Abstract：Abstract In order to investigate the molecular mechanism of salt tolerance, the proteome in leaves of 30 days seedling age southern type alfalfa (Medicago sativa 'Millenium') under control condition were compared with those under 72 h treatment at 250 mmol/L NaCl. Isobaric tags for relative and absolute quantitation(iTRAQ) technique with and 2-dimensional liquid chromatography-tandem mass spectrometry(2D-LC-MS /MS) were used to identify the differentially expressed proteins in southern type alfalfa leaf under salt stress, biological analysis on differentially expressed proteins was conducted to some of the potential target proteins to salt stress. The results indicated that 3 712 quantitative proteins were identified, 417 differentially expressed proteins (fold change ratio≥1.2, P≤0.05) were identified, in which 291 proteins were up-regulated and 126 proteins were down-regulated. The identified proteins were then categorized based on their sub-cellular localizations and biological functions using Gene Ontology (GO). Significantly enriched the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway were related to metabolic pathways, biosynthesis of secondary metabolites, pagosome, fatty acid metabolism, photosynthesis and so on (P＜0.05, false discovery rate(FDR)＜0.05). The identified differentially expressed proteins were involved in photosynthesis(7%), signal transmission(3%),oxidation resistance(6%), defense (2%), protein synthesis, processing and degradation(18%), energy and transport(7%), carbohydrate metabolism(11%), amino acid metabolism(7%), lipid metabolism(5%), other metabolism(7%), membrane and intracellular transport(7%) and cell structure, division and cytoskeleton(3%) and function unknown protein(16%). Functional analysis of the differentially expressed protein species showed an generally increased abundance of proteins related to oxidation resistance, energy and transport, defens and signal transmission, metabolism and a decreased generally abundance of proteins related to protein synthesis, processing and degradation, metabolism and photosynthesis. The results also showed that cytochrome P450, oxygen-evolving enhancer protein, phosphatidylinositol-specific phospholipase C, fructose-1,6-bisphosphate aldolase, trehalose-6-phosphate synthase, aspartate aminotransferase, E3 ubiquitin ligase, vacuolar H+-ATPase subunit C and so on were potential target proteins in alfalfa in response to salt stress. Our results suggested that differentially expressed proteins in southern type alfalfa leaf to salt stress can be effectively screened by iTRAQ technique combined with 2D-LC-MS/MS, may play roles in alfalfa adaptation to salt stress. Further study of these proteins will provide theory evidences into the molecular mechanisms of salt stress in alfalfa.
