Wu Jun, Xu Jingliang, Hong Qing, Li Shunpeng
Key Lab of Microbiological Engineering Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Wei Sheng Wu Xue Bao. 2008 Jan;48(1):45-50.
The complete dehydrochlorinase gene linA of a gamma-hexachlorocyclohexane (gamma-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 (Km(r)) 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 gamma-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 microg/mL initial gamma-HCH concentration. The linA of the strain DJL-6A was genetically stabile after successive plating DJL-6A for 30 days on nonselective media.
通过PCR扩增了γ-六氯环己烷（γ-HCH）降解菌株鞘氨醇单胞菌属（Sphingomonas sp.）BHC-A的完整脱氯化氢酶基因linA，其包含启动子和Shine-Dalgarno序列（SD序列）。将linA基因插入经NotI酶切的转座子载体pUT/mini-Tn5（Km(r)）中，得到新型转座子载体pUT/mini-Tn5-linA。借助辅助质粒RK600，通过三亲本杂交将转座子载体pUT/mini-Tn5-linA导入一株多菌灵降解革兰氏阳性菌株红球菌属（Rhodococcus sp.）DJL-6中，然后通过转座子mini-Tn5将脱氯化氢酶基因linA整合到红球菌属DJL-6的染色体中。筛选出的多功能基因工程菌株DJL-6A能够同时降解γ-HCH和多菌灵。在初始γ-HCH浓度为0.05和5μg/mL时，DJL-6A的脱氯化氢酶活性与鞘氨醇单胞菌属BHC-A的活性相当。在非选择性培养基上连续传代培养DJL-6A 30天后，该菌株的linA基因遗传稳定。
