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.
SUN Bing-bing
,
ZHANG Wei
,
DUAN Hong-ying
,
LI Wei
,
TIAN Tao
,
ZHANG Li-qun
. Cloning of EnvZ/OmpR Two-component System from Pseudomonas fluorescens 2P24 and Prokaryotic Expression of OmpR[J]. Acta Agriculturae Boreali-Sinica, 2014
, 29(3)
: 41
-45
.
DOI: 10.7668/hbnxb.2014.03.009
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