| 聚磷激酶基因在假单胞菌中的整合和表达 |
| 摘要点击 2668 全文点击 1971 投稿时间：2008-11-13 修订日期：2009-01-16 |
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| 中文关键词 聚磷 聚磷激酶（PPK） 三亲接合 恶臭假单胞菌 |
| 英文关键词 poly-phosphate poly-phosphate kinase(PPK) triparetal conjugation Pseudomonas putida |
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| 中文摘要 |
| 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 putida KT2440，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 putida KT2440-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. |
| 英文摘要 |
| In order to construct high accumulating-phosphate microorganism, the ppk gene from E.coliwas 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 3.05 mg/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. |
