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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。
In order to elucidate the interaction between ARC1 and Exo70A1, a self-incompatible signal transmission factor, the yeast two-hybrid system was used for detection and analysis. The sequences of Bo ARC1, Bo ARC1-N (1 ~ 279 aa with UND domain) and Bo ARC1-C terminal (280 ~ 663 aa with U-box domain and Arm repeat domain) were constructed Into the pGBKT7 vector, BD-ARC1, BD-ARC1-N and BD-ARC1-C recombinant plasmids were obtained. The sequences of Kale Bo Exo70A1, Bo Exo70A1-Δ1 (1-85 aa), Exo70A1-Δ2 (1-270 aa) and Exo70A1-Δ3 (271-638 aa, containing Exo70 domain) were constructed into pGADT7 vector , AD-Exo70A1, AD-Exo70A1-Δ1, AD-Exo70A1-Δ2 and AD-Exo70A1-Δ3 recombinant plasmids were obtained. The obtained recombinant plasmids were respectively co-transformed into yeast Y187 strain and cultured and grown in double-deficient (-Leu-Trp) yeast culture medium. The β-gal colorimetric assay showed that the yeast co-transfected with BD-ARC1-N / Bo Exo70A1-Δ1 or BD-ARC1-N / Bo Exo70A1-Δ2 displayed blue on the filter paper indicating that the N-terminus of Bo ARC1 binds to Bo The N-terminus of Exo70A1 mediates the interaction between the two.