农业生物技术学报2012，Vol.20Issue(10)：1135-1142,8.DOI:10.3969/j.issn.1674-7968.2012.10.005
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 (MxHA 2) from Malus xiaojinensis
摘要
Abstract
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 MxHA 2 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 MxHA 2 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 MxHA 2 gene, we transformed MxHA 2 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 application prospect for the molecular breeding of resistance to iron deficiency in fruit trees. Besides this study provides foundation for further study of the mechanism of iron efficient in Malus xiaojinensis.关键词
小金海棠/缺铁胁迫/表达分析/亚细胞定位/酵母转化Key words
Malus xiaojinensis,Iron-deficiency stress,Expression analysis,Subcellular localization,Yeast transformation引用本文复制引用
槐心体,王忆,张倩,张新忠,韩振海..小金海棠质膜H+-ATPase基因(MxHA2)的克隆及功能分析[J].农业生物技术学报,2012,20(10):1135-1142,8.基金项目
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)和国家转基因生物新品种培育科技重大专项(2009ZX08009-122B) （863）
