磷酸化修饰对大豆PM18（LEA3）蛋白增强植物耐旱性的影响

项目名称： 磷酸化修饰对大豆PM18（LEA3）蛋白增强植物耐旱性的影响

项目编号： No.31300215

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 刘昀

作者单位： 深圳大学

项目金额： 22万元

中文摘要： 抗逆蛋白可通过可逆磷酸化修饰参与植物耐旱保护。作为胚胎晚期富集蛋白（LEA）的重要成员，LEA3蛋白可显著增强植物的抗旱能力。然而目前仍未见LEA3蛋白存在磷酸化修饰的报道，磷酸化对LEA蛋白增强植物抗旱性的机制仍不清楚。我们前期研究发现大豆胚根PM18蛋白（LEA3）存在5个磷酸化修饰的异构体。本项目拟将正常培养、干旱和恢复大豆幼苗根的磷酸化热稳定蛋白进行双向电泳。分析干旱时PM18各异构体含量变化。利用液相-串联质谱鉴定各异构体发生磷酸化修饰的氨基酸位点。由此获知PM18蛋白中与抗旱密切相关的磷酸化位点。针对重要磷酸化位点设计突变基因mPM18，分析磷酸化对PM18蛋白增强拟南芥抗旱性的影响。利用圆二色谱以及酶活测定等方法研究磷酸化修饰对PM18蛋白二级结构和蛋白保护功能的影响。通过上述研究，阐明磷酸化修饰对LEA蛋白发挥抗旱保护功能的贡献，揭示LEA蛋白提高植物抗旱性的分子机制。

中文关键词： 胚胎晚期富集蛋白；磷酸化修饰；蛋白质聚集；二级结构；抗逆功能

英文摘要： Reversible phosphorylation of proteins is involved in the drought response of plant. The expression of late embryogenesis abundant protein (LEA) ，especially group 3(LEA3) protein,could enhance the drought-tolerance of transgenic plant.Although reversible phosphorylation of proteins is important for plant responsing to droutht stress, it is still far unclear that how phosphorylation affect the function of LEA proteins on protecting plant from drought stress. In our pilot studies, the PM18 protein (LEA3) was proved to be phosphorylated at Tyr-136. We have noticed that there are five isoforms of PM18 protein in soybean radicles. In the present study, the phospho-, and heat-stable proteome from the roots of soybean seedlings was separated by 2-DE.The content of each PM18 isoform in normal, drought and rehydation soybean seedling roots was compared. By using LC-MS/MS, the phosphorylated sites of each isoforms was determined. Taken together,the important drought-associate phosphorylated sites of PM18 protein could be identified. Moreover, reversed genetics was used to compare the drought-tolerace of transgenic Arabidopsis thaliana expressing PM18 and mPM18, which gene was mutant at key phosphorylated sites. The effect of phosphorylation on secondary structure of PM18 protein was analyzed in water, dehydration and reh

英文关键词： late embryogenesis abundant protein；phosphorylation；protein aggregation；secondary structure；stress-tolerant