羽衣甘蓝ARC1与Exo70A1蛋白相互作用结构域的分析与鉴定
Acta Horticulturae Sinica(2015)
Abstract
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BoARC1、BoARC1-N端(1~ 279 aa,含有UND结构域)及BoARC1-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pGBKT7载体中,获得BD-ARC1、BD-ARC1-N和BD-ARC1-C重组质粒.将羽衣甘蓝BoExo70A1、BoExo70A1-△1(1~85aa)、Exo70A1-△2(1~270aa)及Exo70A1-△3(271~638aa,含有Exo70结构域)的序列分别构建到pGADT7载体中,获得AD-Exo70A1、AD-Exo70A1-△1、AD-Exo70A1-△2和AD-Exo70A1-△3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-ARC 1-N/BoExo70A1-△1或BD-ARC 1-N/BoExo70A1-△2的酵母在滤纸上显示蓝色,这说明BoARC1的N端与BoExo70A1的N端介导了两者的相互作用.
MoreKey words
self-incompatibility,domain,ARC1,Brassica oleracea var.acephala,Exo70A1
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