谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析

Cloning and analysis of promoter-active fragments from Corynebacterium glutamicum 10147

【目的】获得谷氨酸棒杆菌10147基因组中具有启动子活性片段的结构序列,为构建表达载体做准备。【方法】利用启动子探测载体pAKC6,采用鸟枪法克隆经过限制性内切酶Sau3A I完全酶切的谷氨酸棒杆菌10147染色体DNA片段,并测定pAKC6上报告基因编码的氯霉素乙酰转移酶(CAT)的比活力,以筛选有启动子功能的片段。【结果】共克隆到30个具有启动子功能的片段。其中有3个插入片段启动的氯霉素乙酰转移酶比活力大于24 U/mg,插入片段F57启动的CAT比活力为32.50 U/mg;而插入有启动子Ptrc的阳性对照的CAT比活力为26.33 U/mg。【结论】获得3个DNA插入片段具有与已知启动子Ptrc相当的启动活性,这些片段可以用于构建谷氨酸棒杆菌表达载体。

[ Objective ] To clone promoter-active fragments from Corynebacterium glutamicum for further construction of expression vectors. [ Methods] Random Sau3A I digested fragments of C. glutamicum 10147 chromosome were shot-gun cloned into the promoter-probe vector pAKC6 and promoter activity of the inserted fragments was selected by chloramphenicol resistance of transformed C. glutamicum cells. [ Results] Thirty promoter-carrying fragments were isolated. Three C. glutamicum clones harboring pAKC6 with promoter fragments displayed chloramphenicol acetyltransferase （CAT） activity of more than 24 U/mg. The fragment F57 led to the highest CAT activity of 32.50 U/mg, even more than that produced by the promoter Ptre, 26.33 U/mg. [ Conclusion] The strength of promoter on fragments F21, F54 and F57 is as strong as promoter Ptrc in C. glutamicum. These fragments can be used to construct expression vector.