结球甘蓝花粉钙调素基因的克隆与表达分析  

Cloning and Expression Analysis of Calmodulin Gene in Brassica oleracea L. var. capitata L.

作　　者：孙梓健[1] 韦静宜[1] 王小佳[1] 宋明[1] 汤青林[1] 王志敏[1] 任雪松[1] 

机构地区：[1]西南大学园艺园林学院,重庆市蔬菜学重点实验室,南方山地园艺学教育部重点实验室,重庆400715

出　　处：《园艺学报》2012年第4期677-686,共10页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31071802;31000908);重庆市自然科学基金重点项目(2011BA1002);中央高校基本科研业务费专项(XDJK2009C126;XDJK2012B020);教育部博士点基金项目(20090182120003)

摘　　要：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。Calmodulin（CaM）mediates lots of cellular processes in eukaryotes,it is a pivotal transducer of calcium signal. A Brassica oleracea CaM gene was isolated using homology based on the cloning method. The ORF（open reading frame）of this gene was 450 bp encoding 149 amino acids with four conserved EF-hand domains. There is no transmembrane domain or signal peptide sequence in tentative protein sequence. The recombinant CaM proteins（three mutants included）were expressed in E. coli,and subsequently separated on SDS-PAGE in the presence of EGTA,their electrophoresis motilities were related with that of mutant EF-Hand motifs. The activity assay showed that the CaM activity relied on the presence of Ca2＋. Gene expression in the pollen germination was investigated using qPCR,the results indicated that the gene expression increased first and then descended in pollen of self-incompatible line,but decreased continuously in self-compatible line,both of which were down-regulated significantly when TFP was added in pollens;There was no significant difference in the beginning or at the end of pollen germination when W-7 sepharose was added.

关 键 词：甘蓝 花粉 钙调素 克隆 表达分析 

分 类 号：S635.1[农业科学—蔬菜学]