【摘要】 【目的】探究紫花苜蓿磷转运体MsPT5的功能，为解析苜蓿响应低磷胁迫及培育磷营养高效紫花苜蓿提供理论依据和基因资源。【方法】以紫花苜蓿为材料分别进行足磷和低磷胁迫处理,选取根系进行转录组测序，利用加权基因相关性网络分析筛选苜蓿低磷胁迫响应关键基因MsPT5；克隆获得MsPT5的CDS序列，利用DNAMAN和TMHMM在线网站对MsPT5蛋白跨膜结构域进行分析；以Super1300:GFP植物表达载体为骨架，通过同源重组法构建过表达载体Super1300:MsPT5-GFP；采用冻融法将重组载体Super1300:MsPT5-GFP转化至农杆菌GV3101，将农杆菌注射到烟草中进行亚细胞定位观察；通过农杆菌介导的花序侵染法将Super1300:MsPT5-GFP转入拟南芥，筛选纯合株系进行无机磷含量测定、生物量统计和砷酸盐表型检测；利用组织培养的方法获得MsPT5转基因紫花苜蓿，筛选阳性植株进行生物量统计、磷含量和蛋白质含量测定。【结果】MsPT5为PHT1家族成员，具有12个跨膜结构域，定位于细胞质膜；拟南芥生理指标测定显示，MsPT5过量表达株系无机磷含量分别为23.02和22.30 nmol·mg^-1，野生型对照无机磷含量为19.97 nmol·mg^-1。MsPT5过量表达株系生物量分别为0.047和0.054g/plant，野生型对照生物量为0.026 g/plant。MsPT5过量表达能够提高拟南芥无机磷含量和生物量；砷酸盐表型检测结果显示，MsPT5过表达材料具有明显砷毒害表型；紫花苜蓿生理指标测定显示，MsPT5转基因紫花苜蓿生物量分别为19.39g/plant、18.62 g/plant、16.65 g/plant，野生型对照生物量为15.14 g/plant。MsPT5转基因紫花苜蓿磷含量分别为0.37%、0.39%、0.38%，野生型对照磷含量为0.30%。MsPT5转基因紫花苜蓿蛋白质含量分别为21.37%、21.54%、19.91%，野生型对照蛋白质含量为18.04%。MsPT5转基因紫花苜蓿生物量显著提高，同时磷含量和蛋白质含量显著高于对照。【结论】MsPT5是苜蓿应对低磷胁迫过程中的重要基因，过量表达MsPT5可提高植物磷吸收能力和磷含量，提高苜蓿产量和品质，MsPT5在培育高产优质苜蓿品种中具有重要应用价值。更多还原

【Abstract】 【Objective】 This study aimed to explore the function of MsPT5, a phosphate transporter in alfalfa, so as to provide the theoretical basis and genetic resources for analyzing the response of alfalfa to low phosphate stress and cultivating phosphate nutrition efficient alfalfa. 【Method】 Alfalfa was subjected to both full phosphate and low phosphate stress treatments, and root systems were selected for transcriptome sequencing. Weighted gene correlation network analysis was used to screen for the key gene Ms PT5 in response to low phosphate stress in alfalfa; the experiment was carried out to clone the CDS sequence of MsPT5 gene, and the transmembrane domain of MsPT5 protein was analyzed using DNAMAN and TMHMM online websites; Super1300: GFP plant expression vector was used as the backbone, and the overexpression vector Super1300:Ms PT5-GFP was constructed through enzyme digestion and homologous recombination; the recombinant vector Super1300:Ms PT5-GFP was transformed into Agrobacterium GV3101 using the freeze-thaw method, and Agrobacterium was injected into tobacco for subcellular localization observation; Super1300:Ms PT5-GFP was transferred into Arabidopsis through Agrobacterium mediated inflorescence infection, and homozygous strains were screened for inorganic phosphate content determination, biomass statistics, and arsenate phenotype detection; the tissue culture method was employed to obtain Ms PT5 transgenic alfalfa, and the positive plants were screened for biomass statistics, phosphate content, and protein content determination. 【Result】 Ms PT5 was a member of the PHT1 family, with 12 transmembrane domains located on the cytoplasmic membrane; the physiological indicators of Arabidopsis thaliana showed that the inorganic phosphate content of Ms PT5 overexpressing strains was 23.02 and 22.30 nmol·mg^-1, respectively, while the inorganic phosphate content of the wild-type control was 19.97 nmol·mg^-1. The biomass of Ms PT5 overexpressing strains were 0.047 g/plant and 0.054 g/plant, respectively, while the biomass of the wild-type control was 0.026 g/plant. Overexpression of Ms PT5 could increase the inorganic phosphate content and biomass of Arabidopsis; the results of arsenate phenotype detection showed that the Ms PT5 overexpression material exhibited a significant arsenic toxicity phenotype; the physiological indicators of alfalfa showed that the biomass of Ms PT5 transgenic alfalfa was 19.39 g/plant, 18.62 g/plant, and 16.65 g/plant, respectively, while the biomass of the wild-type control was 15.14 g/plant. The phosphate content of Ms PT5 transgenic alfalfa was 0.37%, 0.39%, and 0.38%, respectively, while the wild-type control had a phosphate content of 0.30%. The protein content of Ms PT5 transgenic alfalfa was 21.37%, 21.54%, and 19.91%, respectively, while the protein content of the wild-type control was 18.04%. The biomass of Ms PT5 transgenic alfalfa was significantly increased, while the phosphate and protein content were significantly higher than the control. 【Conclusion】 Ms PT5 was an important gene in alfalfa’s response to low phosphate stress. Overexpression of MsPT5 could increase plant phosphate absorption capacity and phosphate content, and improve alfalfa yield and quality. Therefore, Ms PT5 was important for cultivating high-yield and high-quality alfalfa.更多还原