Molecular Cloning,Expression Analysis And Functional Characterization Of A Hexokinase Gene MdHXK1 In Apple

A hexokinase gene named MdHXK1（ MDP0000309677） was cloned from’Gala’ apple（ Malus × domestica Borkh.）. Its bioinformatics and southern blotting were analyzed. Its expression patterns in different tissues and in fruit at different developmental stages as well as in response to various stresses were examined using qRT-PCR. In addition, Glc phosphorylation relative activity of MdHXK1 and glucose concentration were detected during apple fruit development. Recombinant protein was obtained with procaryotic expression, and transgenic Arabidopsis lines that overexpressing MdHXK1 were obtained and used for further investigation. The main results were shown as follows.（1） Sequence analysis showed that the length of MdHXK1 gene was 1497 bp, which encodes 499 amino acids. Key amino acid motifs and residues are very conservative among sequences of AtHXK1, AtHXK2 and MdHXK1. Domain analysis demonstrated that MdHXK1 contained two conserved domains: Hexokinase₁ and Hexokinase₂. Silico analysis suggested that the promoter sequence of MdHXK1 contains defense responsive elements, sugar signaling responsive elements and phytohormone responsive elements.（2） The result of Southern blotting showed that MdHXK1 existed as a single copy in apple genome. MdHXK1 is located on the chromosome 15 and has 9 exons, just like the most homologous genes. The prediction of subcellular localization suggested that MdHXK1 protein is localized in most subcellular structures.（3） qRT-PCR analysis showed that the MdHXK1 gene was mainly expressed in stem and flower. During the development of apple fruits, the expression of MdHXK1 gene, Glc phosphorylation relative activity and glucose concentrations showed the same trend that they increased at first and then decreased. Besides, the expression of this gene was induced by salt stress, low temperature and ABA.（4） Under the treatment of 6% glucose, transgenic Arabidopsis lines overexpressing MdHXK1 exhibited hypersensitivity to glucose, showing severely stunted cotyledon, hypocotyl and root, indicating that MdHXK1 involved in the glucose-mediated regulatory role on seedling growth after seed germination.