AtSARK关键自磷酸化位点Tyr-350的点突变及其功能分析

Site-directed mutagenesis and functional analysis of key autophosphorylation site Tyr-350 of AtSARK

本实验室前期在拟南芥中发现了一个可以正向调控叶片衰老的LRR型丝/苏氨酸和酪氨酸双底物特异性类受体蛋白激酶At SARK(Arabidopsis thaliana senescence-associated receptor-like kinase),并且经软件预测和质谱分析发现其有多个自磷酸化位点。本文把其中位于C末端的一个自磷酸化位点Tyr-350分别突变为谷氨酸(E)以模拟磷酸化状态或突变为苯丙氨酸(F)以模拟非磷酸化状态,用诱导型启动子GVG系统来控制点突变的At SARKm的表达,然后以表达水平与正对照GVG:At SARKwt相近的转基因株系为实验对象,分别观察不同点突变对转基因拟南芥成苗和幼苗表型的影响,同时检测一些衰老相关标识基因的表达,从而研究Tyr-350自磷酸化状态变化对At SARK功能的影响。实验结果显示,对Tyr-350位点进行模拟磷酸化点突变会减弱过表达At SARK引起的早衰,而该位点模拟非磷酸化点突变则不会影响At SARK的功能。这些结果暗示着C末端磷酸化可能对At SARK的功能起抑制作用,而Tyr-350是其中一个重要调节位点。

Our lab previously identified an Arabidopsis dual-specificity leucine-rich repeat receptor-like kinase At SARK as a positive regulator of leaf senescence. This kinase was further found to have many phosphorylation sites by prediction and mass spectral analysis. In current study,we focused on Tyr-350,a potentialphosphorylation site on C-terminus,and examined the effects of site-directed mutagenesis on the function of At SARK. We substituted Tyr-350 with either Phenylalanine（F） to mimic non-phosphorylation stateor Glutamic acid（D） to mimic phosphorylation state. The GVG system was employed to induce the expression of At SARK. Phenotypes of GVG：At SARKY350 F and GVG：At SARKY350 E transgenic adults plants and seedlings that had similar expression levels of At SARK with positive control plants GVG：At SARK were recorded and compared as well as the transcript levels of several senescence-related marker genes. It was demonstrated that Tyr-350 of phosphorylation state weakened the function of At SARK as a positive regulator of leaf senescence,while Tyr-350 of non-phosphorylation state did not make any significant difference,suggesting that phosphorylation on C-terminus may inhibit the function of At SARK,and more importantly Tyr-350 appeared as one of the key regulation sites.