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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。
The coding regions of ARC1 and EXO70A1 genes were cloned from the stigma of Brassica napus and Kale, and the deduced amino acid sequence of ARC1 and ARC1 was less than that of ARC1. ARC1 protein was present in both kale and Brassica napus The deduced amino acid sequence of EXO70A1 showed only 6 amino acid differences in the two plants with 99.4% similarity and 98.9% identity, respectively. The EXO70A1 protein is highly conserved among species and species and is more conserved than ARC1. Using yeast two-hybrid system to detect the interaction of the two proteins, we found that full-length ARC1 interacts with EXO70A1 in diploid yeast cells and activates four reporter genes (ADE2, HIS3, AUR1- C and MEL1); ARC1N and EXO70A1 after removing 316 amino acid residues at the C-terminal (including the arm repeat region) showed weaker interaction with only 3 reporter genes (ADE2, AUR1-C And MEL1), indicating that ARC1 arm repeat region may not be located in the ARC1 and EXO70A1 interaction interface core segment N-terminal domain of ARC1 ARC1 and EXO70A1 interaction play a key role; also found that different ARC1- The possible interaction between the EXO70A1 combinations is that the sequence differences between ARC1 and EXO70A1 in Brassica napus and Brassica napus did not affect the conformation of the ARC1-EXO70A1 interaction interface.