Cellular Ultrastructure Analysis And Proteomics Study In Soybean Infected By Soybean Mosaic Virus

Soybean mosaic virus disease, caused by soybean mosaic virus(SMV), is a serious disease of soybean [Glycine max(L.) Merr].To control the disease, the most effective and safe way is developing resistant varieties. Various resistant factors expressed in plants after infected by pathogeny. Resistance related protein is a significant resistant factor. Therefore, study of host ultrastucture and proteome in pathogen-host interactions at subcellular level is significant to resistance mechanism researche.This research observed the alteration of leaf ultrastucture after infected by SMV and found some proteins relate to the disease resitance. We also analyzed the metabolic pathway of differentially expressed proteins participated. Precise gene localization might found resistant candidate genes to verify the proteome results.The study build the basis for understanding of virus resistance mechanism.The main research results as follows:1. The leaf ultrastructure after inoculated SMV strain SC3The leaf ultrastructure of resistant lines and susceptible lines were studied lOd and 20d after inoculation with SMV strain SC3. In resistant lines, there were no visible external symptoms and obvious damage of mesophyll cell ultrastructure during the two periods. But the virus particles were obseverd at 20d after inoculation.In susceptible lines, mosaic symptom appeared 10d after inoculation, and lots of thread-like SMV partcles, cylinderical inclusion bodies and pinwheel inclusion bodies were obsevered at 20d after inoculation. Great damages of chloroplasts and mitochondria emerged in the cell, the chloroplast lamellar structure distorted and swelled, the mitochondrial crest swelled and appeared vague. As the disease developed, the chloroplast and mitochondrial membrane were damaged and degradated.Shaped growths were found on outer membranes of virus, and increased afterwards. Virus were surrounded by large number of vacuoles and vesicles, which may be relevant to transfer or assembling of the virus.2. Resistant lines and susceptible lines of NILs (Near isogenic lines) comparative proteomics researchIn this study, some factors of the 2-DE system were optimized. We combined 2-DE (Two-Dimensional Gel Electrophoresis) and MALDI_TOF/TOF technology to analyze the different expressions of total proteins between resistant lines and susceptible lines.Samples were collected at 4,6 & 12 hrs and 1,3,5,7&14 after inoculated SMV strain SC3.105 specific-expressed proteins were identified, with a significant difference of relative abundance more than 2 times according to the T-test (p<0.05).77 protein spots were up-regulated expression and 29 protein spots were down-regulated.Proteins differentially expressed were analized by GO (Gene Ontology) analysis.Most proteins participate in response to stress and primary metabolic processes, and mostly located in plastid or membrane system, revelant to molecular synthesis or catalytic activity. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed significant changes in 22 metabolic pathways, mainly for carbon fixation and glycolysis.Among these differentially expressed proteins,18.80% were related to protein binding and assembly, 17.95% to glycolysis and energy metabolism,14.53% to photosynthesis,11.97% to resistance-related,9.40% to cell defense,9.40% to amino acid metabolism,7.69% to transcriptional regulation and signal transduction,5.98% to antioxidant,and 4.28% to cell wall synthesis.3. Analysis of inheritance and linkage of resistance genes for SC7 and SC8 strains in Kefeng No.lCrosses were made between the soybean cultivars Kefeng No.1 and Nannong 1138-2 and they were selfed to construct the F1, F2 and F2:3 populations. The individuals and populations were used to know the inheritance for resistance to SMV strain SC7 and SC8. All F1 plants showed resistant, F2 generation segregated on 3 resistant:1 susceptible ratio, and 1R:2Seg.:1S in F2:3 families by Chi-square tests. It indicates Kefeng No.1 carried a single resistant gene for SC7 and SC8 strain.At the same time,23 of 150 F2:3 families with resistance to SMV strains SC7 and SC8 performance inconsistently to these two strains.By speculate F2 genotype chi-square test was not clearly meet the 9:3:3:1 segregation ratio and it indicates two resistance genes are not the same gene. We found that these two resistant genes linked, and the genetic distance of the two resistance genes is 14.66cM.Further researches of resistant gene localization is to be done.