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以南方型紫花苜蓿(Medicago sativa’Millennium’)30 d苗龄的实生苗为实验材料,分析正常培养和250 mmol/L Na Cl处理72 h后叶片中的蛋白表达变化,以期从蛋白质水平上揭示南方型紫花苜蓿适应盐胁迫的分子机制。采用同位素相对标记与绝对定量技术(isobaric tags for relative and absolute quantitation,i TRAQ)结合双向液相色谱与串联质谱(2-dimensional liquid chromatography-tandem mass spectrometry,2D-LC-MS/MS)定量蛋白质组技术鉴定南方型紫花苜蓿叶片响应盐胁迫的差异表达蛋白,对所获得差异蛋白进行生物信息学分析,筛选出可能的耐盐潜在靶标蛋白。结果表明,共鉴定3 712个定量蛋白,417个表达量有显著差异的蛋白质(变化倍数≥1.2,P≤0.05),其中包含291个表达上调的蛋白,126个表达下调的蛋白。按照基因本体(Gene Ontology,GO)分类体系,对差异蛋白归类分析,揭示其亚细胞定位和分子功能。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路显著性富集于代谢途径、次生代谢产物生物合成、吞噬体、脂肪酸代谢和光合作用等(P<0.05,错误发现率(false discovery rate,FDR)<0.05)。成功鉴定的差异蛋白分别涉及到光合作用(7%)、信号传递(3%)、防御(2%)、碳水化合物代谢(11%)、氨基酸代谢(7%)、脂类代谢(5%)、其它代谢(7%)、蛋白质合成、加工与降解(18%)、细胞结构、分裂和细胞骨架(3%)、抗氧化物(6%)、能量产生与转运(7%)、膜和胞内运输(7%)及未知功能蛋白类(16%)。差异表达蛋白中与抗氧化物、能量产生与转运、防御和信号传递及代谢等相关的蛋白表达量总体上调,而与蛋白质合成、加工与降解和光合途径相关蛋白表达量总体下调。研究发现,细胞色素P450、PSⅡ放氧增强蛋白、磷脂酰肌醇特异性磷脂酶C、果糖-1,6-二磷酸醛缩酶、甘露糖-6-磷酸还原酶、海藻糖-6-磷酸合酶、天冬氨酸转氨酶、E3泛素连接酶和H+-ATP酶C亚基蛋白等可能是紫花苜蓿耐盐潜在靶标蛋白。采用i TRAQ结合2D-LC-MS/MS技术,能有效地筛选出南方型紫花苜蓿叶片响应盐胁迫差异表达蛋白,这些差异表达蛋白可能在紫花苜蓿耐盐调控过程中发挥重要作用,该研究为深入认识紫花苜蓿盐胁迫的应答分子调控机制提供理论依据。
The seedlings of 30-day-old seedlings of Medicago sativa’Millennium were used as experimental materials to analyze the changes of protein expression in leaves of normal and 72 h after treatment with 250 mmol / L NaCl, in order to reveal the protein level Molecular Mechanism of Southern Alfalfa Adaptation to Salt Stress. The proteome was quantified by isobaric tags for relative and absolute quantitation (i TRAQ) combined with 2-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS / MS) Technical identification Southern alfalfa leaves in response to salt stress differentially expressed proteins, bioinformatics analysis of the differential proteins obtained to screen potential salt potential target protein. The results showed that 3 712 proteins were identified and 417 proteins with significant differences (change multiple≥1.2, P≤0.05) were identified, including 291 proteins with up-regulated expression and 126 proteins with down-regulated expression. According to Gene Ontology (GO) classification system, differential proteins were classified and analyzed to reveal subcellular localization and molecular function. The pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) were significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, phagosomes, fatty acid metabolism and photosynthesis (P <0.05, false discovery rate false discovery rate, FDR) <0.05). The successful identification of differential proteins involved photosynthesis (7%), signal transduction (3%), defense (2%), carbohydrate metabolism (11%), amino acid metabolism (7%), lipid metabolism , Other Metabolism (7%), Protein Synthesis, Processing and Degradation (18%), Cell Structure, Fission and Cytoskeleton (3%), Antioxidant (6%), Energy Production and Transport (7%), Membrane and Intracellular transport (7%) and unknown functional proteins (16%). Among the differentially expressed proteins, the expression levels of proteins related to antioxidants, energy production and transport, defense and signal transduction and metabolism were generally up-regulated, while those related to protein synthesis, processing and degradation and photosynthetic pathway were down-regulated. Studies have found that cytochrome P450, PS Ⅱ oxygen-enhancing protein, phosphatidylinositol-specific phospholipase C, fructose-1,6-bisphosphate aldolase, mannose-6-phosphate reductase, trehalose- Synthetic enzymes, aspartate aminotransferase, E3 ubiquitin ligase and H + -ATPase C subunit protein may be potential target alfalfa salt tolerance protein. Using i TRAQ combined with 2D-LC-MS / MS technology, the differentially expressed proteins of S. alfalfa in response to salt stress can be effectively screened. These differentially expressed proteins may play an important role in salt tolerance regulation of alfalfa In-depth understanding of alfalfa salt stress response regulation mechanism provide a theoretical basis.