结球甘蓝花粉膜联蛋白基因的克隆及其表达分析

Molecular Cloning and Expression Analysis of Annexin Gene from Cabbage

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。

Plant annexins constitute a multigene family having suggested roles in a variety of cellular processes. We have isolated and characterized an annexin cDNA of cabbage （Brassica oleracea L. var. capitata L.） gene （BoAnnexin2） encoding annexin proteins using RT-PCR and RACE-PCR after 2-D electrophoresis of cabbage pollen total proteins, which found that armexin protein was downregulated when pollens were germinated. The BoAnnexin2 gene was 1 157 bps, which contained 951 bps open reading frame. The predicted molecular mass of BoAnnexin2 is 36.02 kD with acidic pI of 6.33. At the amino acid level, BoAnnexin2 contains two type II Caz＋ binding domain repeats, there was a GXXXGXS （T） /DXXG motif in the fourth domain. The quantitative RT-PCR revealed that the BoAnnexin2 gene expression in mature pollen is eight times higher than that in germinated pollen. These results indicate that BoAnnexin2 gene may play an important role in cabbage pollen germination.