利用蛋白质组学策略分析星星草根盐胁迫应答氧化还原敏感蛋白质

Puccinellia tenuiflora is a monocotyledonous halophyte species belonging to the Gramineae and has outstanding nutritional value for livestocks. It can thrive in the saline-alkali soil of the Songnen plain in northeastern China. Some physiological and proteomics mechanisms in leaves of P. tenuiflora in response to salt/alkali treatments have been highlighted in our previous proteomics investigation. However, the salt tolerance mechanism in roots is still unclear so far. In the fine-tuned sophisticate molecular networks for salt tolerance, the anti-oxidative mechanism and the thiol redox-regulated modification of proteins in roots play key roles. Further investigation of the thiol redox-sensitive proteins involved in salt response will provide valuable information for screening of salt-tolerant genes and improving crops tolerance. In this study, we plan to investigate the salt-responsive expression patterns (e.g. abundance and redox state) of thiol redox-sensitive proteins in P. tenuiflora roots using high throughput quantitative redox proteomics approaches. Three complementary labeling strategies will be used for analyzing thiol redox-sensitive proteins in roots. They are Cy3/Cy5 labeling method, isotope coded affinity tag (ICAT) method, and cysteine-reactive tandem mass tag (CysTMT) technology. Based on these methods, we can find out the reversible and/or irreversible redox-sensitive proteins to cope with salinity. In addition, the salt-responsive thioredoxin target proteins will be identified using CysTMT method combined with mass spectrometry analysis. Moreover, some of the key representative redox-sensitive proteins will be selected to verify their salt-/redox-responsive functions by transferring their encoding genes to Arabidopsis. The target proteins extracted from stable transgenic Arabidopsis plants under salt and H2O2 treatment will be characterized and quantified using Cy3/Cy5 staining gel electrophoresis, Western blot and modified protein electrophoretic mobility shift assay (PESMA) method. Based on these studies, we will integrate the expressional patterns of redox-sensitive proteins and various cellular and physiological characteristics of roots under salts treatment to present more novel detailed information and clues underlying the complex salinity-responsive signaling and metabolic network.

星星草（P.tenuiflora）是具有很强耐盐能力的禾本科牧草。研究其盐胁迫应答机制对于筛选重要耐盐基因、提高作物耐盐性具有重要意义。星星草根中的抗氧化系统与蛋白质氧化还原状态转换在盐胁迫应答过程中起着关键作用。本研究拟利用新发展起来的Cy3/Cy5荧光标记技术、同位素标记的亲和标签（ICAT）技术和半胱氨酸反应串联质量标签（CysTMT）技术3种彼此互补的氧化还原蛋白质组学研究策略，解析星星草根应答不同类型/强度盐胁迫过程中硫醇氧化还原敏感蛋白质的表达丰度与修饰状态，并将重要蛋白质编码基因转入拟南芥中，在盐和过氧化氢胁迫条件下，利用Cy3/Cy5染色、Western杂交、蛋白质凝胶阻滞等技术验证蛋白质应对氧化胁迫的功能。通过与根氧化胁迫应答相关细胞学与生理学特征整合分析，构建硫醇氧化还原敏感蛋白质参与调控的受不同盐信号诱导的盐胁迫应答网络机制，为深入解析植物耐盐机制提供更多证据与线索。

土壤盐碱化严重影响作物产量和农业生产效率。碱性盐给植物造成离子、渗透和高pH 胁迫。盐生植物星星草根的盐碱胁迫响应的分子机制目前并不十分清楚。本研究利用生理学与iTRAQ定量蛋白质组学技术分析了星星草根的Na2CO3和H2O2适应机制。我们建立了含有3876种非冗余蛋白质的星星草根部蛋白质数据集。研究结果初步表明，Na2CO3轻微抑制根的生长，导致ROS积累，细胞膜损伤和离子失衡，干扰蛋白质转运、合成与周转。胁迫响应蛋白质的丰度变化模式揭示了星星草根部应答Na2CO3胁迫的特殊信号与代谢途径。钙离子结合蛋白的聚集暗示着钙离子信号通路被激活从而完成盐胁迫信号转导。根部利用提高过氧化物酶的活性来提高H2O2等ROS的清除效率，通过限制钠离子吸收并提高钠离子区室化，并利用合成甜菜碱调节渗透平衡。更重要的是，我们发现多种激酶和两种转录因子的丰度明显上升，它们很可能参与了重要的盐碱胁迫应答的基因表达调控过程。碳代谢与其它代谢相关的多种酶系丰度的变化为胁迫应答过程中能量与碳骨架的供应提供了有力保障。多种H2O2响应蛋白质的发现为深入研究蛋白质氧化还原调控在星星草根部盐碱胁迫应答中的作用提供了重要信息。