Cloning,expression and enzymatic characteristics of HpHXK gene in red pitaya(Hylocereus polyrhizus)
[Objective]To analyze the cloning,expression and enzymatic characteristics of hexokinase(HXK)gene,a key enzyme in the hexose phosphorylation glycolysis pathway of Hylocereus polyrhizus,and to provide a theoretical re-ference for studying the mechanism of HXK gene in the accumulation of soluble sugar in fruits and improving the quality of H.polyrhizus.[Method]The mature stem tissues of H.polyrhizus cv.'Zihonglong'and its fruits at 20,23,25,27 and 30 d postanthesis were used as the research objects.The HXK protein sequence was obtained by transcriptome sequen-cing,and the HXK gene was cloned by reverse transcription PCR.The bioinformatics analysis of HpHXK protein was car-ried out by online tools such as ExPASy,Plant-PLoc,Phobius and SMART.The amino acid sequence was multiple aligned by Clustal W,and the phylogenetic tree was constructed by Neighbor-joining(NJ)method of MEGA 7.0.The pGEX-4T-HpHXK vector was constructed and transformed into tobacco leaves.The expression pattern of HpHXK gene in different tissues was analyzed and the enzyme activity of the recombinant protein was measured by colorimetry.[Result]The open reading frame(ORF)of HpHXK gene was 1410 bp in length,encoding 469 amino acid residues.Bioinforma-tics analysis showed that the relative molecular weight of HpHXK protein was 52.47 kD,the theoretical isoelectric point(pI)was 5.42,and the protein had the characteristic domain of HXK.The results of phylogenetic tree showed that HpHXK was clustered with HXKs of rice,Arabidopsis,apple,pear,grape and loquat.The results of real-time fluore-scence quantitative PCR showed that the relative expression of HpHXK gene was the highest in fruit at 20 d postanthesis,which was significantly higher than that in fruit and mature stem at 25,27 and 30 d postanthesis(P<0.05).Subcellular lo-calization results showed that HpHXK protein was localized in the cytoplasm and cell membrane.The results of SDS-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the expression product of the recombinant plasmid was con-sistent with the expected relative molecular weight after induction with isopropylthio-β-D-galactoside(IPTG),indicating that HpHXK recombinant protein was successfully obtained.The affinity of HpHXK protein for glucose Michaelis con-stant(Km=0.61 mmol/L),was higher than that of fructose(Km=2.92 mmol/L).[Conclusion]HpHXK protein has a con-served domain of HXK family.During the fruit development of H.polyrhizus,HpHXK gene negatively regulates the accu-mulation of soluble sugar in fruit and catalyzes the phosphorylation of hexose dominated by glucose in cytoplasm.
pitayaHXK geneexpression characteristicsenzymatic characteristicsvector construction
万方数据
维普
