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基于TMT质谱分析技术筛选水稻根尖响应汞胁迫差异表达蛋白

祁忠达1,2,3,韦艳1,2,龙水庭1,2,3,代佑罡1,2,仇广乐3*   

  1. (1贵州医科大学公共卫生学院职业卫生与环境卫生学系, 贵阳 550000;2贵州医科大学环境污染与疾病监控教育部重点实验室, 贵阳 550000;3中国科学院地球化学研究所环境地球化学国家重点实验室, 贵阳 550081)
  • 出版日期:2019-06-10 发布日期:2019-06-10

Screening differentially expressed proteins in response to mercury stress in rice roots by proteomic quantification based on stable isotope labeling and parallel reaction monitoring.

QI Zhong-da1,2,3, WEI Yan1,2, LONG Shui-ting1,2,3, DAI You-gang1,2, QIU Guang-le3*   

  1. (1Department of Occupational and Environmental Health, School of Public Health, Guizhou Medical University, Guiyang 550000, China; 2Key Laboratory of Ministry of Education for Environmental Pollution and Disease Surveillance, Guizhou Medical University, Guiyang 550000, China; 3State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550081, China).
  • Online:2019-06-10 Published:2019-06-10

摘要: 稻米是人群汞暴露的最主要途径之一,为了从蛋白质水平上揭示水稻(Oryza sativa L.)适应重金属汞胁迫的分子机制,本研究以拔节期金优431水稻品种为材料,对比分析对照组和50、150、300 mg·kg-1氯化汞胁迫组,采用同位素相对标记与绝对定量TMT(Tandem Mass Tag)技术,结合定量蛋白质组平行反应监测(parallel reaction monitoring, PRM)技术,鉴定水稻根尖部位响应汞胁迫差异表达蛋白,对所获得差异蛋白进行生物信息学分析,从而筛选出汞胁迫响应显著的潜在靶标蛋白。同时,选择20个差异表达蛋白通过平行反应监测试验进行了蛋白验证。结果表明,在变化倍数≥1.3、P<0.05条件下,共鉴定5253种定量蛋白,包含364种差异蛋白,其中258种表达上调,106种表达下调。基因本体分子功能提示,这些差异性蛋白主要参与催化活性、绑定转运活性和抗氧化活性。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、苯丙烷类生物合成等,错误发现率(false discovery rate, FDR)<0.05。成功鉴定并验证到的差异蛋白分别涉及抗氧化还原蛋白、金属螯合肽合成蛋白、金属硫蛋白相关蛋白等。差异表达蛋白中,与抗氧化、重金属胁迫防御响应和信号通路等相关的蛋白总体上调,而与代谢和能量产生与转运相关蛋白表达量总体下调。

关键词: 24-表油菜素内酯, 弱光, 番茄, 形态, 光合特性

Abstract: Mercury (Hg) is highly toxic. Rice (Oryza sativaL.) is one of the most important pathways for Hg exposure to populations. To reveal the molecular mechanism of rice adaptation to Hg stress, the Jinyou 431 rice cultivar grown under supplementation of 0 mg·kg-1 (CK), 50 mg·kg-1 (low), 150 mg·kg-1 (middle), and 300 mg·kg-1 (high) HgCl2 was selected to investigate root tips’ differentially expressed proteins in response to Hg stress using the isotope relative labeling and the absolute quantitative technique TMT (Tandem Mass Tag) combined with parallel reaction monitoring (PRM) quantitative proteomic technique. Bioinformatics analysis of the obtained differential proteins was performed to screen the target proteins responding significantly to Hg stress. A total of 20 differentially expressed proteins identified were selected for protein validation. The results showed that 5253 quantitative proteins and 364 differential proteins were identified (variation fold≥1.3, P<0.05), out of which 258 were up-regulated and 106 downregulated. The molecular function of gene ontology suggests that differential proteins are mainly involved in catalytic activity, binding transport activity, and antioxidant activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significantly enriches in metabolic pathways, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis (with false discovery rates of <0.05). The identified and validated differential proteins are related to antioxidant reductase, metal chelate peptide synthesis protein, and metallothioneinrelated proteins like. The expression levels of proteins involved in responding and signaling pathway to antioxidants and heavy metal stress defense were generally up-regulated, while the expression levels of those involved in metabolism, energy production and transport were generally down regulated.

Key words: 24-epibrassinolide, low light stress, tomato, morphology, photosynthetic performance.