A Genetically Encoded Fluorescent Probe For In Vitro And In Vivo Detection Of T6P

Trehalose-6-phosphate?T6P?is an intermediate product of trehalose metabolism and a signaling molecule that regulates plant sugar metabolism and physiological processes.It is critical to coordinate T6P production and degradation to ensure the adaptability of the organism towards environmental and nutritional changes.In recent years,with the deepening of research,it has been found that T6P,as a signal molecule,plays an important role in regulating plant embryo development,stomatal opening,leaf senescence and flowering time.However,the detection of T6P is a major difficulty.The concentration of cellular T6P is low,especially in plants,which is reported to be only 0.1-15?M.Moreover,the presence of the T6P isomer sucrose-6-phosphate?S6P?in vivo cause difficulty in baseline separating when using liquid chromatography-tandem mass spectrometry.In addition,HILIC-MS/MS method cannot measure intracellular free T6P selectively or monitor dynamic changes of cellular T6P.In order to better explore the dynamic changes and physiological effects of T6P in vivo,it is particularly important to explore a new method to quantify T6P.In recent years,a genetically encoded fluorescent probe based on fluorescent resonance energy transfer?FRET?have been widely used to detect various signal molecules and metabolites in a living cell,such as Ca²⁺,ROS,ATP,amino acids,H₂O₂,carbohydrates and hormone.The real-time quantitative monitoring of these small intracellular molecules is of great significance for people to study in vivo signaling.Inspired by this,herein,we designed a genetically encoded fluorescent probe based on FRET to monitor the dynamic changes of T6P in vivo.The genetically encoded fluorescent probe consists of a chimeric protein composed of an intermediate ligand-binding protein fused to two different wavelengths of fluorescent proteins.The binding of the target molecule causes a conformational change of the intermediate reporter protein,which changes the fluorescence.The distance between the groups,which produces the transfer of energy.In this study,trehalose-6-phosphate phosphatase?AtTPPG?,which had no activity after mutation but could specifically bind to T6P,was used as a specific recognition element of T6P.AtTPPG mutant was inserted between the donor green fluorescent protein?Clover?and the receptor red fluorescent protein?mRuby2?to construct a fluorescent probe for monitoring the dynamic changes of T6P.Using laser confocal microscopy imaging and in vitro fluorescent protein lifetime detection,we screened three fluorescent probes with AtTPPG site-directed mutagenesis for monitoring T6P and named as CR-Mu1,CR-Mu2,and CR-Mu3,respectively.When we applied these three probes in E.coli,they can specifically recognize T6P and monitor the changes of T6P at different concentrations.The fluorescence intensity was changed with the concentration of T6P,which was the donor fluorescence gradually decreased,and the receptor fluorescence gradually strengthened.A comprehensive comparison of several probes,we found that CR-Mu3 had the best effect.In this study,the performance of T6P probe was characterized and verified in vitro and in E.coli and yeast,indicating that the T6P probe designed in this experiment can monitor the change of T6P content,which can be used has important guiding significance in studying the metabolic process of T6P in vivo and the response mechanism.