Cloning and prokaryotic expression of hexokinase gene from Dimocarpus longan

【Objective】Hexokinase gene( named Dl HXK) was cloned from longan,Dimocarpus longan cv.Shixia,fruit. The bioinformatics and prokaryotic expression of Dl HXK were analyzed. 【Method 】Total RNA extracted from longan fruit was used as a template. The full length c DNA of the gene was cloned using a combination of RT-PCR and RACE-PCR. Prokaryotic expression vector p ET-32a-Dl HXK was constructed and transformed into Escherichia coli Rosetta( DE3) to induce expression.【Result and conclusion】The full length c DNA of Dl HXK was cloned and its NCBI Gene Bank 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 Dl HXK 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 Dl HXK. Therefore,the full length of Dl HXK has been cloned and its prokaryotic expression vector has been constructed successfully. It was induced to express effectively in E. coli Rosetta( DE3).