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S位点受体激酶(SRK)和臂重复蛋白(ARC1)是芸薹属植物自交不亲和反应的两种重要的信号元件,SRK-ARC1之间的互作决定着自交不亲和反应的进程。本研究利用PCR技术获得结球甘蓝(Brassicaoleracea var.capitata)SRK激酶结构域(SRKJ)的编码区以及羽衣甘蓝(B.oleracea var.acephala)ARC1基因不同片段长度的基因组DNA与编码区,分别构建以pGBKT7为载体的长度依次递减的ARC1a、ARC1b、ARC1c和ARC1d的重组“诱饵”质粒和以pGADT7为载体的SRKJ的重组猎物质粒,并将重组质粒转化酵母菌株进行蛋白质相互作用检测。结果显示:羽衣甘蓝ARC1存在5个连续的臂重复区,与结球甘蓝ARC1的氨基酸序列一致性达到98%;构建的重组表达载体在酵母细胞中无毒性和自激活作用产生;3个实验组Y2HGold[pGBKT7-ARC1a]×Y187[pGADT7-SRKJ]、Y2HGold[pGBKT7-ARC1c]×Y187[pGADT7-SRKJ]和Y2HGold[pGBKT7-ARC1d]×Y187[pGADT7-SRKJ]同时激活了4种报告基因AUR1-C、MEL1、HIS3和ADE2;表明羽衣甘蓝ARC1与结球甘蓝的SRK激酶结构域能够发生相互作用,且互作的区域位于连续的臂重复区,其与结球甘蓝ARC1的氨基酸差异不足以引起互作区正确构象的改变。研究结果为芸薹属植物自交不亲和反应的分子机理提供新内容。
S-site receptor kinase (SRK) and arm repeat protein (ARC1) are two important signaling elements in the self-incompatibility response of Brassica. The interaction between SRK-ARC1 determines the self-incompatibility The reaction process. In this study, the coding region of the SRKJ domain of Brassica oleracea var. Japonica var. Japonica var. Japonica var. Japonica was obtained by PCR and the genomic DNA and coding region of different fragments of the ARC1 gene of B. oleracea var. Acephala were constructed respectively The recombinant prey plasmids of ARC1a, ARC1b, ARC1c and ARC1d with pGBKT7 as the decreasing length and SRKJ with pGADT7 as the carrier were constructed. The recombinant plasmids were transformed into yeast strains for protein interaction detection. The results showed that there were 5 consecutive arm repeat regions in ARC1 and 98% identity with ARC1 in Brassica oleracea. The constructed recombinant expression vector was non-toxic and self-activating in yeast cells. Three experimental groups Y2HGold [pGBKT7-ARC1a] × Y187 [pGADT7-SRKJ], Y2HGold [pGBKT7-ARC1c] × Y187 [pGADT7-SRKJ] and Y2HGold [pGBKT7-ARC1d] × Y187 [pGADT7-SRKJ] activated four reporter genes AUR1- C, MEL1, HIS3 and ADE2, respectively. It is indicated that the koala ARC1 interacts with the SRK kinase domain of the cabbage and the interaction region is located in the continuous arm repeat region, which is not enough to cause the amino acid difference with the ARC1 of cabbage Changes in the correct conformation of the area of interaction. The results provide new information on the molecular mechanism of self-incompatibility of Brassica plants.