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  

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

作　　者：孙冰冰[1] 张伟[2] 段红英 李伟[1] 田涛[1] 张力群[2] 

机构地区：[1]天津市植物保护研究所,天津300381 [2]中国农业大学植物病理学系,北京100193 [3]天津市西青区植物保护站,天津300380

出　　处：《华北农学报》2014年第3期41-45,共5页Acta Agriculturae Boreali-Sinica

基　　金：天津市自然科学基金重点项目(12JCZDJC23100);国家高技术研究发展计划项目(2011AA10A205);天津市农科院院长基金项目(11004;13011)

摘　　要：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。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.

关 键 词：荧光假单胞菌 随机突变 EnvZ/OmpR 双因子系统 

分 类 号：Q785[生物学—分子生物学]