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对结球甘蓝(Brassica oleracea L.var.capitata L.)花粉总蛋白双向电泳及差异蛋白质谱分析,发现膜联蛋白在花粉萌发后较萌发前表达下调。通过同源扩增和RACE等方法首次在结球甘蓝花粉中扩增得到BoAnnexin2基因的cDNA序列,该基因cDNA全长1157bp,开放阅读框为951bp,编码316个氨基酸残基,预测分子量为36.02kD,等电点6.33。BoAnnexin2编码蛋白C端有4个膜联蛋白重复序列,共2个Ⅱ型钙结合区域,在第4个钙结合区位点包含GXXXGXS(T)/DXXG基序。实时荧光定量试验表明,BoAnnexin2在甘蓝成熟未萌发花粉中的表达量是萌发45min后表达量的8倍,表明BoAnnexin2在甘蓝花粉萌发起始阶段起到重要调控作用。
Two-dimensional gel electrophoresis and differential proteomic analysis of total protein in Brassica oleracea L.var.capitata L. showed that annexin was downregulated before germination after pollen germination. The cDNA sequence of BoAnnexin2 was amplified by homologous amplification and RACE for the first time. The cDNA of BoAnnexin2 is 1157bp in length and 951 bp in open reading frame (ORF) encoding 316 amino acid residues with a predicted molecular mass of 36.02 kD , Isoelectric point 6.33. BoAnnexin2 encodes a protein with four annexin repeats at the C-terminus and a total of two type II calcium-binding domains, including the GXXXGXS (T) / DXXG motif at the fourth calcium binding site. Real-time fluorescence quantitative assay showed that the expression level of BoAnnexin2 in mature and non-germinating pollens of Brassica oleracea was eight times of that in 45 min after germination, indicating that BoAnnexin2 plays an important regulatory role in the initial stage of pollen germination.