羽衣甘蓝ARC1蛋白的原核表达、纯化及泛素连接酶活性分析  

Prokaryotic Expression,Purification and in Vitro Ubiquitination Assay of BoARC1 from Ornamental Kale

作　　者：蓝兴国[1] 李晓屿 杨佳[1] 李玉花[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》2013年第12期2472-2478,共7页Acta Horticulturae Sinica

基　　金：中央高校基本科研业务费专项基金项目(DL13CA13);国家自然科学基金项目(30900115;31070275)

摘　　要：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, which belonging to the plant-specific U-box/ARM protein, acts as a positive mediator of self-incompatibility signaling in Brassica. Here, the BoARC1 coding region sequence was amplified and inserted into the prokaryotic expression vector pET-14b. The pET-14b-BoARC 1 constructs were confirmed by the double restriction enzyme and sequencing analysis. The E. coli BL21 （DE3） pLysS cell was transformed pET- 14b-BoARC 1. SDS-PAGE results showed that the recombinant BoARC 1 fusion protein about 69 kD was induced by IPTG and purification by affinity chromatography using Ni2＋-NTA resin. In vitro ubiquination assays were performed using a yeast E1 enzyme, a E2 enzyme His6-UBC7, ubiquitin and His6-BoARC1. Western blot results showed that His6-BoARC1 mediated the poly- ubiquitination of proteins with anti-ubiquitin antibodies. Further, a point mutation of U-box domain at amino acid position 323 substituting Pro for Ala or omission of any of the components resulted in a loss of protein ubiquitination.

关 键 词：羽衣甘蓝 自交不亲和性 ARC1 原核表达 泛素连接酶 

分 类 号：S681.9[农业科学—观赏园艺]