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从莲种子发育中期胚cDNA文库中克隆得到的防御素基因cDNA全长561bp,GenBank登录号为EF421192。分析发现该基因234bp的开放读码框编码77个氨基酸的多肽序列,除信号肽序列外与其它不同来源的植物防御素基因有较高同源性,将其命名为NnDefensin。NnDefensin蛋白带有30个氨基酸的信号肽,具有8个半胱氨酸的高度保守结构等典型的植物防御素特征,成熟肽部分含有γ–硫素蛋白功能结构域。在系统进化上NnDefensin与单子叶植物中的粳稻和小麦的同源蛋白,以及车前草同源蛋白亲缘关系较为接近。通过PCR扩增克隆莲胚防御素基因片段并连接到载体pBI121和带有花生油体蛋白种子特异启动子的载体pAhOleo17.8︰GUS中,成功构建了35S启动子控制的植物表达双元载体pBI121-NnDef和种子特异表达双元载体pAhOleo-NnDef,为该基因的功能鉴定及通过基因工程方法提高转基因植物以及种子的抗病能力奠定基础。
The full-length cDNA of defensin gene cloned from the lotus seed embryo cDNA library was 561bp in length and GenBank accession number was EF421192. The 234bp open reading frame (ORF) of the gene encodes a polypeptide sequence of 77 amino acids with high homology with other plant defensin genes except for the signal peptide sequence, and named it NnDefensin. The NnDefensin protein is characterized by a typical plant defensin with a 30 amino acid signal peptide and a highly conserved structure of 8 cysteines. The mature peptide portion contains the γ-thioin functional domain. Phylogenetic relationship between NnDefensin and monocotyledonous japonica and wheat homologous proteins, as well as plantago homologus protein closer relationship. The cloned lotus root defensin gene fragment was amplified by PCR and ligated into the vector pBI121 and the vector pAhOleo17.8: GUS carrying the peanut oil seed protein specific promoter. The 35S promoter-controlled plant expression binary vector pBI121- NnDef and seed-specific binary vector pAhOleo-NnDef laid the foundation for the functional identification of the gene and the improvement of the resistance of transgenic plants and seeds by genetic engineering methods.