Proteomic Analysis In The Process Of Puccinia Striiformis F.SP.Tritici Infected Wheat And Subcellular Localization Research Of Effector

Wheat stripe rust is a worldwide disease,which caused by Puccinia striiformis f.sp.tritici damaged to the world's food production seriously.As a kind of obligate parasitic fungi,puccinia striiformis invade the host from the stomatal,it will be form the primary hypha in the intercellular space of the host,absorb nutrients from the host cell by haustoria.Then,establish a parasitic relationship with the host by generate a lot of secondary hyphae in the intercellular space.In the process,puccinia striiformis secrete virulence factors into the intercellular space,and resistance protein secreted by host are in the intercellular space also.Therefore,looking for intercellular protein to reveal the interaction mechanism between puccinia striiformis have great significance.Therefore,this study established a method of extraction of intercellular fluid protein,based on the extracted intercellular protein mass spectrometry analysis,as well as the subcellular localization of puccinia striiformis effector,the results are as follows:This research is based on the methods of intercellular fluid protein extraction that has already reported,by means of the determination of protein concentration and the appraisal experiment of glucose 6-phosphate dehydrogenase,will eventually determine the extraction method for: 50 mMol/L PBS buffer,-0.1 MPa pressure processing 5 min,4 ? 2000 rpm centrifugal 5 min,collect intercellular fluid protein after centrifugal acetone precipitation and freeze drying.The inoculation of puccinia striiformis in wheat leaf as experimental group,the wheat leaf with distilled water as the experimental group,in 12 h,48 h,7 d collect samples,extracted intercellular fluid protein.Sequencing the protein by trypsin digestion,TMT markers,LC-MS analysis,get 772 protein after bioinformatics analysis,of which 612 are wheat protein,160 are puccinia striiformis protein.Analyze the article wheat and puccinia striiformis respectively,in 12 h,48 h,7 d three time points the the number of raised expression wheat protein were 200,215,133.According to GO analysis respectively,the results show that raised expressed protein of wheat in three points are in the extracellular area,cell wall,and plastid stroma etc,and that may be associated with disease resistance response.In the 160 of the fungus protein,there are115 hypothetical proteins,29 ribosomal protein and adenosine homocysteine enzyme etc.The prediction result of signal peptide and the functional domain of hypothetical protein show that20 protein with signal peptide,89 protein have functional domain.We need to predict whether these proteins is puccinia striiformis effector.The subcellular localization experiment of puccinia striiformis effector is build target gene with no signal peptide to the 1302-eGFP fusion expression vector,with the help of agrobacterium infect system fusion protein expression in the tobacco,under the fluorescence microscope BW,YFP and white light observation the location,to explore the effector located in the cell.The result of localization for five effector with no signal peptide as: PST-Hasp97,PST-Hasp260,PST-Hasp257 located in cell membrane and cytoplasm,PST-Hasp68 nosp are located in cytoplasm,cell membrane,and nucleus,PST – 8603 located in cell nucleus,it suggest that the way of puccinia striiformis is variety.