Construction of a genetically engineered and stable strain of degrading γ-hexachlorocyclohexane and carbendazim by transposon mini-Tn5

Abstract:

The complete dehydrochlorinase gene linA of a γ-hexachlorocyclohexane (γ-HCH) degrading strain Sphingomonas sp. BHC-A, containing promoter and Shine-Dalgarno sequence (SD sequence), was amplified by PCR. The linA gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Kmr) to get a novel transposon vector pUT/mini-Tn5-linA. With the helper plasmid RK600, the transposon vector pUT/mini-Tn5-linA was introduced into one carbendazim degrading gram-positive strain Rhodococcus sp. DJL-6 by triparental conjugation and then the dehydrochlorinase gene linA was integrated into the chromosome of Rhodococcus sp. DJL-6 by the transposon mini-Tn5. The selected multifunctional genetically engineered strain DJL-6A could degrade γ-HCH and carbendazim simultaneously. The dehydrochlorinase activity of DJL-6A was as strong as that of Sphingomonas sp. BHC-A in 0.05 and 5 μg/mL initial γ-HCH concentration. The linA of the strain DJL-6A was genetically stabile after successive plating DJL-6A for 30 days on nonselective media.