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2025年4月5日 星期六
  2016, Vol. 24 Issue (11): 1629-1642    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
南方型紫花苜蓿苗期叶片盐胁迫差异蛋白分析
裴翠明,张振亚,马进
浙江农林大学风景园林与建筑学院
Differentially Expressed Proteins Analysis of Seedling Leaf of Southern Type Alfalfa ( Medicago sativa 'Millenium' ) Under Salt Stress
2,
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摘要 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 NaCl 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, iTRAQ)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iTRAQ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。
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裴翠明
张振亚
马进
关键词 紫花苜蓿盐胁迫差异蛋白同位素相对标记与绝对定量技术(iTRAQ)    
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.
Key wordsAlfalfa    Salt stress    Differentially expressed protein    Tsobaric tags for relative and absolute quantitation (iTRAQ)
收稿日期: 2016-06-23      出版日期: 2016-10-01
基金资助:南方型紫花苜蓿盐诱导蛋白基因克隆及功能分析;南方型紫花苜蓿耐盐相关miRNAs及靶基因挖掘与功能解析
通讯作者: 马进     E-mail: majinzjl@163.com
引用本文:   
裴翠明 张振亚 马进. 南方型紫花苜蓿苗期叶片盐胁迫差异蛋白分析[J]. , 2016, 24(11): 1629-1642.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I11/1629
 
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