Cloning of EnvZ/OmpR Two-component System from Pseudomonas fluorescens 2P24 and Prokaryotic Expressi

来源 :中国植物病理学会2014年学术年会 | 被引量 : 0次 | 上传用户:sgcc7186
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  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) is disrupted in mutant strain Sesu-25.A DNA fragment harbouring an intact EnvZ/OmpR TCS was obtained by subcloning.The envZ and ompR of P.fluorescens 2P24 shared an 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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