《植物生理学报》 2017, 53(1): 45-51

AtSARK关键自磷酸化位点Tyr-350的点突变及其功能分析

龚雅茹, 郭朝霞, 王丹, 梅圆圆*, 王宁宁^*

南开大学生命科学学院, 天津300071

通信作者：王宁宁；E-mail: yuanyuan.mei@nankai.edu.cn; wangnn@nankai.edu.cn

摘　要：

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AtSARK (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AtSARKm的表达, 然后以表达水平与正对照GVG:AtSARKwt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AtSARK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AtSARK引起的早衰, 而该位点模拟非磷酸化点突变则不会影响AtSARK的功能。这些结果暗示着C末端磷酸化可能对AtSARK的功能起抑制作用, 而Tyr-350是其中一个重要调节位点。

关键词：点突变; 磷酸化; AtSARK; 类受体蛋白激酶; 衰老

收稿：2016-12-01   修定：2016-12-06

资助：国家自然科学基金(31570293)、教育部博士点基金优先发展领域课题(20130031130003)和天津市自然科学基金(16JCQNJC09300)。

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

GONG Ya-Ru, GUO Zhao-Xia, WANG Dan, MEI Yuan-Yuan*, WANG Ning-Ning^*

College of Life Sciences, Nankai University, Tianjin 300071, China

Corresponding author: MEI Yuan-Yuan; E-mail: yuanyuan.mei@nankai.edu.cn; wangnn@nankai.edu.cn

Abstract:

Our lab previously identified an Arabidopsis dual-specificity leucine-rich repeat receptor-like kinase AtSARK 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 AtSARK. 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 AtSARK. Phenotypes of GVG:AtSARKY350F and GVG:AtSARKY350E transgenic adults plants and seedlings that had similar expression levels of AtSARK with positive control plants GVG:AtSARK 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 AtSARK 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 AtSARK, and more importantly Tyr-350 appeared as one of the key regulation sites.

Key words: site-directed mutagenesis; phosphorylation; AtSARK; receptor-like kinase; senescence