Abstract: In order to study the regulation mechanism of hexokinase Ⅱ (HK Ⅱ) to mitochondrial permeability transition pore (MPTP) opening,a cDNA encoding HK Ⅱ protein was isolated from Feicheng peach fruits by RT-PCR and rapid amplification of cDNA ends (RACE).Induced by IPTG and determinated by SDS-PAGE,the expression vector pET-30a-HK Ⅱ was expressed by BL21.The HK Ⅱ protein was purified through nickel chelating affinity chromatography.The full-length cDNA of HK Ⅱ was 1 835 bp including 1 496 bp of coding sequence encoding a polypeptide of 497 amino acids.The predicted protein molecular formula of HK Ⅱ was C₂₃₇₉H₃₈₄₆N₆₅₂O₇₁₇S₁₇,containing 7 611 atoms.For HK Ⅱ,the molecular weight was 53.6 kDa,the theoretical isoelectric point was about 6.17,and the total average hydrophobicity index was 0.023,which indicated that HK Ⅱ protein was a hydrophobic protein.TMHMM transmembrane structure predicted that HK Ⅱ was a transmembrane protein with 1 transmembrane,most of which were outside the membrane.According to the results of the two level prediction,the HK Ⅱ protein was composed of 15 segments of α helix and 11 segments of β folding and the irregular curl.The three level structure of peach HK Ⅱ protein in Feicheng showed a V font,there were a large number of folded structure.It showed the highest homology with the loquat (96.98%) through the phylogenetic tree and multiple sequences alignment analysis.The high homology of amino acids of HK in peach fruit and other plants,indicating that HK Ⅱ is more conservative.The results would lay a foundation for further analysis of the structure and function of HK Ⅱ protein to explore the mechanism of HK Ⅱ regulation of mitochondrial MPTP.

Key words: Feicheng peach fruits, Hexokinase, Gene cloning, Bioinformatics analysis, Prokaryotic expression

CLC Number: 

• S662.03

• Q78