Screening Differentially Expressed Proteins in Southern Type Alfalfa (Medicago sativa 'Millenium') Root upon Salt Stress by iTRAQ Protein Mass Spectrometry
Abstract：In order to investigate the molecular mechanism of salt tolerance, the proteome in roots of 30 d seedling age southern type Alfalfa (Medicago sativa 'Millenium') under control condition were compared with those under 72 h treatment at 250 mmol/L NaCl. Proteins were abstracted from root under control and NaCl- treated conditions, reduction and enzymolysis of proteins were performed. Isobaric tags for relative and absolute quantitation (iTRAQ) technique and 2-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS /MS) were used to identify the differentially expressed proteins in southern type alfalfa root upon salt stress, biological analysis on differentially expressed proteins was conducted to some of the potential target proteins to salt stress. Quantitative Real-time PCR (qRT-PCR) technique was used to validate the expression data of five randomly selected differentially expressed proteins. The results indicated that 3 857 quantitative proteins were identified 534 differentially expressed proteins(fold change ratio≥1.2, P＜0.05) were identified, in which 281 proteins were up-regulated and 253 proteins were down-regulated. Gene ontology (GO) molecular functional analysis showed that the differentially expressed proteins were mainly involved in transporter activity, catalytic activity and enzyme regulator activity. Significantly enriched KEGG pathway were related to metabolic pathways, biosynthesis of secondary metabolites, phenylalanine metabolism and ribosome and so on (P＜0.05, False discovery rate (FDR)＜0.05). The identified differentially expressed proteins were involved in signal transmission (8.99%), oxidation resistance (7.68%), defense(5.61%), protein synthesis, processing and degradation (14.79%), energy and transport (5.81%), metabolism (26.97%), membrane and intracellular transport (5.62%) and cell structure, division and cytoskeleton (2.06%) and so on. Functional analysis of the differentially expressed protein species showed an generally increased abundance of proteins related to signal transmission, oxidation resistance and defense and a decreased generally abundance of proteins related to metabolism and energy and transport. The expression levels of differentially expressed proteins were verified by real-time PCR and the results were not consistent with those of the quantitative mass spectrometry (P＜0.05).The results also showed that histidine phosphotransfer protein, mitogenactivated protein kinase, h-type Thioredoxin, plastidial methionine sulfoxide reductase, guanosine diphosphate (GDP) dissociation inhibitor, Lipid transfer protein, β-1, 2-xylosyltransferase and core histone H2A/H2B/H3/H4 and so on were potential target proteins in alfalfa in response to salt stress. Differentially expressed proteins in southern type alfalfa root to salt stress can be effectively screened by iTRAQ technique combined with 2D-LC-MS /MS in order to lay a solid basis for a holistic understanding of the molecular regulatory mechanism in southern type alfalfa to salt stress.
