| 多聚磷酸盐激酶基因在污水生物除磷中的功能 |
| 摘要点击 1785 全文点击 825 投稿时间：2016-06-23 修订日期：2016-11-14 |
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| 中文关键词 污水生物除磷 大肠杆菌 无痕敲除 多聚磷酸盐激酶 除磷性能 |
| 英文关键词 biological phosphate removal in waste water E coli. scarless gene deletion polyphosphate kinase (PPK) phosphate removal characteristics |
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| 中文摘要 |
| 为验证多聚磷酸盐激酶基因（ppk）在污水生物除磷中的功能.采用Red敲除系统，以pKD4质粒为模板，设计同源短臂，扩增外源线性DNA片段，将外源线性DNA片段电转化整合入已导入pKD46的大肠杆菌ATCC25922野生型菌株.获得重组菌E.coli/ppk- Kan+.将pCP20导入大肠杆菌E.coli/ppk- Kan+以消除卡那霉素抗性基因，通过负抗性筛选及正反向引物验证，构建无抗生素抗性的ppk基因缺失工程菌株E.coli/ppk- Kan-.比较工程菌株和野生型菌株的生长特性，并比较两者在缺磷诱导/富磷及多次厌氧/好氧诱导条件下的除磷性能.结果表明采用Red重组系统，通过无痕敲除，成功构建了大肠杆菌ppk基因缺失菌株E.coli/ppk- Kan-.敲除后的工程菌株和野生型菌株生长整体没有差异，但是4 h前对数期工程菌株生长快于野生型菌株，8 h后稳定期工程菌株生长慢于野生型菌株，表明ppk影响菌体的生长；缺磷诱导/富磷条件下，工程菌株并未表现出因ppk缺失而影响其除磷能力；经过5次厌氧/好氧诱导，两菌菌体含磷量保持在1%~2%，没有因诱导次数的增加而表现出菌体含磷量增加的趋势，也未发现厌氧有PHB好氧有聚磷颗粒生成，表明ppk基因的缺失并没有引起菌体除磷能力的下降.ppk并未表现出明显的与污水生物除磷相关的功能. |
| 英文摘要 |
| This study aimed to identify the function of polyphosphate kinase gene (ppk) in phosphorus removal. With the Red system, the target DNA with the homologous short arms was amplified in the plasmid pKD4. Then the target DNA was transformed into E. coli ATCC25922 which already had the suicide plasmid pKD46 by electroporation. The plasmid pCP20 was transformed into the recombinant strains to delete the kanamycin resistance gene. With the screening by negative resistance, together with verification using positive and negative primers, the construction of ppk gene deletion strain E. coli/ppk- Kan- was confirmed. The growth characteristics of both the wild-type strain and the mutant strain were determined, and the phosphate accumulating characteristics were compared when cultured in the phosphate luxuriant medium after induced in the phosphate lacking medium. Also the phosphate accumulating characteristics of the two strains were compared after cultured in the anaerobic and aerobic alternating conditions for 5 times. The results showed that the ppk deletion strain E. coli/ppk- Kan- was successfully constructed. There was no growth difference between the mutant strain and the wild-type strain. But in the first 4 hours of log phase, the mutant strain grew faster than the wild-type strain. And 8h later, when both strains were in stationary phase, the mutant strain grew slower than the wild type, indicating that ppk affected the growth of the bacteria. Cultured in the phosphate lacking medium and the phosphate luxuriant medium, the mutant strain's ability of phosphate accumulating didn't decrease in spite of having no ppk gene. After 5 times induction, the amounts of phosphorus in both strains were about 1%-2%. The phosphate amounts in the cells did not increase with increasing inducing times. Polyphosphate or PHB was detected neither at anaerobic phase nor at the aerobic phase. It indicated that the deletion of ppk did not affect the phosphorus removal in wastewater treatment process, and the ppk gene did not show the function of phosphorus removal. |
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