Cloning and prokaryotic expression of hexokinase gene from Dimocarpus longan

Objective Hexokinase gene (named DlHXK) was cloned from longan, Dimocarpus longan cv. Shixia, fruit. The bioinformatics and prokaryotic expression of DlHXK were analyzed.

Method Total RNA extracted from longan fruit was used as a template. The full length cDNA of the gene was cloned using a combination of RT-PCR and RACE-PCR. Prokaryotic expression vector pET-32a-DlHXK was constructed and transformed into Escherichia coli Rosetta (DE3) to induce expression.

Result and conclusion The full length cDNA of DlHXK was cloned and its NCBI GeneBank accession number was KF776906.1, which has 2 101 bp nucleotides including a 1 488 bp ORF. It was predicted to encode 495 amino acids. It had the highest homology with the Citrus unshiu (82%) through the NCBI and evolutionary tree analysis. The results of quantitative RT-PCR showed that DlHXK gene expression increased gradually during the longan fruit maturation. Induced by IPTG and determinated by SDS-PAGE, the inducing expressed recombinant protein from prokaryotic expression vector was consistent with the putative protein of DlHXK. Therefore, the full length of DlHXK has been cloned and its prokaryotic expression vector has been constructed successfully. It was induced to express effectively in E. coli Rosetta (DE3).