野生大豆GsPP2CA和GsPKA对GsSnRK1.1蛋白活性的调控  

Co-regulation of GsSnRK1.1 Activity by Wild Soybean GsPP2CA and GsPKA

作　　者：刘圆明 尤宏光 卢浩然 丁晓东[1] LIU Yuanming;YOU Hongguang;LU Haoran;DING Xiaodong(College of Life Sciences,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《西北植物学报》2020年第8期1277-1286,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31670272);黑龙江省自然基金(C2017014);东北农业大学高级人才引进启动基金。

摘　　要：为了明确GsSnRK1.1蛋白激酶在野生大豆生长发育中的具体调控机制,该研究利用酵母二元杂交技术发现了蛋白激酶GsSnRK1.1的互作蛋白GsPP2CA和GsPKA,利用原核表达系统对GsSnRK1.1、GsPP2CA和GsPKA进行了表达和纯化用于Pull-down和体外磷酸化分析,并在酵母中研究了GsPP2CA和GsPKA对GsSnRK1.1蛋白活性的调控功能。结果表明:(1)GsSnRK1.1与GsPP2CA和GsPKA具有物理互作关系,Phos-Tag和pPKDsub特异性磷酸化抗体检测发现,GsSnRK1.1的Thr176磷酸化可以被蛋白磷酸酶GsPP2CA去磷酸化,GsPKA可能会磷酸化GsSnRK1.1的其他潜在磷酸化位点,进而竞争性抑制GsSnRK1.1的Thr176位点的磷酸化水平。(2)将这些基因回补进入酵母ARY330(-snf1/-reg1/-sit4)突变株系中,发现共转化GsSnRK1.1和GsPP2CA或GsPKA的转化子可在非葡萄糖碳源和高葡萄糖碳源的选择培养基上正常生长,GsPP2CA、GsPKA可以替代Reg1和Sit4降低GsSnRK1.1过度磷酸化对酵母细胞产生的毒害作用,进而调控GsSnRK1.1对非发酵型碳源的利用。The functions of plant protein kinase SnRK1s are regulated by many factors.In this work,we used protein kinase GsSnRK1.1 as bait to screen the wild soybean cDNA libray and identified GsPP2CA and GsPKA as interactors of GsSnRK1.1 by yeast two-hybrid.To further investigate the mechanism of how GsPP2CA and GsPKA regulate GsSnRK1.1 kinase activity,we expressed and purified these recombinant proteins in E.coli system for pull-down and phosphorylation assays.The results of pull-down analyses indicated that GsSnRK1.1 had physical interactions with GsPP2CA and GsPKA,and the Phos-tag assays and western blottings using specific pPKDsub antibody showed that the Thr176 of GsSnRK1.1 could be phosphorylated by GsPKA and could be dephosphorylated GsPP2CA.When we complemented these genes into ARY330(-snf1/-reg1/-sit4)yeast mutant strain,we found that GsSnRK1.1 could play the function of SNF1 kinase to make yeast cells grow on the media with non-fermented sugars.The phosphorylation level of GsSnRK1.1 protein kinase can be regulated by GsPP2CA and GsPKA,which may replace Reg1 and Sit4 in yeast and further to regulate the use of non-fermentative carbon sources by GsSnRK1.1.

关 键 词：GsSnRK1.1 蛋白激酶 蛋白磷酸酶 磷酸化 酵母 

分 类 号：Q785[生物学—分子生物学] Q786