羽衣甘蓝ARC1的抗体制备及蛋白表达分析

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。

Preparation of Monoclonal Antibodies Against BoARC1 and Expression Analysis of BoARC1 in Ornamental Kale

Self-incompatibility is an effective way to avoid inbreeding depression and promote out-crossing in flowering plants.Armadillo Repeat Containing protein 1 (ARC 1),the downstream factor in SI pathway,can specifically mediated SI pathway.In Brassica oleracea var.acephala,we constructed the fullprotein (1-663aa,BoARC1-F),NUDdomain (1-279aa,BoARC1-N) and ARM domain (361-663 aa,BoARC 1-C) prokaryotic expression plasmids.SDS-PAGE results showed that the BoARC 1-F (68 kD),BoARC1-N (33 kD) and BoARC1-C (38 kD) could be induced by IPTG and purified by affinity chromatography using Ni2+-NTA resin.We prepared the monoclonal antibodies against BoARC1-C through immune mouse and cell fusion technology.Cross reaction analysis showed that the monoclonal antibodies could specific react with BoARC1.The expression level of BoARC1 in various tissue and different developmental stigmas were detected.Western blot results revealed BoARC 1 mainly expressed in stigma and its expression level reached the maximum at opening flower.