用Mini-Tn5转座子随机突变荧光假单胞菌野生型菌株2P24，得到抗生素2，4二乙酰基间苯三酚(2，4-diacetylphloroglucinol，2，4-DAPG)产量提高的突变体Sesu-25。对转座子侧翼序列分析表明，转座子插入到EnvZ/OmpR双因子系统的反应调节因子 ompR 基因。克隆得到包含完整EnvZ/OmpR系统，全长约为5.9 kb的DNA片段。该双因子系统中的 envZ 基因和 ompR 基因与 P.fluorescens F113的 envZ 基因和 ompR 基因同源性分别为93%和96%。将 ompR 基因克隆到表达载体pET-22b(+)中，得到重组表达载体pET-ompR。将pET-ompR转入 Escherichia coli BL21中得到重组菌株BL21-ompR，用IPTG诱导表达和亲和柱层析法纯化的OmpR蛋白，经SDS-PAGE电泳分析表明 ompR 基因得到成功表达和纯化。
孙冰冰
,
张伟
,
段红英
,
李伟
,
田涛
,
张力群
. 生防荧光假单胞菌2P24中EnvZ/OmpR双因子系统克隆与OmpR原核表达[J]. 华北农学报, 2014
, 29(3)
: 41
-45
.
DOI: 10.7668/hbnxb.2014.03.009
Using the colour of colony as the selective marker, the random mutagenesis of mini-Tn5 was performed to wild-type biocontrol strain Pseudomonas fluorescens 2P24 for the screening of mutants, which might have altered yields of polyketide metabolite 2, 4-diacetylphloroglucinol(2, 4-DAPG).The strain Sesu-25 with a increased yield of 2, 4-DAPG was screened from the mutant pool.The analysis of flank sequence of transposon implied that the response regulator OmpR of EnvZ/OmpR two-component regulatory system(TCS) was disrupted in mutant strain Sesu-25.A DNA fragment harbouring a intact EnvZ/OmpR TCS was obtained by subcloning.The envZ and ompR of P.fluorescens 2P24 shared a identity of 93% and 96% with P.fluorescens F113, respectively.The ompR was ligated into the expression vector pET-22b(+) to obtain the recombinated plasmid pET-ompR, and the plasmid pET-ompR was transformed into E.coli BL21 to generate the strain BL21-ompR.The His-taged OmpR was purified by affinity chromatography and verified by SDS-PAGE electrophoresis analysis.
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