磷酸化修饰对大豆PM18（LEA3）蛋白增强植物耐旱性的影响

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 rehydration by Circular Dichroism (CD) and Fourier Transform Infrared Spectrometer(FT-IR). Meawhile,how the phosphorylation affect the function of PM18 protein on protecting enzyme(lactate dehydrogenase and citrate synthase) or plant soluble proteome from dessication stress were analyzed. From the above, these results will help to elucidate the dynamic changes of LEA phosphorylation under drought conditions,and try to reveal the mechanism of LEA protein phosphorylation on enhancing the drought tolerance of plant.

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。

磷酸化修饰调控蛋白质的构象、活性、稳定性和亚细胞定位，在真核生物的生命活动中发挥关键作用。胚胎晚期富集蛋白（LEA），尤其是第三组LEA蛋白（LEA3）可显著增强植物的抗逆能力。然而目前仍未见LEA3蛋白存在磷酸化修饰的报道，LEA蛋白的抗逆机理仍不清楚。PM18为LEA3蛋白，我们前期实验证明PM18蛋白可显著提高拟南芥对干旱胁迫的耐受性。通过本项目的研究，我们首次发现大豆PM18蛋白存在3个磷酸化位点，分别位于S90、Y136和T266。将磷酸化位点进行点突变并转化酵母，通过比较酵母耐盐能力证明了S90和Y136位点的磷酸化对PM18蛋白抗逆功能具有重要贡献。在对PM18蛋白的抗逆机制研究时，我们发现PM18蛋白可防止polyQ蛋白在烟草细胞中的聚集。进一步将原核表达的PM18蛋白进行磷酸化和去磷酸化处理，再经圆二色谱和酶活保护功能研究，结果显示磷酸化修饰对PM18蛋白的二级结构具有一定的影响，并可增强PM18蛋白对LDH酶的保护作用。此外，为进一步揭示PM18蛋白的抗逆机理，我们在该项目获得的组学数据基础上，深入分析发现了大量其他与抗逆相关的蛋白。其中多个蛋白的抗逆功能为首次发现。这些蛋白与PM18一起，共同参与植物抗逆。以上结果揭示了磷酸化修饰对LEA蛋白发挥抗逆保护功能的贡献，为阐明大豆PM18蛋白参与植物抗逆的分子机制奠定了基础。