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