目的 克隆山豆根Sophora tonkinensis StNAC46基因，并对其进行生物信息学、亚细胞定位及功能验证分析，探究StNAC46基因在山豆根中的作用。方法 以山豆根总RNA反转录得到的cDNA作为模板，通过PCR技术克隆StNAC46基因。利用生物信息学分析、融合报告基因定位、实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）、农杆菌介导的遗传转化以及高效液相色谱电喷雾串联质谱法（ESI-HPLC-MS/MS）等方法，分析StNAC46蛋白的结构特性、启动子序列、亚细胞定位以及转基因功能。结果 StNAC46基因全长1 083 bp，编码360个氨基酸，其蛋白质相对分子质量为39 800，等电点为8.67，属于亲水性蛋白，含有32个丝氨酸磷酸化位点。StNAC46包含1个NAM结构域，与毒羊豆、相思子以及密花豆等多种植物中的NAC（NAM、ATAF1/2、CUC1/2）蛋白显示出较高的同源性。StNAC46基因启动子包含核心元件（CAAT-box和TATA-box）以及响应植物激素—脱落酸（abscisic acid，ABA）、赤霉素（gibberellic acid，GA）和茉莉酸甲酯（methyl jasmonate，MeJA）的特定反应元件。亚细胞定位结果显示，StNAC46蛋白定位于细胞核与细胞质中。酵母单杂交验证发现，StNAC46与StCAO基因启动子区域存在相互作用。与野生型（wide type，WT）相比，StNAC46转基因烟草中苦参碱和氧化苦参碱含量增加。结论 山豆根StNAC46基因能够促进植物的生物碱合成，为山豆根的品种选育以及深入解析山豆根生物碱生物合成途径的分子机制奠定了重要基础。

Objective To clone the gene StNAC46 from Sophora tonkinensisfollowed by bioinformatics, subcellular localization and functional verification were performed, so as to explore the role of StNAC46 gene.Methods The StNAC46 gene was cloned by polymerase chain reaction (PCR) using the cDNA obtained by reverse transcription of total RNA from S. tonkinensis as a template. Subsequently, the structural characteristics, promoter sequence, subcellular localization, and transgenic function of StNAC46 protein were analyzed by bioinformatics analysis, fusion reporter gene localization, quantitative real-time PCR (qRT-PCR), transgenic technology, and High performance liquid chromatography electrospray tandem mass spectrometry (ESI-HPLC-MS/MS).Results The full length of StNAC46 gene was 1083 bp, encoding 360 amino acids. The molecular weight of StNAC46 protein was 3.98 × 10⁴, and the isoelectric point was 8.67. It was a hydrophilic protein with 32 serine phosphorylation sites. The StNAC46 protein contained a NAM domain, which showed high homology with NAC proteins from a diversity of plants such as Gastrolobium bilobum, Abrus precatorius, and Spatholobus suberectus. The StNAC46 gene promoter had core elements (CAAT-box and TATA-box), as well as specific response elements in response to phytohormones abscisic acid (ABA), gibberellenic acid (GA) and methyl Jasmonate (MeJA). Subcellular localization results demonstrated that StNAC46 protein was localized in the nucleus and cytoplasm. Yeast one-hybrid assay indicated that StNAC6 protein could interact with StCAO gene promoter. Moreover, matrine and oxymatrine were increased in transgenic tobacco compared to wild type (WT).Conclusion StNAC46 gene in S. tonkinensis could stimulate alkaloid synthesis in plants. It lays a foundation for the selection of varieties of S. tonkinensis and further analyzing the molecular mechanism of alkaloid biosynthesis pathway in S. tonkinensis.

R286.12

国家自然科学基金项目（82260747）；广西自然科学基金项目（2025GXNSFAA069971）；广西中药材品质创新研究团队（GZKJ2305）