甘蓝型油菜AnnBn1基因的克隆和表达分析

摘要

利用电子克隆和RT- PCR技术从甘蓝型油菜抗旱品系Q2中克隆了膜联蛋白(annexin)基因,命名为AnnBn1(GenBank登录号HM244482),并对其进行了表达分析.AnnBn1的开放阅读框长度为954bp,编码317个氨基酸.序列分析推测AnnBn1基因编码的氨基酸序列含有4个重复的结构域及钙离子结合位点,与拟南芥、番茄、棉花和玉米等物种的膜联蛋白具有较高的同源性.荧光定量PCR结果发现AnnBn1基因在Q2的根、茎、叶、芽等组织中均有表达,而且表达量基本相同.对3叶期幼苗进行10％ PEG(聚乙二醇)溶液模拟干旱时发现,干旱胁迫后30h内,AnnBn1基因在茎、芽中的表达量升高了2～4倍,而在叶、根中显著升高了10倍以上.AnnBn1基因表达峰值也具有时空特点,干旱胁迫后20h茎和芽中表达量高,而在30h之后叶和根中表达量高.%An annexin coding gene, designated as AnnBnl ( GenBank assession number HM244482 ) , was cloned from Brassica napus L. var Q2 (tolerant to drought) by in silico cloning and RT - PCR ( reverse transcription PCR). The open reading frame (ORF) of AnnBnl was 954bp, encoding 317 amino acid residues. The deduced a-raino acid of AnnBnl have four repeated domains and a calcium binding site, and were highly similar to annexin from other species (I. E. Arabidopsis thaliana, Lycopersicon esculentum, Gossypium hirsutum and Zea mays) . Expression patterns of AnnBnl were studied by real - time quantitative PCR. Result showed that AnnBnl was expressed in leaf, root, stem and shoot at a similar level. However, when the seedling roots were under drought stress [imitated by 10% polyethylene glycol (PEG) soak at 3 —leaf seedling stage] , AnnBnl expressions increased 2 to 4 folds in stem and shoot and 10 folds in leaf and root within 30h. The expression peaks occurred at 20h in stem and shoot and more than 30h in leaf and root.