Magnetic Nanoparticles Preparation And Application In Separation Of TIR1 Protein In Arabidopsis Thaliana

The isolation and identification of herbicide targets is one of the key problems to the development of high efficient herbicides.It is very important to establish simple,rapid and accurate methods for the isolation and identification of herbicide targets.The TIR1 protein is the key hormone receptor in Arabidopsis Thaliana and the action target of hormone-type herbicides,and its signaling pathways have aroused great attention in academic research in recent years.In this thesis,we aim at developing methods for the preparation of functionalized magnet nanoparticles for the use as active carriers to enrich and isolate the TIR1 protein in Arabidopsis,with the intention to establish a foundation for the development of new herbicides targeting the TIR1 protein.The work in this thesis has three parts:1.The first part reviews the relevant background knowledges about the work in this thesis,including the synthesis and application of magnetic nanoparticles,the auxin action principle,the TIR1 protein,the SDS-PAGE protein isolation method,and the dot blot immunological protein identification.Then the design of the research project of this thesis is put forward.2.In the second part,the magnetic nanoparticles(MNPs)with three differently modified surfaces were synthesized.The three surface modifiers are as the following:citric acid,dopamine and a trifunctional polymer DSZ containing terminal azide groups in the side chains that was synthesized in our research group.These MNPs were characterized with surface morphology,zeta potential,TEM,DLS and infrared spectroscopy(FTIR).The toxicity of MNPS was tested by MTT experiment.The results showed that MNPs prepared in this work have good dispersion stability,narrow size distribution and low toxicity.3.Using HRP enzyme and HSA protein as example establish the reaction of magnetic nanoparticles enrich protein.4.In the third part,we synthesized the magnetic separation carriers through the coupling of functionalized MNPs and the primary antibody of the TIR1 protein.Then the carriers were incubated in the total protein extracts from different tissues of Arabidopsis thaliana at room temperature.After magnetic separation,the TIR1 protein was released from the MNPs surface by Tris-HCl washing.The protein was characterized by SDS-PAGE and dot blot.The results showed that both Arabidopsis roots and leaves have protein TIR1,and the root contains more protein TIR1.After pH adjustment,dot blot detection demonstrated that the activity of the eluted protein was well preserved.The work in this thesis has set up a method that can be used for the TIR1 protein enrichment by magnetic nanoparticles,forming a foundation for developing new herbicides targeting the TIR1 protein in plants.