目的 基于砷（As）胁迫下三七Panax notoginseng根的转录组数据，克隆液泡膜内在蛋白基因PnTIP1;3，通过生物信息学分析和酵母异源表达实验，解析其在As转运过程中的功能特性。方法 利用生物信息学工具分析基因PnTIP1;3的序列特征及其启动子区顺式作用元件；构建酵母异源表达体系，通过表型实验、ICP-MS测定细胞内As含量与亚细胞分布，并检测相关抗氧化指标，系统评估PnTIP1;3在细胞As转运中的功能。结果 克隆获得三七液泡膜内在蛋白基因PnTIP1;3，其开放阅读框为750 bp，编码250个氨基酸，系统进化分析表明其属于TIP1亚族。启动子分析显示其含有多个激素、生长发育及胁迫响应相关的顺式作用元件。As耐性实验表明，异源表达PnTIP1;3的酵母转化子（TPnTIP1;3）的EC₅₀为5.36 mmol/L，略高于空载对照（5.03 mmol/L）。As处理对2种菌株生长均产生抑制，但对空载菌株的抑制更为显著。ICP-MS分析显示，TPnTIP1;3菌株中总As积累量显著高于对照，亚细胞组分中As含量表现为细胞壁＞液泡＞细胞质。在As胁迫下，TPnTIP1;3中活性氧（reactive oxygen species，ROS）水平显著降低（P＜0.001），而超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）活性以及谷胱甘肽（glutathione，GSH）和金属硫蛋白（metallothionein，MT）含量均显著上升（P＜0.05）。相关性分析进一步表明，这些抗氧化指标与总As含量及细胞壁As浓度呈显著正相关。结论 三七液泡膜内在蛋白基因PnTIP1;3属于TIP1亚族，其异源表达可促进酵母对As的积累与液泡区隔化储存，并通过激活宿主抗氧化系统增强对As胁迫的耐受能力。

Objective Based on transcriptome data from roots of Panax notoginseng under arsenic (As) stress, the tonoplast intrinsic protein gene PnTIP1;3 was cloned. Its functional characteristics in As transport were investigated through bioinformatic analysis and heterologous expression in yeast. Methods Bioinformatic tools were used to analyze the sequence features and promoter cis-acting elements of PnTIP1;3. A yeast heterologous expression system was constructed, and the role of PnTIP1;3 in cellular As transport was systematically evaluated through phenotypic assays, intracellular As content and subcellular distribution measurement by ICP-MS, and analysis of relevant antioxidant indicators. Results The tonoplast intrinsic protein gene PnTIP1;3 was cloned, with an open reading frame of 750 bp encoding 250 amino acids. Phylogenetic analysis indicated that it belongs to the TIP1 subfamily. Promoter analysis revealed the presence of multiple cis-acting elements related to hormones, growth and development, and stress responses. As tolerance assays showed that the half-maximal effective concentration (EC₅₀) of yeast transformants heterologously expressing PnTIP1;3 (TPnTIP1;3) was 5.36 mmol/L, higher than that of the empty vector control (5.03 mmol/L). As treatment inhibited the growth of both strains, but the empty vector control was more severely affected. ICP-MS analysis indicated that total As accumulation was significantly higher in TPnTIP1;3 than in the control, and the subcellular distribution of As followed the pattern: cell wall > vacuole > cytoplasm. Under As stress, reactive oxygen species (ROS) levels were significantly decreased (P < 0.001) in TPnTIP1;3, while the activities of superoxide dismutase (SOD) and catalase (CAT), as well as the contents of glutathione (GSH) and metallothionein (MT), were significantly increased (P < 0.05). Correlation analysis further demonstrated significant positive relationships between these antioxidant indicators and both total As content and cell wall As concentration. Conclusion The P. notoginseng tonoplast intrinsic protein gene PnTIP1;3, a member of the TIP1 subfamily, enhances yeast tolerance to As stress by promoting As accumulation and vacuolar sequestration, and through activation of the host antioxidant system.

R282.12

云南省基础研究计划重点项目（202501AS070141）；国家自然科学基金项目（82260743）；国家自然科学基金项目（82360750）；中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”（2060302-2404-05）；云南省“万人计划”青年拔尖人才专项（YNWR-QNBJ-2020-279）；云南省“兴滇英才支持计划”青年人才项目（XDYC-QNRC-2023-0526）；云南省科技厅-云南省国际科技特派员（个人）项目（202403AK140080）；云南省中医药应用基础研究联合专项（202101AZ070001-014,202001AZ070001-009）；云南省教育厅科学研究基金研究生项目（2025Y0604）