苹果砧木山定子MbNas1的克隆、表达与亚细胞定位  

Cloning, Expression Analysis and Subcellular Localization of Nicotianamine Synthase Gene 1(Mb Nas1) in Apple Stock Malus domestica

作　　者：杨国慧[1] 石岩[1] 吕冰玉[1] 王冰[1] 于洋[1] 韩德果[1] 

机构地区：[1]东北农业大学园艺学院/农业部东北地区园艺作物生物学与种质创制重点实验室,哈尔滨150030

出　　处：《湖北农业科学》2015年第9期2260-2263,共4页Hubei Agricultural Sciences

基　　金：黑龙江省教育厅科学技术研究项目(12541004)

摘　　要：以苹果砧木山定子(Malus baccata)为试材,根据小金海棠(Malus xiaojinensis Cheng et Jiang)中克隆得到的尼克烟酰胺合成酶基因Mx Nas1(登录号:DQ403256)的全长序列设计特异引物,通过RT-PCR方法从山定子c DNA中克隆到Nas1基因并命名为Mb Nas1,该基因全长为978 bp。预测该基因可编码325个氨基酸,理论等电点为5.26,相对分子质量为36.09 ku。Real-time PCR结果表明,正常供铁(EDTA-Na Fe,40μmol/L)时,该基因在山定子水培苗的新叶、老叶、根、茎的韧皮部中均有表达,在木质部表达量较低;缺铁处理(4μmol/L)时,该基因在根和新叶中的表达加强,第6天达到最高值,之后开始下降;在老叶中表达逐渐减弱;高铁处理(160μmol/L)时,该基因在根和新叶中的表达减弱,在第9天达到最小值,之后有所上升;在老叶中表达逐渐增强。基因枪亚细胞定位试验表明,Mb Nas1蛋白质定位在细胞质膜上。The apple stock Malus baccata was used as test materials, the specific primers were designed according to sequence of Malus xiaojinensis Cheng et Jiang nicotianamine synthase gene MxNas1 （accession number：DQ403256）. Nas1 gene was cloned from cDNA of Malus baccata by RT-PCR method and named as MbNas1. Sequence analysis showed that the MbNasl gene contains a complete sequence of 978 bp, the predicted protein of MbNas1 comprises 325 amino acids with a theoretical isoelectric point of 5.26 and a predicted molecular weight of 36.09 kD. Real-time RT-PCR was operated for investigating the expressions level of the MdNas1 in various Malus baccata tissues. In normal Fe level （EDTA-NaFe,40 μmol/L）, MdNas1 was expressed preferentially in new leaf, old leaf, root, and also in the phloem of stem of Malus baccata seedlings, however, the expression in the xylem was very low. The expression levels of MdNas1 increased in root and new leaf, reached the maximums at 6 d, then decreased slightly when dealt with low Fe stress （4μmol/L）, the expression level of MdNasl in mature leaf decreased gradually. On the contrast, the expression level decreased in root and new leaf when dealt with high Fe stress （160 μmol/L）, reached the minimum at 9 d, then increased slightly, the expression level of MdNas1 in mature leaf increased gradually. The subcellular localization experiment with gene gun showed that MbNas1 protein localized to plasma membrane.

关 键 词：山定子(Malus baccata) 尼克烟酰胺合成酶基因(Mb Nas1) 表达分析 亚细胞定位 

分 类 号：Q7[生物学—分子生物学]