The Preliminary Study Of Ricehigh Affinity Nitrate Transporter OsNRT2.3 Transmembrane Structure

Nitrate transporter geneOsNRT2.3 have two protein products,OsNRT2.3a and OsNRT2.3b.Overexpression of OsNRT2.3b can increase rice yield and nitrogen use efficiency.But overexpression of OsNRT2.3a doesn't hane the same effect.In order to explore the difference between the mechanism of these two proteins in rice,we adopted prokaryotic protein expression system,protoplast transient expression system,and using fluorescence microscope asssay,microplate reader assay,flow cytometry assay to detect the structural differences between these two transmembrane proteins.The preliminary analysis results are as follows:1)After expressing the fusion ofOsNRT2.3a/b original gene fragments and reporter gene GFP/PhoA,I did not detected out theGFP fluorenscenceinN134-GFP,N199-GFP of OsNRT2.3a.And same inN104-GFP?N169-GFP of OsNRT2.3b.Besides,I did not detected out thePhoA signal inN 134-PhoA,N199-PhoA of OsNR T2.3a andinN 104-PhoA?N169-PhoA of OsNRT2.3b.The results indicated that thesegene fragments didn't expressin the cell membrane.Therefore I used synthetizedprokaryoticcodingOsNRT2.3a/bgene to express into E.Coli.2)The expressed protein of OsNRT2.3a/b synthetized gene showed OsNRT2.3a-N-GFP,OsNR T2.3 b-N-GFP were detected GFP fluorenscence.OsNRT2.3a-N-PhoA,OsNRT2.3b-N-PhoA were not detected PhoA enzyme activity.In one side,this indicated condon synthetization cuold make a higher expression efficiency.In another side,it proved OsNRT2.3a/bN teminal were all in cytoplasm.But the reporter gene signal of OsNRT2.3a/b specific sites and C terminal fusion proteins had no differences.This was not conform with the predicted topology model.This may be due to its imprecised location of transmembrane stucture.So we decided make OsNRT2.3a/b expressed in rice protoplasm.3)OsNRT2.3a/bwhole gene C terminals and specific sites fused 6×His-tag(His-His-His-His-His-His).The results showed that the FI(Fluorence index = the number of cells with GFP/the number of whole cells)of unpermeated OsNRT2.3a(201 His)was high and the permeated was high too,which was same to positive control.So this site was in extracellular.The FI of unpernmeated OsNRT2.3b(171His)was low and the permeated was high which was same to negative control.So this site was in cytoplasm.The detected result of OsNRT2.3aC fusion protein was same to negative control.So this site was in cytoplasm.The FI of unpermeated OsNRT2.3b-C was high and the permeated was high too,so this site was in extracellular.These detected results were all conformed to OsNRT2.3a/b topology prediction model.In summary,Iusedreporter gene to detecttransmembrane structure of OsNRT2.3a/b in N terminal,C terminal and middle transmembrane domains.Preliminary results of OsNRT2.3a/bwas conformed to OsNRT2.3a/b topology prediction model.Showed that there are notable differences between OsNRT2.3a and OsNRT2.3b,which indicated that this difference may effect the function of protein.And this might finally effect the yield of rice in feild.Our experiment also provided topology evidence to the study of OsNRT2.3b pH site.So it proved OsNRT2.3b plays a key role in the regulation of intracellular pH homeostasis of rice.