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根据芸薹属植物钙调素基因保守区域设计引物,采用同源克隆的方法从结球甘蓝自交不亲和系和自交系花粉中克隆得到一个钙调素开放阅读框cDNA序列,该序列长450bp,编码149个氨基酸;编码蛋白不含跨膜区,无信号肽,具有4个完整的EF-hand结构域。构建了结球甘蓝花粉钙调素原核表达系统,钙调素基因及其3个突变体在E.coli中得到表达,均获得分子量约为16kD的可溶性融合蛋白,在EGTA存在的条件下,各融合蛋白具有各自独特的凝胶迁移现象,活性检测表明甘蓝花粉钙调素活性依赖于钙离子。该基因在甘蓝自交不亲和系花粉萌发过程中表达量先上升后下降,在自交系中随花粉萌发增大而降低;在含有钙调素拮抗剂TFP的培养基中,自交不亲和系和自交系花粉中钙调素基因表达量均受到抑制;在含有W-7琼脂糖的培养基中无明显差异。
According to the conserved regions of Brassica calmodulin gene, primers were designed and a cDNA sequence encoding the calmodulin was cloned by homologous cloning from the inbred lines of cabbage and inbred lines. The sequence of the open reading frame Length 450bp, encoding 149 amino acids; encoding protein does not contain transmembrane region, no signal peptide, with four complete EF-hand domain. The prokaryotic expression system of pollen calmodulin was constructed. The calmodulin gene and its three mutants were expressed in E.coli, and the soluble fusion protein with a molecular weight of about 16 kD was obtained. Under the conditions of EGTA, each fusion The proteins have their own unique gel migration phenomena. Activity tests showed that the activity of calmodulin in cabbage pollen is dependent on calcium ion. The expression of this gene firstly increased and then decreased in the process of pollen germination of self-incompatible cabbage and decreased with the increase of pollen germination in inbred lines. In the medium containing calmodulin antagonist TFP, The expression levels of calmodulin gene in pollen of both inbred lines and inbred lines were inhibited, but there was no significant difference in the medium containing W-7 agarose.