聚磷激酶基因在假单胞菌中的整合和表达

Integration and Expression of Polyphosphate Kinase Gene in Pseudomonas putida

为了构建高效除磷的微生物,将来源于大肠杆菌的聚磷激酶基因(ppk)插入广宿主载体pBBR1MCS-2多克隆位点区,得到质粒pBBR1MCS-2-ppk.以该质粒为模板,通过PCR扩增出携带有载体启动子和终止子序列的ppk基因,插入自杀型质粒pUTmini-Tn5中得到重组质粒pUTmini-Tn5-ppk.pUTmini-Tn5-ppk经三亲接合作用进入Pseudomonas putidaKT2440,同时mini-Tn5通过转座作用将ppk整合到宿主菌株的染色体DNA中,获得基因工程菌Pseudomonas putidaKT2440-PPK,用于表达ppk.RT-PCR结果显示,ppk基因在KT2440-PPK中得到较高量的表达,而在原始菌株KT2440中表达微弱.人工模拟污水实验结果表明,接种1 h时KT2440-PPK中聚磷含量达到最大,为3.05 mg/g,约是对照菌株KT2440的15倍.测定模拟污水中磷酸盐的含量表明,KT2440-PPK可以去除该模拟污水中90%以上的磷酸盐.

In order to construct high accumulating-phosphate microorganism,the ppk gene from E.coli was inserted to the broad-host-range plasmid pBBR1MCS-2 to form plasmid pBBR1MCS-2-ppk.The complete ppk gene with promoter and terminator sequences from pBBR1MCS-2-ppk was then cloned and inserted to suicide plasmid pUTmini-Tn5 to form plasmid pUTmini-Tn5-ppk,which was transformed into Pseudomonas putida KT2440 by triparental conjugation.Finally,ppk gene was integrated into the chromosomal DNA of KT2440.The results of RT-PCR showed that the selected genetically engineered bacterium KT2440-PPK expressed ppk efficiently,while KT2440 as control expressed weakly.The results of artificial wastewater treatment showed after 1h inoculation,the concentration of poly-phosphate in KT2440-PPK came to the maximum approximately 3.05mg/g, which was 15 times higher than that in KT2440 at the same experimental condition.And KT2440-PPK can remove more than 90% phosphate in artificial wastewater.