Study On The Involvement Of LHT1 In Transporting Glyphosate Based On FRET Technology

Guided pesticide is a new type of pesticide which is coupled with the guide carrier to make the pesticide have good transport in the plant and can be accumulated in a specific site.A large amount of work has been done in the early stage of the laboratory,including synthesized a series of guided pesticide conjugating glycosyl or amino acid,the evaluation of insecticidal activity,the evaluation of the inbirobiability of the transport and the mechanism of the related transport.Glyphosate is an early internal sucking and killing herbicide,which has good conductivity in plants.The molecular structure of glyphosate contains a phosphate group and has shown that the phosphate transporter is involved in the transport of glyphosate;the structure of glyphosate molecular structure is similar to that of glycine.Glyphosate is transported to the vacuole by the amino acid transporter of the ABC family in the glyphosate resistant weeds.Glyphosate can also be absorpted across epithelium by the LAT1 transporter.The amino acid transporter LHT1 of Arabidopsis has been proved to be the key transporter in the root absorption of neutral and acidic amino acids,and its loss affects the absorption and distribution of amino acids in plants.LHT1 can also transport Glycinergic-Chlorantraniliprole conjugate(CAP-Gly-2)with amino acid structure.This paper takes Arabidopsis amino acid transporter LHT1 as the main research object,combined with FRET technology,further studies whether LHT1 is involved in glyphosate transport.First,the "sandwich" probe of CFP-LHT1-YFP was constructed,and the probes were connected to pRSET-A and pDR196 vectors respectively,and the probes were expressed in BL21 prokaryotic cells and BY4741 yeast cells respectively.The purified prokaryotic expression probe protein was mixed with the tested amino acid and glyphosate respectively.The results showed that the positive control group treated with glycine and glutamic acid had obvious FRET changes;the change trend after glyphosate treatment was similar to that of glycine and glutamic acid;arginine and PBS treated.There was no significant change in FRET in the negative control group.And the change of FRET was influenced by the concentration of substrate and pH.Correctly expressing FRET probe yeast cells was used for photobleaching test to detect FRET changes in single cells.Photobleaching method was used to study the FRET changes of single yeast cells after amino acid or glyphosate treatment,and the results were consistent with those detected by fluorescence labelling instrument.In this paper,the FRET probe based on amino acid transporter LHT1 of Arabidopsis thaliana was successfully constructed.Through the prokaryotic expression system and the yeast eukaryotic expression system,it is proved that LHT1 has the ability to combine with glyphosate,and it is speculated that glyphosate can be transported by LHT1 base on plant level,and the probe can be further used for the screening of other guided pesticides.