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

作　　者：武俊[1] 许敬亮[1] 洪青[1] 李顺鹏[1] 

机构地区：[1]南京农业大学生命科学学院,农业部农业环境微生物工程重点开放实验室,南京210095

出　　处：《微生物学报》2008年第1期45-50,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(40471073and30400013);国家“863计划”[2003AA241150,2004AA246070,2004(249)and2004(514)];江苏省科技厅高技术研究项目(BE2002345,BE2003343,JHZD06-2andBG2005322)

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0.05μg/mL和5μg/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。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 （Km^t） to get a novel transposon vector pUTImini-Tn5-1inA. With the helper plasmid RK600, the transposon vector pUT/mini-Tn5-1inA 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.

关 键 词：脱氯化氢酶基因linA 转座载体pUT/mini-Tn5 多功能工程菌 

分 类 号：X172[环境科学与工程—环境科学]