利用酵母双杂交系统筛选水稻类受体激酶OsWAK50胞内域相互作用蛋白

细胞壁连接的类受体激酶(wall-associated kinase，WAK)是植物细胞中一类特有的类受体激酶基因亚家族，因其胞外域与细胞壁紧密相连而得名．水稻中共有125个OsWAK基因，OsWAK50编码的蛋白质具有胞外域、跨膜域和激酶域，呈现典型的WAK样受体激酶特征．首先通过对OsWAK50-GFP融合蛋白的观察发现OsWAK50定位于细胞膜并且与细胞壁偶联．进而通过酵母双杂交系统筛选到了20个可能与OsWAK50胞内域相互作用的候选蛋白，并通过一对一酵母转化验证了OsSK4、OsSWIB和OsSWI3C全长均可与OsWAK50胞内域相互作用．进一步分析显示，OsSWIB能够直接与OsWAK50激酶域互作，而OsSK4和OsSWI3C与OsWAK50胞内域的互作是依赖于OsWAK50 C端的．研究还表明，OsSK4和OsSWIB亦能与OsWAK50同源基因OsWAK53a结合，而OsSWI3C则不能与OsWAK53a结合．双分子荧光互补实验证明，OsSK4与OsWAK50和OsWAK53a能够在植物体内发生互作．以上结果为阐明OsWAK50发挥功能的分子机制提供了重要线索．

Screen of Receptor-like Kinase OsWAK50 Intracellular Interacting Proteins by Yeast Two-hybrid System

The wall-associated kinases (WAKs) belong to a unique subfamily of the receptor-like kinases (RLKs) in plants, named for its tight association with the cell wall. There are 125 OsWAK genes in rice (Oryza sativa), of which OsWAK50 is a typical RLK with an extracellular domain, a transmembrane domain and an intracellular kinase domain. OsWAK50-GFP fusion protein is localized on the cell surface and associated with the cell wall. By the yeast two-hybrid screen, we identified 20 possible OsWAK50 intracellular interacting proteins. Further one to one reverse hybridization indicates that OsSK4, OsSWIB and OsSWI3C interact with OsWAK50 intracellular domain. OsSWIB interacts directly with the kinase domain of OsWAK50, while OsSK4 and OsSWI3C interact with OsWAK50 intracellular domain in a C terminal dependent manner. OsSK4 and OsSWIB can also interact with OsWAK53a, the closest homolog of OsWAK50 in rice, whereas OsSWI3C can not interact with OsWAK53a. The interaction between OsWAK50 and OsSK4, OsWAK53a and OsSK4 was further confirmed by in vivo BiFC assay. These results provide important clues for elucidating the molecular mechanisms of OsWAK50 function in rice.