基于FRET原理构建AtLHT1转运底物筛选探针

Construction of AtLHT1 probe for its transport substrate screening based on FRET principle

【目的】为提高导向药物筛选通量,加快导向农药分子设计、合成、筛选等研发速度,构建基于拟南芥氨基酸转运蛋白AtLHT1的荧光共振能量转移(Fluorescence resonance energy transfer,FRET)探针作为导向农药筛选平台。【方法】构建CFP-LHT1-YFP的三明治探针,分别在大肠埃希菌Escherichia coli BL21(DE3)原核细胞和BY4741酵母细胞中进行表达、鉴定和纯化。采用荧光酶标仪和激光共聚焦检测FRET效率的变化。【结果】CFPAtLHT1-YFP融合蛋白探针纯化后,分别与供试氨基酸和草甘膦混合,供试氨基酸和草甘膦的加入导致探针蛋白的FRET比率发生明显变化,草甘膦处理使D535 nm/D480 nm升高7%~12%,其变化趋势与阳性配体甘氨酸和谷氨酸接近,而加入阴性配体精氨酸则没有明显变化。同时,FRET比率呈现底物浓度依赖。样品中分别加入1、5和30 mmol/L的草甘膦溶液,20 min后检测到D535 nm/D480 nm分别升高3%、1%和13%,阳性配体也存在FRET比率随着底物浓度上升而升高的趋势,而阴性配体变化不规则。利用光漂白法检测到氨基酸或草甘膦处理后单个酵母细胞的FRET效率发生明显变化。加入阳性对照的甘氨酸和谷氨酸后,FRET效率变化明显,其中,甘氨酸和谷氨酸的FRET效率分别达到30%和26%;加入草甘膦处理的FRET效率与谷氨酸接近,达26%;含PBS的空白对照组没有明显的FRET效率变化。【结论】AtLHT1-FRET探针可以与中性氨基酸甘氨酸和酸性氨基酸谷氨酸结合,但不结合碱性氨基酸精氨酸;FRET效率因不同底物有所差异,且具有一定的底物浓度依赖性;同时,证明了草甘膦能够被氨基酸转运蛋白AtLHT1转运。

【Objective】In order to improve the efficiency of guided-pesticides screening and accelerate the research and development of molecular design,synthesis and screening of guided-pesticides,a fluorescence resonance energy transfer(FRET)probe based on Arabidopsis thaliana amino acid transporter AtLHT1 was constructed as guided-pesticide screening platform in this study.【Method】The sandwich molecular probe of CFP-LHT1-YFP was constructed and expressed in prokaryotic cell of Escherichia coli BL21(DE3)and BY4741 yeast cell,respectively,then identified and purified.The change of FRET efficiency was detected with fluorescence microplate reader and laser scanning confocal microscope.【Result】The CFP-AtLHT1-YFP fusion protein probe was purified and mixed with the tested amino acids and glyphosate,respectively.The addition of the tested amino acids and glyphosate resulted in significant changes in the FRET ratio of the probe protein.Glyphosate treatment resulted in the D535 nm/D480 nm increase of 7%to 12%.The similar tendency was also observed in the treatments of positive ligands using glycine and glutamic acid,but no obvious change was observed in the treatment of negative ligand using arginine.The FRET ratio showed substrate concentration dependence.At 20 min after the addition of 1,5 and 30 mmol/L glycine,the increase of D535 nm/D480 nm were 3%,1%and 13%respectively.FRET ratio increased with the increase of subatrate concentration in treatments with positive ligand,while the changes were not regular in treatments with negative ligand.Significant changes in FRET efficiency were also detected in individual yeast cells after treatment with amino acids or glyphosate using the photobleaching method.Changes in FRET efficiency were more pronounced with the addition of positive controls of glycine and glutamic acid,with 30%FRET efficiency for glycine and 26%for glutamic acid.The addition of glyphosate resulted in 26%FRER efficiency which was close to that of glutamic acid.The blank control containing PBS buffer showed no significant change in FRET efficiency.【Conclusion】The AtLHT1-FRET probe can bind to the neutral amino acid glycine and the acidic amino acid glutamic acid,but not to the basic amino acid arginine.The FRET efficiency varies among different substrates with some concentration dependence.It is demonstrated that glyphosate can be transported by the amino acid transporter protein AtLHT1.