遗传稳定型六六六、多菌灵降解基因工程菌构建（英文）

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

摘要:

通过PCR的方法从六六六降解菌Sphingomonas sp. BHC-A 扩增出完整的脱氯化氢酶基因linA。将其克隆到含有mini-Tn5 的自杀性质粒pUT4K上, 构建成质粒pUT/mini-Tn5-linA. 通过三亲杂交, 在辅助质粒RK600的帮助下, 将pUT/mini-Tn5-linA 转移到一株高效降解多菌灵菌株Rhodococcus sp. DJL-6 中。利用mini-Tn5的转座作用将linA基因整合到DJL-6的染色体DNA上, 得到工程菌株DJL-6A。该工程菌具有同时降解多菌灵和六六六的功能, 且对于初始浓度为0.05 μg/mL 和 5 μg/mL 的六六六的降解活性与亲本菌株BHC-A相当。在不加任何选择压力的条件下工程菌株进行连续传代, 结果证明linA基因可以持续稳定的存在于宿主的染色体DNA上。

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.