Studies On The Extraction,Separation And Enrichment Technology Of Rice Leaf Proteomics

Proteomics has become an indispensable subject for studying biological sciences,which can connect three disciplines including genomics,genetics and physiology.In proteomics research,steps such as protein extraction,separation,and enrichment of low abundance proteins directly affect subsequent protein identification results.Low-abundance proteins are important active substances in plants,and they are involved in various physiological and pathological processes such as metabolism,transcription,and translation.Thus,it is of great significance to enrich low-abundance protein for the study of plant proteomics.The dynamic range between high abundance proteins and low abundance proteins can reach 12 orders of magnitude,making detection of low-abundance proteins difficult.The combinatorial peptide ligand library technology(CPLL)is a novel method for enriching low abundance proteins,which can reach the purpose of reducing high-abundance proteins and enriching low-abundance proteins,and it has been applied to proteomics research in various fields.In this paper,the researchs on the extraction,separation and enrichment technology of rice leaf proteome was studied,which provided technical support for rice proteome research and provided a theoretical basis for the further study of rice proteome.1.A two-dimensional liquid chromatography(2-D LC)system was developed to separate proteins from rice leaves,which was extracted by phenol method.This extraction method provides technical support for subsequent protein enrichment studies.The impact of protein extraction methods(phenol method,sodium dodecyl sulfate method(SDS method)and trichloroacetic acid/acetone method(TCA/acetone method)),liquid chromatography separation system(1-D LC and 2-D LC)on the number of identified proteins were compared.Proteins identified by 2-D LC after proteins were extracted by the above three extraction methods were 2712,2415 and 1914,respectively,the proteins were 2.7-fold,2.5-fold and 1.9-fold the number of proteins identified by 1-D LC respectively.Compared with the SDS method and the TCA/acetone precipitation method,the phenol method not only identified a large number of proteins but also identified some extreme proteins.In addition,proteins,which were extracted by different extraction methods and separated by 2-D LC,were classified.It was found that protein functions by the three extraction methods were complementary.The method provides technological support for rice proteomics and reference for research techniques of other crop proteomics.2.Using the CPLL to enrich low-abundant proteins in rice leaves,and this method is suitable for the optimization of enrichment process of rice leaf protein.The effects of three buffer pHs(pH 7 Tris buffer solution,pH 9 Tris buffer solution and pH 4 acetate buffer solution),three eluents(8 M urea/2% CHAPS/5% acetic acid,boiling 4% SDS/50 mmol/L DTT and 9 mol/L urea/5% acetic acid)on the enrichment efficiency of the CPLL was examined.It is clear that the CPLL has a better enrichment effect at pH 7 and pH 9,and the complementarity was found between the identified crude protein and the protein in different pH components.In addition,8 mol/L urea/2% CHAPS/5% acetic acid eluted proteins most thoroughly.This method provides technical support for the application of CPLL technology in the study of plant proteomics as well as the separation and purification of low abundance proteins.3.The differences in expression of proteomes of rice leaves identified after treatment with different cadmium concentrations were studied.The number of identified proteins in the control sample,cadmium ion concentration 0.1 ?mol/L and cadmium ion concentration 5 ?mol/L were 1277,1236,and 1171,respectively,which indicated that there were differences in protein expression in rice plants exposed to different concentrations of cadmium stress and different cadmium ion concentrations have different effects on the number of rice proteins.The analysis of the number of proteins identified after different cadmium treatment concentrations and the newly identified protein functions after cadmium treatment provided the theoretical basis for elucidating the relationship between heavy metal stress and protein changes in rice.