关键词: 甘蓝, 花粉, 钙调素, 克隆, 表达分析

Abstract: 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 significantlywhen 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.

Key words: cabbage, pollen, calmodulin, cloning, expression analysis