小金海棠质膜H^+-ATPase基因(MxHA2)的克隆及功能分析  

Cloning and Function Analysis of Plasma Membrane H^+-ATPase Gene (MxHA2) from Malus xiaojinensis

作　　者：槐心体[1] 王忆[1] 张倩[1] 张新忠[1] 韩振海[1] 

机构地区：[1]中国农业大学园艺植物研究所/北京市果树逆境生理与分子生物学重点实验室,北京100193

出　　处：《农业生物技术学报》2012年第10期1135-1142,共8页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(2011AA001204);现代农业产业技术体系岗位专家项目(CARS-28);国家转基因生物新品种培育科技重大专项(2009ZX08009-122B)

摘　　要：植物质膜H+-ATPase能把质子泵出根外酸化土壤,增加铁的溶解度,利于植物对铁的吸收。小金海棠是一个苹果铁高效基因型砧木,为研究质膜H+-ATPase在小金海棠铁吸收过程中的功能,本研究通过同源克隆的方法从小金海棠(Malus xiaojinensis)中克隆到了一条全长2865bp含有完整编码区的质膜H+-ATPase基因(MxHA2),编码954个氨基酸(GenBank登录号:JQ867095)。系统进化分析结果表明MxHA2蛋白与碧桃(Prunus persica)的PPA2蛋白亲缘关系较近。基因枪法在洋葱(Allium cepa)表皮内进行亚细胞定位分析,结果表明,MxHA2与GFP的融合蛋白定位于细胞膜上。荧光定量PCR分析表明,缺铁胁迫后,MxHA2基因在根和叶中均有不同程度的增强。把MxHA2基因转化到酿酒酵母(Saccharomyces cerevisiae)(BJ2168)后,转MxHA2的酿酒酵母H+-ATPase活性极显著增强,且缺铁处理后其生长量持续高于对照,表明该基因编码的蛋白具有H+-ATPase活性,其过量表达能提高酵母菌株的耐缺铁性。结果提示,MxHA2可能在小金海棠的耐缺铁胁迫过程中起重要作用,为进一步研究小金海棠的铁高效机理提供了依据,在果树耐缺铁分子育种上有重要的应用前景。In plants, plasma membrane H^＋-ATPase can mediate proton excretion into the rhizosphere to acidify the soil, increase the solubility of iron and be beneficial to the iron absorption for plants. In order to research function of MxHA2 gene in the process of iron absorption in Malus xiaojinensis, which is a high Fe-utility-efficiency apple genotype, the full length of the MxHA2 gene was cloned from Malus xiaojinensis based on homologous sequence from online Blast results. The MxHA2 gene had an completed ORF（open reading flame） of 2 865 bp, encoding 954 amino acids （GenBank accession No. JQ867095）. Phylogenetic tree analysis revealed that MxHA2 protein had a closer relationship to PPA2 of Prunus persica. Subcellular localization assay of the MxHA2 protein, which used transient expression of MxHA2：：GFP fusion protein in onion （Allium cepa） cells by particle bombardment, revealed plasma membrane localization of the MxHA2 protein. The expression profiles of the MxHA2 gene were analyzed with Real-time PCR in leaves and roots. The results showed that the MxHA2 gene was expressed both in roots and leaves. And the MxHA2 gene expression was increased by 3 and 6-fold in roots after imposing Fe deficiency. While in the leaves, the MxHA2 gene expression was increased by 2-fold after imposing Fe deficiency for 3 d, continued until 6 d. In order to study the function of MxHA2 gene, we transformed MxHA2 gene into Saccharomyces cerevisiae（BJ2168）. The plasma membrane H^＋-ATPase activity of transgenic yeast was increased very significantly in normal medium, and the growth of transgenic yeasts was higher than controls continually in Fe-deficiency medium. The results obtained in this study suggested that MxHA2 protein had H^＋-ATPase activity, and its high level expression could improve the Fe-deficiency resistance of Saccharomyces cerevisiae. These results image that MxHA2 gene may play an important role in the process of resistance to iron deficiency stresss in Malus xiaojinensis and have an important applic

关 键 词：小金海棠 缺铁胁迫 表达分析 亚细胞定位 酵母转化 

分 类 号：S661.1[农业科学—果树学]