and regulatory mechanism in Salvia miltiorrhiza. Overexpression and knockout vectors for SmYTH3 were constructed and used to generate transgenic hairy roots. This resulted in the establishment of three overexpression hairy root lines (OE-SmYTH3-line2, OE-SmYTH3-line8, OE-SmYTH3-line10) and one homozygous mutant line (smyth3-line10). Phenotypic analysis revealed that, compared to wild-type (WT) hairy roots, the smyth3 mutant exhibited increased lateral branching and accelerated growth during suspension culture. Conversely, the overexpression lines showed reduced lateral branching and slower growth under suspension culture conditions. High-Performance Liquid Chromatography (HPLC) analysis demonstrated a significant increase in the accumulation of salvianolic acid B and rosmarinic acid in the smyth3 mutant. In contrast, the levels of these bioactive compounds were significantly reduced in the overexpression lines. Furthermore, quantitative real-time PCR (qPCR) assays indicated that the expression levels of two key genes in the salvianolic acid biosynthetic pathway, SmRAS and SmCYP98A14, were upregulated in the mutant but downregulated in the overexpression lines.

In summary, this study identified the m⁶A-binding YTH domain protein family within the medicinal plant Salvia miltiorrhiza, characterized its evolutionary relationships and expression patterns, and conducted preliminary functional investigations on SmYTH3 as a representative member. The findings demonstrate that SmYTH3 regulates secondary metabolism in Salvia miltiorrhiza, thereby providing insights into the post-transcriptional regulation of its secondary metabolic network.