多胺缓解黄瓜盐胁迫伤害的磷酸化蛋白质组学研究

Against to the increasingly serious harm by salt stress in vegetable protected cultivation, vegetable crop growth was often inhibited, yield and quality decreased, and the research on polyamine to alleviate salt stress injury become more and more attention.In this study, we will use the nutrient solution cultivation with the normal nutrient solution, salt stress and exogenous polyamine on cucumber seedlings, using IMAC and TiO2 chromatographic to enrich the phosphorylated proteins, and combined with iTRAQ technology to analyze the phosphorylated proteomics of cucumber apical protein,in order to observe the dynamic change of protein phosphorylated levels with treatment time prolonged by salt stress and polyamine,looking for the signals or metabolic pathways regulated by protein phosphorylation;the function of differentially expressed phosphorylated proteins will be suggested by Go, Pathway and protein interaction analysis; using the qRT-PCR and Western blotting to analyze the expression of phosphorylated proteins and its key gene/protein involved in signals or metabolic pathways, further to reveal the protein phosphorylation induced by polyamine under salt stress and its signals or metabolic pathways; our researches will reveal the biological mechanism of polyamine on salt stress alleviation from protein phosphorylation modification level, and have important academic value to enrich the theory of plant stress physiology.

针对设施蔬菜栽培中土壤盐渍化严重，经常导致蔬菜作物生长发育受到抑制、产量和品质下降等问题，多胺缓解植物盐胁迫伤害的机理研究愈来愈深入。本项目以黄瓜为试材，通过正常营养液、盐胁迫、外源多胺处理黄瓜幼苗，利用IMAC和TiO2色谱富集磷酸化蛋白，结合iTRAQ技术研究黄瓜根尖蛋白的磷酸化蛋白质组学，观察随盐胁迫及多胺处理时间的延长，黄瓜根尖蛋白磷酸化水平的动态变化，寻找蛋白磷酸化调节所参与的信号或代谢途径；利用生物信息学方法对差异积累的磷酸化蛋白进行结构与功能分析，获得其GO、Pathway、蛋白互作信息；利用qRT-PCR和Western blotting对差异积累的磷酸化蛋白及其所参与的信号或代谢通路中的关键蛋白基因进行表达分析，阐明盐胁迫下多胺诱导蛋白磷酸化及参与的信号或代谢途径，从蛋白质磷酸化修饰方面揭示多胺缓解黄瓜盐胁迫伤害的生物学机制，对丰富植物逆境生理理论具有重要的学术价值。

采用营养液栽培，准确控制根际生长环境，通过正常营养液、盐胁迫、外源多胺处理黄瓜幼苗，研究了多胺缓解黄瓜盐胁迫伤害的磷酸化蛋白质组差异。结果表明：（1）采用TiO2色谱富集磷酸化蛋白，利用iTRAQ技术分离鉴定了盐胁迫及外源Spd诱导的黄瓜根尖磷酸化差异蛋白共计157个；（2）磷酸化差异蛋白参与的关键信号或代谢途径包括植物耐盐SOS途径、蛋白合成修饰与降解途径、DNA复制与转录因子、细胞分裂相关途径、糖酵解、氮代谢、纤维素合成等代谢途径；（3）盐胁迫诱导激酶类蛋白和跨膜转运蛋白表达上调、糖酵解代谢和氮代谢上调，抑制DNA复制修复和蛋白的合成以及细胞分裂，促进蛋白质的降解；而外源多胺处理后，进一步上调了部分激酶类蛋白和转运蛋白的表达，诱导了一些转录因子上调和蛋白合成增加，减弱了糖酵解代谢，促进纤维素合成；（4）盐胁迫下丝氨酸/苏氨酸蛋白激酶（STK）、MAPK、Ca2+相关激酶、蛋白磷酸酶、受体激酶基因表达均显著上调，而盐胁迫下多胺处理5d后，诱导STK、MAPK和受体激酶基因表达上调，Western blot发现STK、MAPK、SAMs均进一步上调。上述结果表明，盐胁迫下多胺能通过促进DNA复制和蛋白合成以及细胞分裂，减弱糖酵解代谢，重要的是能诱导SOS途径的信号转导分子上调，进而激活SOS途径，抵御盐胁迫伤害。研究结果对丰富植物逆境生理理论具有重要意义，对设施作物抗逆促长优质高效栽培和抗性品种选育均具有重要的学术价值。