College of Pharmacy, Guangxi University of Chinese Medicine, Nanning 530200, China.
Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine, Guangxi University of Chinese Medicine, Nanning 530200, China.
Int J Mol Sci. 2024 Nov 6;25(22):11900. doi: 10.3390/ijms252211900.
(Champ. ex Benth.) Schot is a significant leguminous plant valued for its edible tuberous roots, which are a plentiful source of isoflavonoids. Basic helix-loop-helix (bHLH) transcription factors (TFs) have been reported to regulate secondary metabolism in plants, especially flavonoid biosynthesis. However, the genes in have not yet been reported, and their regulatory role in isoflavonoid biosynthesis remains unexplored. Here, 146 genes were identified in the genome, classifying them into 23 subfamilies based on the gene structures and phylogenetic relationships. All the CsbHLH proteins contained both motifs 1 and 2, whereas motif 8 was only distributed in subgroup III (d + e). Collinearity analysis demonstrated that fragmental replications are the primary driver of CsbHLH evolution, with the majority of duplicated gene pairs experiencing selective pressure. Nine candidate genes were found to play a potential role in regulating isoflavonoid biosynthesis through a combination of gene-to-metabolite correlation analysis and weighted gene co-expression network analysis (WGCNA). Additionally, the -regulatory elements and response to MeJA of these nine genes were characterized and confirmed through quantitative real-time PCR (qRT-PCR) analysis. Among them, three CsbHLHs (CsbHLH9, CsbHLH89, and CsbHLH95) were selected for further investigation. Yeast two-hybrid (Y2H), dual-luciferase (LUC) assays, bimolecular fluorescence complementation (BiFC) assays, and transient transformation demonstrated that CsbHLH9 acted as a transcriptional activator through its interaction with CsMYB36 and binding to the promoters of isoflavonoid biosynthesis genes in a MeJA-induced manner, such as , , and , to promote isoflavonoid (calycosin, calycosin-7-o-glucoside, and formononetin) accumulation. Our results establish a basis for the functional analysis of genes and investigations into the molecular mechanisms underlying isoflavonoid biosynthesis in .
（Champ. ex Benth.）Schot 是一种重要的豆科植物，其可食用的块根富含异黄酮，具有重要的经济价值。碱性螺旋-环-螺旋（bHLH）转录因子（TFs）已被报道在植物中调节次生代谢，特别是类黄酮的生物合成。然而，在 中尚未报道其相关基因，其在异黄酮生物合成中的调控作用仍有待探索。本研究在 基因组中鉴定出 146 个基因，根据基因结构和系统发育关系将其分为 23 个亚家族。所有的 CsbHLH 蛋白都包含基序 1 和 2，而基序 8 仅分布在亚组 III（d + e）中。共线性分析表明，片段复制是 CsbHLH 进化的主要驱动力，大多数重复的 基因对都经历了选择压力。通过基因-代谢物相关性分析和加权基因共表达网络分析（WGCNA）相结合，发现 9 个候选 基因可能在调节异黄酮生物合成中发挥作用。此外，通过定量实时 PCR（qRT-PCR）分析，对这 9 个基因的 -调控元件和对 MeJA 的响应进行了特征分析和验证。其中，选择了 3 个 CsbHLH（CsbHLH9、CsbHLH89 和 CsbHLH95）进行进一步研究。酵母双杂交（Y2H）、双荧光素酶（LUC）测定、双分子荧光互补（BiFC）测定和瞬时转化表明，CsbHLH9 通过与 CsMYB36 相互作用并在 MeJA 诱导下与异黄酮生物合成基因（如 、 和 ）的启动子结合，作为转录激活因子发挥作用，以促进异黄酮（毛蕊异黄酮、毛蕊异黄酮-7-O-葡萄糖苷和芒柄花素）的积累。本研究为 基因的功能分析和异黄酮生物合成的分子机制研究奠定了基础。
