Crystallization Studies Of Plant Phosphorus Sensitive Protein Spx1 And Phr Complexes

Phosphorus is an essential element in the growth and development of plants.The occurrence of phosphorus in the soil is widespread,however,the absorption capacity of the plants is limited as compared to their need.Hence,it is a very important pivotal factor for the crop productions and yields.PHR is a transcriptional reg?Lator in plants and is responsible for reg?Lating the transcription of phosphorus starvation-responsive genes in plants.The PHR gene reg?Lates the function of SPX1,it is a phosphorus-responsive protein in plants.There is two proteins complex which can interact and play an important role in reg?Lating the level of phosphorus in plants.Therefore,it is very important to resolve the structure of these two protein complexes,which can further laid foundation for the better understanding their reg?Lation mechanism of phosphorus balance in plants.It will be helpf?L for to shed light on the theoretical support for the c?Ltivation of crops that can efficiently absorb and use limited phosphorus mineral in soil.In this thesis,three recombinant plasmids of PHR1(208-362aa),PHR2(243-426aa)and SPX1(1-295aa)were successf?Lly constructed.By linking the histidine tag at the N-terminus of the protein,the three proteins were successf?Lly expressed in the E.coli expression system.Proteins with a purity of more than 95%were successf?Lly obtained by Ni-column affinity chromatography and gel filtration chromatography.After purification,their binding mechanism was obtained.After m?Ltiple incubations,we obtained AtPHR1/OsSPX1 and OsPHR2/OsSPX 1 protein complexes.The crystals of the AtPHRl and AtPHRl/OsSPXl proteins were obtained at different conditions during initial screening of conditions.The AtPHR1 obtained best diffraction crystals were obtained after a series of crystal optimization conditions i.e.pH screening and varying precipitant concentration.Finally,we obtained the co-crystallized ligand(selenomethionine)with the proteins through X-ray diffraction technique.In the conclusion,the final data processing and SDS-PAGE res?Lts showed that the protein was degraded during the crystallization process.It was confirmed that we obtained a half-length sequence of the AtPHR1 crystals.In summary,the crystallization conditions of the AtPHRl and AtPHR/OsSPX1 protein complexes are obtained in this thesis,which will laid an important information for the crystal optimization and structural analysis of the two proteins.