利用酵母双杂交系统鉴定羽衣甘蓝臂重复蛋白(ARC1)与结球甘蓝S位点受体激酶(SRK)激酶结构域的相互作用

摘要

S-receptor kinase (SRK) and ARM repeat conaining(ARCl) are two important sigal componets, the interaction between SRK and ARC1 determines the process of the self-incopatibility. The sequences of SRK kinase domain (SRKJ) and different-length ARCla, ARClb , ARC1C and ARCld of Brassica oleracea were amplified by PCR, then SRKJ was subcloned into pGADT7 and different ARC1 were subcloned into pGBKT7 vectors. After sequencing, the plasmids were transformed into the yeast cells, and the interaction between the truncated fragments of ARC1 from B. oleracea var. acephala and kinase domain of SRK from B. oleracea var. capitata L were tested by yeast two-hybrid System; The results indicated that there existed five ARM repeats in ARC1, which had 98% similarity at amino acid level between B. oleracea var. acephala L and B. oleracea var. apitata. The recombinant expression vectors did not proform autoactivation and toxicity. Three experimental groups Y2HGold [pGBKT7-ARCla] xY187 [pGADT7-SRKJ], Y2HGold [pGBKT7-ARC1C] xY187 [pGADT7-SRKJ] and Y2HGold [pGBKT7-ARCld]xYl87 [pGADTl-SRKJ] could grow on QDO/x/A nutritional media with transcription activation of the reporter genes A UR1-C, MEL1, HIS3 and ADE2, it indicated that there existed interaction between ARC1 from B. oleracea var. acephala and SRK from B. oleracea var. capitata and the interaction domain was located on ARM repeats, and the fifference at amino acid level with the ARC1 of B. oleracea var. capitata was not enough to change the conformation in the interaction region. All above mentioned provides some insights into the molecular mechanism of self-incompatibility in Brassica.%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(Brasica oleracea 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-A RC1a] ×Y 187 [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.