假单胞菌M18rsmA^-突变株的构建及其对Plt和PCA合成的区别性调控作用

Construction of Pseudomonas fluorescent sp. M18 rsmA^- Mutant and Its Different Regulation on Biosynthesis of PCA and Plt

假单胞菌 (Pseudomonassp .)M18是促进植物生长的根际细菌 ,能产生吩嗪 1 羧酸 (PCA)和藤黄绿菌素 (Plt)两种不同的抗生素抑制植物病原菌 ,保护植物免受病害。运用PCR方法 ,从M18基因组中 ,扩增出rsmA基因部分片段 ,并以该片段为探针 ,从M18的基因组柯斯文库中筛出阳性克隆 ,切取带有rsmA基因及两侧序列的 1 5kb片段 ,中间插入编码Kmr 的DNA片段 ,获得rsmA- 体外突变体。运用同源重组剔除技术 ,构建了M18菌株的rsmA突变株M18R- 。突变株M18R- 生物合成Plt的能力比野生型M18提高 4倍 ,但是 ,PCA产量仅为野生型的 2 0 %。研究结果表明 ,全局性调控基因rsmA可能通过不同的机制区别性地影响Plt和PCA的生物合成。

Pseudomonas fluorescent sp. M18, one of plant-growth-promoting rhizobacteria, can inhibit several soilborn phytopathogens by producing secondary metabolites including phenazine-1-carboxylic acid(PCA)and pyoluteorin (Plt). Partial segment of rsmA was amplified from M18 genome and used as probe to screen M18 gene library, the 1.5kb sequence containing rsmA gene was obtained by digesting one of positive clones, then kanamycin resistant cassette was inserted into rsmA gene to inactivate it in vitro. Through homologous recombination, mutant M18R -was constructed. Result of dynamic analysis shows that Plt yield in the mutant increases four times and PCA is only 20% of the level of wild type. This study suggests that rsmA gene in Pseudomonas fluorescent sp. M18 regulates PCA and Plt production differently.