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园艺学报 ›› 2013, Vol. 40 ›› Issue (12): 2472-2478.

• 观赏植物 • 上一篇    下一篇

羽衣甘蓝ARC1 蛋白的原核表达、纯化及泛素连接酶活性分析

蓝兴国, 李晓屿, 杨 佳, 李玉花   

  1. (东北林业大学生命科学学院,哈尔滨 150040)
  • 出版日期:2013-12-25 发布日期:2013-12-25

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

LAN Xing-Guo , LI  Xiao-Yu, YANG   Jia, LI  Yu-Hua   

  1. (College of Life Sciences,Northeast Forestry University,Harbin 150040,China)
  • Online:2013-12-25 Published:2013-12-25

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-14b-
BoARC1 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。

关键词: 羽衣甘蓝, 自交不亲和性, ARC1, 原核表达, 泛素连接酶

Abstract: 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-BoARC1 constructs
were confirmed by the double restriction enzyme and sequencing analysis. The E. coli BL21(DE3)pLysS
cell was transformed pET-14b-BoARC1. SDS-PAGE results showed that the recombinant BoARC1 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 polyubiquitination
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.

Key words: Brassica oleracea var. acephala, self-incompatibility, ARC1, prokaryotic expression, ubiquitin ligase E3

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