摘要
【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.
出处
《微生物学报》
CAS
CSCD
北大核心
2009年第7期972-977,共6页
Acta Microbiologica Sinica
关键词
谷氨酸棒杆菌
启动子探测载体
启动子
氯霉素乙酰转移酶
Corynebacterium glutamicum
promoter-probe vector
promoter
chloramphenicol acetyltransferase