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ARC1是芸薹属植物自交不亲和(Self-incompatibility,SI)信号复合体下游传递因子,特异性地介导SI信号传递。利用原核表达系统对羽衣甘蓝(Brassica oleracea var.acephala)ARC1的全长蛋白(1~663 aa,BoARC1-F)、UND结构域蛋白(1~279 aa,BoARC1-N)和ARM结构域蛋白(361~663aa,BoARC1-C)进行重组表达。SDS-PAGE结果显示这3种蛋白分别在分子量68、33和38 kD处特异性地诱导表达。利用Ni~(2+)-NTA树脂亲和层析技术获得重组蛋白。将BoARC1-C蛋白免疫小鼠并通过细胞融合技术制备单克隆抗体,获得2株单克隆抗体。交叉反应试验结果显示制备的单克隆抗体具有ARC1识别特异性。通过免疫印迹技术检测BoARC1在不同组织和不同发育阶段柱头中的表达,结果显示BoARC1特异性地在羽衣甘蓝柱头中表达,而且在开花阶段的柱头表达量最高。
ARC1 is a transcription factor downstream of the self-incompatibility (SI) signaling complex of Brassica, which specifically mediates SI signaling. The full-length protein (1 ~ 663 aa, BoARC1-F), UND domain protein (1 ~ 279 aa, BoARC1-N) and ARM domain protein of ARC1 in Brassica oleracea var.acephala 361 ~ 663aa, BoARC1-C) for recombinant expression. The result of SDS-PAGE showed that these three proteins induced expression specifically at the molecular weights of 68, 33 and 38 kD respectively. The recombinant protein was obtained by Ni ~ (2 +) - NTA resin affinity chromatography. Mice were immunized with BoARC1-C protein and monoclonal antibodies were prepared by cell fusion technique to obtain 2 monoclonal antibodies. Cross-reaction test results show that the prepared monoclonal antibody has ARC1 recognition specificity. The expression of BoARC1 in stigma of different tissues and different developmental stages was detected by Western blotting. The results showed that BoARC1 was expressed specifically in stigma head of Brassica oleracea, and had the highest stigma expression in flowering stage.