甘蓝自交不亲和信号转导元件ARC1与EXO70A1的相互作用

摘要

ARC1 and EXO70A1 are important signal elements of self-incompatibility in Brassica. To identify the interaction of ARC1-EXO70A1 during the course of SI, we cloned the coding sequences of ARCl and EXO7QA1 from Brassica napus L. And Brassica oleracea L. Var. Acephala. Sequence analysis showed that ARC1 consisted of 663 amino acids in Brassica oleracea and 661 amino acids in Brassica napus, and there existed 45 amino acids difference between them. Sequence alignment showed that similarity positions and identity positions reached 95.9% and 93.9% between BoARCl and BnARCl, whereas there existed only six-amino-acid difference between BoEXO70Al and BnEXO70Al. The Similarity positions and identity positions reached 99.4% and 98.9% between BoEXO70Al and BnEXO70Al respectively. The homology of EXO70A1 was higher than that of ARCl. Yeast two-hybrid results indicated that the strong interaction existed between ARC1 and EXO70A1, and it could activate the expression of four reporter genes (ADE2, HIS3, AUR1-C, and MELT) in diploid yeasts. However, low interaction existed between EXO70A1 and ARClNwith 316 amino acids deleted from C-terminal, and it only activated the expression of three reporter genes (ADE2, AIJR1-C, and MELT). This provides an insight that the interface of interaction between ARC1 and EXO70A1 may not consist of the domains of arm repeats in ARC1. N-terminal domains of ARC1 play an essential role in the interaction of ARC1-EXO70A1. The influences of the differences in amino-acid composition between BoARCl and BnARCl on the interaction of ARC1-EXO70A1 could not be detected by means of yeast two-hybrid system, which probably resulted from that the binding interface between ARC1 and EXO70A1 was not altered by sequence difference of two proteins in two Brassica species.%从甘蓝型油菜与羽衣甘蓝柱头中克隆出ARC1与EXO70A1基因的编码区,序列分析显示推导的甘蓝型油菜ARC1比羽衣甘蓝ARC1少编码2个氨基酸,ARC1蛋白在羽衣甘蓝与甘蓝型油菜中存在45个氨基酸的差异,其序列相似度与一致性分别达到95.9%和93.9%;推导的EXO70A1在2种植物中仅有6个氨基酸的差异,其序列相似度与一致性分别达到99.4%和98.9%;EXO70A1蛋白在种内和物种间都保持着高度的保守性,其保守的程度高于ARC1.应用酵母双杂交系统检测2种蛋白质的相互作用,发现在二倍体酵母细胞中,全长的ARC1与EXO70A1之间存在较强的相互作用,能激活4个报告基因(ADE2、HIS3、AUR1-C和MEL1)的表达;去除C-端(包括臂重复区)316个氨基酸残基后的ARC1N与EXO70A1之间表现出较弱的相互作用,只能激活3个报告基因(ADE2、AUR1-C和MEL1)的表达,表明ARC1的臂重复区可能并不位于ARC1与EXO70A1的互作界面的核心区段,ARC1的N-端结构域对ARC1与EXO70A1互作起关键作用;同时发现不同ARC1-EXO70A1组合的互作强度相当,其可能原因是ARC1和EXO70A1在甘蓝与甘蓝型油菜中存在的这些序列差异并未影响ARC1-EXO70A1互作界面的构象.