Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-prot...Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-protein interactions.Previously,we obtained five proteins that interact with ethylene-responsive factor(ERF)from the cDNA library of Populus simonii x Populus nigra.To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability,we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay(Y2H).The secondary structures and 3D structures of PsnERF,ERFm,TrunERF,and psnerf^(197/198/202a) were predicted by homology modeling.The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions,while dephosphorylated mutations blocked the interactions.Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation,and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.展开更多
类钙调磷酸酶B亚基蛋白CBL(calcineurinB-likeprotein)与其互作蛋白激酶CIPK(CBL-interacting protein kinase)组成的CBL-CIPK系统,在真核生物钙信号转导及各种胁迫应答途径中发挥重要作用。采用SMART与LD-PCR技术构建了山葡萄‘双丰’(...类钙调磷酸酶B亚基蛋白CBL(calcineurinB-likeprotein)与其互作蛋白激酶CIPK(CBL-interacting protein kinase)组成的CBL-CIPK系统,在真核生物钙信号转导及各种胁迫应答途径中发挥重要作用。采用SMART与LD-PCR技术构建了山葡萄‘双丰’(Vitis amurensis Rupr.‘Shuangfeng’)叶片组织低温胁迫下的均一化酵母双杂交三框cDNA文库,库容量分别为1.7×106、1.3×106与1.9×106cfu·mL(-1),外源基因插入片段长度约为500~2000 bp,重组率为100%,符合后续双杂交筛选要求。以本课题组前期鉴定受低温诱导表达、具有核质亚细胞定位的山葡萄VaCIPK18全长(VaCIPK18)、激酶结构域缺失(VaCIPK18△STKc)以及NAF结构域缺失(VaCIPK18△NAF)为诱饵,进行酵母双杂交筛选,获得了17个候选互作靶蛋白;从中选取并克隆了7个参与非生物胁迫的候选互作蛋白进行酵母回转验证,仅发现1个ABA信号通路上游信号调节因子VaPYL9与VaCIPK18有互作关系。通过酵母双杂交和双分子荧光互补验证,确定了VaPYL9可与VaCIPK18和VaCIPK18△NAF在酵母中物理互作,而在植物拟南芥原生质体中VaPYL9与VaCIPK18△STKc和VaCIPK18△NAF互作,但与全长VaCIPK18不互作。展开更多
基金the Natural Science Foundation of Heilongjiang Province of China(LH2019C059).
文摘Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-protein interactions.Previously,we obtained five proteins that interact with ethylene-responsive factor(ERF)from the cDNA library of Populus simonii x Populus nigra.To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability,we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay(Y2H).The secondary structures and 3D structures of PsnERF,ERFm,TrunERF,and psnerf^(197/198/202a) were predicted by homology modeling.The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions,while dephosphorylated mutations blocked the interactions.Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation,and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.
