关键词: 苹果, 己糖激酶, HXK1, 基因克隆, 表达分析, 原核表达

Abstract: A hexokinase gene named MdHXK1（MDP0000309677）was cloned from‘Gala’apple （Malus × domestica Borkh.）. Sequence analysis showed that the length of MdHXK1 gene was 1 497 bp，which encoded 499 amino acids. It was predicted that the molecular mass of this protein was 54.05 kD，and pI was 5.76. A phylogenetic tree indicated that the HXK1 had a higher sequence conservation among different species，while apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that MdHXK1 protein was mainly localized in cytoplasm. There was an indication that MdHXK1 existed as one copy in apple genome by Southernblotting. In silico analysis suggested that the promoter sequence contained several typical cis-acting elements，including defense responsive elements，sugar signaling responsive elements and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower. During the development of apple fruits，the expression of MdHXK1 gene increased at first and then decreased. The changes on Glc phosphorylation activity（relative）and glucose concentration showed the same trend. Besides，the expression of this gene was induced by salt stress，low temperature and ABA. Finally，we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression，which established the foundation for the further study on the functions of MdHXK1 protein.

Key words: apple, hexokinase, MdHXK1, molecular clone, expression analysis, prokaryotic expression