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LI Zhiwei, LI Benjie, LIU Zhibing, et al. Construction of AtLHT1 probe for its transport substrate screening based on FRET principle[J]. Journal of South China Agricultural University, 2022, 43(1): 77-84. DOI: 10.7671/j.issn.1001-411X.202102006
Citation: LI Zhiwei, LI Benjie, LIU Zhibing, et al. Construction of AtLHT1 probe for its transport substrate screening based on FRET principle[J]. Journal of South China Agricultural University, 2022, 43(1): 77-84. DOI: 10.7671/j.issn.1001-411X.202102006

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

More Information
  • Received Date: February 03, 2021
  • Available Online: May 17, 2023
  • 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.

  • [1]
    徐汉虹. 农药研究的新理念: 导向农药[C]//中国化工学会新农药创制交流会. 沈阳: 中国化工学会, 2003.
    [2]
    DELÉTAGE-GRANDON C, CHOLLET J F, FAUCHER M, et al. Carrier-mediated uptake and phloem systemy of a 350-dalton chlorinated xenobiotic with an alpha-amino acid function[J]. Plant Physiology, 2001, 125(4): 1620-1632. doi: 10.1104/pp.125.4.1620
    [3]
    YOU G, MORRIS M E. Drug transporters: Molecular characterization and role in drug disposition[M]. Canada: Wiley & Sons, 2007.
    [4]
    DEGORTER M K, XIA C Q, YANG J J, et al. Drug transporters in drug efficacy and toxicity[J]. Annual Review of Pharmacology and Toxicology, 2012, 52: 249-273. doi: 10.1146/annurev-pharmtox-010611-134529
    [5]
    WU H, XU H, MARIVINGT-MOUNIR C, et al. Vectorizing agrochemicals: Enhancing bioavailability via carrier-mediated transport[J]. Pest Management Science, 2019, 75(6): 1507-1516. doi: 10.1002/ps.5298
    [6]
    DE MOLINER F, KNOX K, REINDERS A, et al. Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides[J]. Journal of Experimental Botany, 2018, 69(10): 2473-2482. doi: 10.1093/jxb/ery075
    [7]
    JIANG X Y, XIE Y, REN Z F, et al. Design of a new glutamine-fipronil conjugate with alpha-amino acid function and its uptake by A. thaliana lysine histidine transporter 1 (AtLHT1)[J]. Journal of Agricultural and Food Chemistry, 2018, 66(29): 7597-7605.
    [8]
    MAO G L, YAN Y, CHEN Y, et al. Family of Ricinus communis monosaccharide transporters and RcSTP1 in promoting the uptake of a glucose-fipronil conjugate[J]. Journal of Agricultural and Food Chemistry, 2017, 65(30): 6169-6178.
    [9]
    CHEN L, BUSH D R. LHT1, a lysine- and histidine-specific amino acid transporter in Arabidopsis[J]. Plant Physiology, 1997, 115(3): 1127-1134. doi: 10.1104/pp.115.3.1127
    [10]
    SVENNERSTAM H, JÄMTGÅRD S, AHMAD I, et al. Transporters in Arabidopsis roots mediating uptake of amino acids at naturally occurring concentrations[J]. New Phytologist, 2011, 191(2): 459-467. doi: 10.1111/j.1469-8137.2011.03699.x
    [11]
    CHEN Y, YAN Y, REN Z F, et al. AtLHT1 transporter can facilitate the uptake and translocation of a glycinergic-chlorantraniliprole conjugate in Arabidopsis thaliana[J]. Journal of Agricultural and Food Chemistry, 2018, 66(47): 12527-12535.
    [12]
    XIE Y, ZHAO J L, WANG C W, et al. Glycinergic-fipronil uptake is mediated by an amino acid carrier system and induces the expression of amino acid transporter genes in Ricinus communis seedlings[J]. Journal of Agricultural and Food Chemistry, 2016, 64(19): 3810-3818.
    [13]
    张志毅, 周涛, 巩伟丽, 等. 荧光共振能量转移技术在生命科学中的应用及研究进展[J]. 电子显微学报, 2007, 26(6): 620-624. doi: 10.3969/j.issn.1000-6281.2007.06.014
    [14]
    FÖRSTER T. Energiewanderung und fluoreszenz[J]. Naturwissenschaften, 1946, 33(6): 166-175. doi: 10.1007/BF00585226
    [15]
    王盛, 陈典华, 蒋驰洲, 等. 基于荧光蛋白的荧光共振能量转移探针的构建及应用[J]. 中国细胞生物学学报, 2012, 34(12): 1258-1267.
    [16]
    孟坤, 何庆瑜, 王通, 等. 基于C6流式细胞仪平台应用FRET技术在活细胞中研究蛋白质相互作用[J]. 中国生物工程杂志, 2017, 37(5): 45-51.
    [17]
    FEHR M, FROMMER W B, LALONDE S, et al. Visualization of maltose uptake in living yeast cells by fluorescent nanosensors[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(15): 9846-9851. doi: 10.1073/pnas.142089199
    [18]
    LAGER I, LOOGER L L, HILPERT M, et al. Conversion of a putative Agrobacterium sugar-binding protein into a FRET sensor with high selectivity for sucrose[J]. Journal of Biological Chemistry, 2006, 281(41): 30875-30883. doi: 10.1074/jbc.M605257200
    [19]
    SVENNERSTAM H, GANETEG U, BELLINI C, et al. Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids[J]. Plant Physiology, 2007, 143(4): 1853-1860. doi: 10.1104/pp.106.092205
    [20]
    HIRNER A, LADWIG F, STRANSKY H, et al. Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll[J]. Plant Cell, 2006, 18(8): 1931-1946. doi: 10.1105/tpc.106.041012
    [21]
    VANOAICA L, BEHERA A, CAMARGO S M, et al. Real-time functional characterization of cationic amino acid transporters using a new FRET sensor[J]. Pflugers Archiv-European Journal of Physiology, 2016, 468(4): 563-572. doi: 10.1007/s00424-015-1754-9
    [22]
    张建伟, 陈同生. 荧光共振能量转移(FRET)的定量检测及其应用[J]. 华南师范大学学报(自然科学版), 2012, 44(3): 12-17.
    [23]
    KAPER T, LOOGER L L, TAKANAGA H, et al. Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle[J]. PLoS Biology, 2007, 5(10): e257. doi: 10.1371/journal.pbio.0050257.
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