Construction of recombinant plasmid through substituting the alchol dehydrogenase B gene of Zymomonas mobilis for the lactate dehydrogenase gene of Streptococcus mutans

Objective To construct a recombinant plasmid through substituting the adhB gene of Zymomonas mobilis for the ldh gene of Streptococcus mutans with a reservation of upstream and downstream homology region of ldh in pMD18-T-ldh.Methods The adhB gene was amplified from total DNA of Zymomonas mobilis by PCR and was cloned in to pGEMT vector.The entire cloned ldh gene of Streptococcus mutans in pMD18-T-ldh was deleted by inverse PCR.Restriction enzymes NcoI and XhoI were used to digest the DNA fragment obtained from inverse PCR amplification.To acquire recombinant plasmid pMDLA,recombinant plasmid pGEMA and ligate the two fragments were performed.Results The adhB was successfully cloned and sequenced.The adh was replaced by adhB.Recombiant plasmids pGEMTA and pMDLA were constructed.Conclusion Results of the study expanded the possible therapeutic use of Zymomonas mobilis;and demonstrated that inverse PCR technique is a quickly and easily method in inducing gene-deletion mutation.