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为研究甘蓝花粉管钙感应蛋白CaM与SRK相互作用的分子机理及其可能相互作用的区域,从自交不亲和甘蓝材料‘E1’中分别克隆得到CaM12基因450 bp及S位点受体激酶(SRK7)基因全长序列2 118 bp,并亚克隆得到SRK胞外域(eSRK)和胞内激酶域(iSRK),构建原核表达载体pGEX-CaM12、pCold-eSRK和pCold-iSRK,转化E.coli BL21(DE3)进行原核表达,表达产物纯化后进行体外相互作用,结果表明CaM12能够与SRK7进行相互作用,但作用区域是iSRK7而不是eSRK7。为进一步验证其相互作用,本研究利用酵母双杂交系统,构建pGBKT7-CaM12、pGADT7-eSRK7、pGADT7-iSRK7和pGADT7-SRK7酵母表达载体,转化相应酵母Y2HGold和Y187感受态细胞后未出现自激活和毒性现象,相互作用结果与原核表达检测一致。同时将CaM12的3个EF-hands结构域突变体CaM12-2-、CaM12-23-和CaM12-234-与iSRK7分别构建酵母表达载体pGADT7-CAM12-2-、pGADT7-CAM12-23-、pGADT7-CAM12-234-,检测其相互作用。结果表明CaM12 EF-hands突变体CaM12-2-、CaM12-23-和CaM12-234-在酵母双杂交系统中均不能与iSRK7片段发生相互作用,说明CaM12的EF-hands结构域突变后失去结合Ca2+能力而不能与iSRK7相互作用。该研究可为自交不亲和机理提供新的参考依据。
In order to study the molecular mechanism of interaction between CaM and SRK in pollen tube of Brassica campestris and its possible interaction, 450 bp of CaM12 gene and S-site receptor kinase were cloned from self-incompatibility cabbage material ’E1’ (SRK7) gene was 2 118 bp in length and was subcloned to obtain eukaryotic expression vector (eSRK) and intracellular kinase domain (iSRK). The prokaryotic expression vectors pGEX-CaM12, pCold-eSRK and pCold-iSRK were constructed and transformed into E.coli BL21 (DE3) for prokaryotic expression. The expressed product was purified and then interacted in vitro. The results showed that CaM12 could interact with SRK7, but the active region was iSRK7 but not eSRK7. In order to further verify their interaction, the yeast two-hybrid system was used to construct yeast expression vectors pGBKT7-CaM12, pGADT7-eSRK7, pGADT7-iSRK7 and pGADT7-SRK7. After self-activation of Y2HGold and Y187 competent cells, Toxicity, the results of the interaction with the prokaryotic expression test. At the same time, three EF-hands domain mutants CaM12-2-, CaM12-23- and CaM12-234- of CaM12 and iSRK7 were used to construct yeast expression vectors pGADT7-CAM12-2-, pGADT7-CAM12-23-, pGADT7- CAM12-234-, to detect their interaction. The results showed that CaM12 EF-hands mutant CaM12-2-, CaM12-23- and CaM12-234- could not interact with iSRK7 in the yeast two-hybrid system, indicating that the CaM12 EF-hands domain mutated and lost Ca2 + Ability to not interact with iSRK7. This study can provide a new reference for self-incompatibility mechanism.