羽衣甘蓝ARC1与Exo70A1蛋白相互作用结构域的分析与鉴定  

Analysis and Characterization of Interaction Domain Between ARC1 and Exo70A1 from Ornamental Kale(Brassica oleracea var.acephala)

作　　者：曹明明[1] 杨佳[1] 李晓屿 李玉花[1] 蓝兴国[1] 

机构地区：[1]东北林业大学生命科学学院,哈尔滨150040

出　　处：《园艺学报》2015年第4期791-798,共8页Acta Horticulturae Sinica

基　　金：中央高校基本科研业务费专项基金项目(DL13CA13);国家基础科学人才培养基金项目(J1210053)

摘　　要：为阐明羽衣甘蓝自交不亲和信号传递因子ARC1与下游底物Exo70A1相互作用的结构域,利用酵母双杂交系统进行检测与分析。将羽衣甘蓝Bo ARC1、Bo ARC1-N端(1~279 aa,含有UND结构域)及Bo ARC1-C端(280~663 aa,含有U-box结构域和Arm repeat结构域)的序列分别构建到p GBKT7载体中,获得BD-ARC1、BD-ARC1-N和BD-ARC1-C重组质粒。将羽衣甘蓝Bo Exo70A1、Bo Exo70A1-Δ1(1~85 aa)、Exo70A1-Δ2(1~270 aa)及Exo70A1-Δ3(271~638 aa,含有Exo70结构域)的序列分别构建到p GADT7载体中,获得AD-Exo70A1、AD-Exo70A1-Δ1、AD-Exo70A1-Δ2和AD-Exo70A1-Δ3重组质粒。将获得的重组质粒分别共转化到酵母Y187菌株中,在双缺陷(-Leu-Trp)酵母培养基中培养与生长。β-gal显色检测结果表明,共转BD-ARC1-N/Bo Exo70A1-Δ1或BD-ARC1-N/Bo Exo70A1-Δ2的酵母在滤纸上显示蓝色,这说明Bo ARC1的N端与Bo Exo70A1的N端介导了两者的相互作用。The yeast-two hybrid system was used to detect the interaction between ARC1 and Exo70A1 from ornamental kale(Brassica oleracea var. acephala). The nucleotide sequences of full-length Bo ARC1,N terminal of Bo ARC1(1–279 aa,UND domain),C terminal of Bo ARC1(280–663 aa,U-box domain and Arm repeat domain)were constructed into the vector p GBKT7,respectively. Thus,the plasmids BD-ARC1,BD-ARC1-N or BD-ARC1-C were obtained and confirmed. On the other hand,the nucleotide sequences of full-length Bo Exo70A1,Bo Exo70A1-Δ1(1–85 aa),Exo70A1-Δ2(1–270 aa),Exo70A1-Δ3(271–638 aa,Exo70 domain)were constructed into the vector p GADT7,respectively. And the plasmids AD-Exo70A1,AD-Exo70A1-Δ1,AD-Exo70A1-Δ2 or AD-Exo70A1-Δ3 were also obtained and confirmed. These plasmids were used to co-transform yeast strain Y187,which were then plated onto SD/-Leu-Trp(synthetic dropout glucose agar plates lacking leucine and tryptophan). The β-galactosidase assays showed that yeast cells co-transformed with the Bo ARC1-N/AD-Exo70A1-Δ1 plasmids or Bo ARC1-N/BD-Exo70A1-Δ2 plasmids turned blue. These results indicated that N terminal of Bo ARC1 and Bo Exo70A1 mediated interaction between Bo ARC1 and Bo Exo70A1.

关 键 词：羽衣甘蓝 自交不亲和性 ARC1 Exo70A1 结构域 

分 类 号：S635.9[农业科学—蔬菜学]