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为了从蛋白质水平上揭示南方型紫花苜蓿(Medicago sativa’Millennium’)适应盐胁迫的分子机制,本研究以30 d苗龄的南方型紫花苜蓿实生苗为材料,分析正常培养和250 mmol/L Na Cl处理72 h后根中的蛋白表达变化,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根部响应盐胁迫差异表达蛋白,对所获得差异蛋白进行生物信息学分析,筛选出可能耐盐潜在靶标蛋白。同时,选择5个差异表达蛋白进行实时荧光定量PCR(q RT-PCR)验证。结果表明,共鉴定3 857种定量蛋白,534种差异蛋白(变化倍数≥1.2,P<0.05),其中表达上调的281种,表达下调的253种。基因本体(Gene Ontology,GO)分子功能提示这些差异性蛋白主要参与转运活性、催化活性和酶调控活性。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、苯丙氨酸代谢和核糖体等(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信号传递(8.99%)、抗氧化物(7.68%)、防御(5.61%)、蛋白质合成、加工和降解(14.79%)、能量产生与转运(5.81%)、代谢(26.97%)、膜与胞内运输(5.62%)和细胞结构、分裂和细胞骨架(2.06%)等。差异表达蛋白中与信号传递、抗氧化物和防御等相关的蛋白表达量总体上调,而与代谢和能量产生与转运相关蛋白表达量总体下调。q RT-PCR实验发现,m RNA与蛋白质表达水平并不一致。研究发现组氨酸磷酸转移蛋白、丝裂原活化蛋白激酶、h类硫氧还蛋白、蛋氨酸亚砜还原酶基因、鸟苷二磷酸(guanosine diphosphate,GDP)解离抑制因子、脂质转移蛋白、β-1,2-木糖基转移酶和H2A/H2B/H3/H4核心组蛋白等可能是紫花苜蓿耐盐潜在靶标蛋白。本研究采用i TRAQ结合2D-LC-MS/MS技术,有效地筛选出南方型紫花苜蓿根部响应盐胁迫差异表达蛋白,为深入认识紫花苜蓿盐胁迫的应答分子调控机制奠定了坚实的基础。
In order to reveal the molecular mechanism of southern alfalfa (Medicago sativa’Millennium) adapting to salt stress at the protein level, we used 30-day-old southern alfalfa seedlings as materials to analyze the effects of normal and 250 mmol / L Na The changes of protein expression in roots after 72 h of Cl treatment were analyzed by isobaric tags for relative and absolute quantitation (i TRAQ) combined with two-dimensional liquid chromatography-tandem mass spectrometry , 2D-LC-MS / MS) were used to identify the differentially expressed proteins in the roots of southern alfalfa in response to salt stress. Bioinformatic analysis of the differentially expressed proteins was carried out to screen potential target proteins that might be salt tolerant. At the same time, five differentially expressed proteins were selected for real-time quantitative PCR (q RT-PCR) validation. The results showed that 3 857 kinds of quantitative proteins and 534 kinds of differential proteins were identified (change multiple≥1.2, P <0.05), of which 281 were up-regulated and 253 were down-regulated. Gene Ontology (GO) molecular function suggests that these differential proteins are mainly involved in transport activity, catalytic activity and enzyme regulatory activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was significantly enriched in metabolic pathway, secondary metabolite biosynthesis, phenylalanine metabolism and ribosome (P <0.05, false discovery rate false discovery rate, FDR) <0.05). The successful differential proteins involved in signal transduction (8.99%), antioxidant (7.68%), defense (5.61%), protein synthesis, processing and degradation (14.79%), energy production and transport (5.81% (26.97%), membrane and intracellular transport (5.62%) and cell structure, division and cytoskeleton (2.06%). Among the differentially expressed proteins, the expression of proteins related to signal transduction, antioxidants and defense were generally up-regulated, while the expression of proteins related to metabolism and energy production and transport was down-regulated. q RT-PCR experiments found that m RNA and protein expression levels are not consistent. Studies have found that histidine phosphate transfer protein, mitogen-activated protein kinase, h-type thioredoxin, methionine sulfoxide reductase gene, guanosine diphosphate (GDP) dissociation inhibitor, lipid transfer protein, β-1,2-xylosyltransferase and H2A / H2B / H3 / H4 core histone may be potential target proteins of alfalfa salt tolerance. In this study, i TRAQ combined with 2D-LC-MS / MS technology was used to effectively screen the roots of southern alfalfa in response to salt stress, which lays a solid foundation for understanding the regulatory mechanism of alfalfa salt stress.