作　　者： ; ; ; ; ;

机构地区： 中山大学生命科学学院

出　　处： 《园艺学报》 2010年第12期1975-1982,共8页

摘　　要： 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 lotus（Nelumbo nucifera Gaertn）seed defensin gene was identified from the lotus cDNA library of middle development stage seed embryo. The 561 bp full length cDNA have a 234 bp open reading frame which encoding a 77 amino acid peptide and was designated as NnDefensin. The amino acid sequence of NnDefensin showed relatively high match with other plant defensin genes and phylogeny analysis showed that NnDefensin have close relationship with defensin from Oryza sativa japonica group, defensin precursor from Triticum aestivum and defensin from Plantago major. NnDefensin have a strictly conserved γ- thionin domain of eight cysteine which is characteristic in plant defencin. Specific primers containing restriction enzyme site of SmaⅠ and SacⅠ was used to amplify the sequence of NnDefensin by polymerase chain reaction（PCR）. The binary expression vector pBI121 and vector pAhOleo17.8︰GUS which containing a seed specific promoter were digested by the corresponding restricted enzymes respectively,and linked with the NnDefinsin fragment directionally. The resulting construction was obtained and named pBI121-NnDef in which NnDefensin was driven by 35S promoter,and pAhOleo-NnDef in which NnDefensin was driven by seed specific promoter AhOleo17.8 from peanut（Arachis hypogaea L.）. The vectors could be transformed into plants mediated by Agrobacterium tumefaciens for the following up research such as gene functional identification and gene transformation for pathogen resistant improvement in transgenic plants and seeds.

关 键 词： 莲 防御素 载体构建 种子特异

领　　域： [农业科学] [农业科学] [生物学]