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ARC1是植物特有的一类含有U-box/ARM结构域的蛋白,在芸薹属植物自交不亲和(self-incompatibility,SI)信号转导中起着正向调控因子的作用。将羽衣甘蓝(Brassica oleracea var.acephala)BoARC1编码区的序列连接到原核表达载体pET-14b上,通过酶切鉴定和测序分析,构建pET-14bBoARC1表达质粒;将获得的阳性表达质粒转化到大肠杆菌表达菌株BL21(DE3)pLysS中,利用IPTG进行诱导表达。SDS-PAGE结果显示,在分子量69 kD处有BoARC1蛋白特异性地诱导表达;利用Ni2+-NTA树脂通过亲和层析的方法获得BoARC1融合蛋白。在泛素激活酶(E1)、泛素结合酶UBC7(E2)和泛素体外泛素化反应后,通过免疫印迹的方法检测,显示出BoARC1融合蛋白能够将底物进行多泛素化修饰;当U-box中保守位点第323位Pro突变为Ala或其他泛素化组分缺少时,底物不能被泛素化修饰。
ARC1 is a plant-specific protein containing the U-box / ARM domain and plays a role of a positive regulator in Brassica self-incompatibility (SI) signal transduction. The sequence of BoARC1 encoding region of Brassica oleracea var. Acephala was ligated into the prokaryotic expression vector pET-14b. The recombinant plasmid pET-14bBoARC1 was constructed by restriction enzyme digestion and sequencing analysis. The positive expression plasmid was transformed into Escherichia coli The expression strain BL21 (DE3) pLysS was induced by IPTG. SDS-PAGE showed that BoARC1 protein was specifically induced at a molecular weight of 69 kD. BoARC1 fusion protein was obtained by affinity chromatography using Ni2 + -NTA resin. Ubiquitin-activating enzyme (Ubiquitin-binding enzyme UBC7 (E2)) and ubiquitin in vitro ubiquitination reaction by Western blot analysis showed that BoARC1 fusion protein substrate can be multi-ubiquitinated modification; Substrates can not be ubiquitinated when the Pro at position 323 in the U-box is mutated to Ala or other ubiquitinated components are missing.