| 摘要: |
| 【目的】敲除布洛芬福斯质粒文库克隆菌株4F6中的邻苯二酚-2,3-双加氧酶基因（C23O），构建其Tn5插入突变体，为布洛芬降解调节因子的深入研究奠定基础。【方法】以构建好的3G7 Tn5突变体菌株为基础，借助pKD46质粒的λ-red同源重组酶，将转座子Tn5从菌株3G7中电击转化至4F6，消除pKD46质粒，敲除4F6菌株中的C23O基因，测定出发菌株3G7、4F6及Tn5插入突变体菌株3G7-G9、3G7-H5、4F6-G9和4F6-H5间位苯环的裂解活性。【结果】42 ℃条件下消除了Tn5插入突变体受体菌4F6中的pKD46质粒，构建了4F6-G9和4F6-H5突变体。Tn5转座子插入突变株3G7-G9、3G7-H5、4F6-G9和4F6-H5的间位苯环裂解活性无显著差异；菌株4F6的间位苯环裂解活性虽明显高于菌株3G7，但也仅有1个C23O基因，其活性差异可能与布洛芬降解调控因子有关。【结论】提供了一种简便有效定位并敲除C23O基因的方法。 |
| 关键词: 可降解布洛芬福斯质粒 Tn5突变体 基因敲除 |
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| 基金项目:国家自然科学基金面上项目(31070453)；陕西省国际科技合作项目(2013KW-29)；西北农林科技大学科技创新专项(QN2011113)；西北农林科技大学国际科技合作项目(A213021311) |
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| Construction of transposon insertion mutants for ibuprofen degradable cloning strains from Fosmid Library |
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WEI Yahong,DUAN Min,XIANG Zhaoju,et al
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| Abstract: |
| 【Objective】The C23O gene was knocked out from ibuprofen clone strain 4F6 and its Tn5 transposon insertion mutants were constructed to provide basis for understanding factors affecting ibuprofen degradation.【Method】Tn5 transposon was transferred from clone 3G7 to 4F6 through electroporation mediated with λ-red homologous recombinase of pKD46 plasmid.After curing pKD46 plasmid and knocking out C23O gene from 4F6,the meta-cleavage activities of Tn5 mutants of 3G7-G9,3G7-H5,4F6-G9 and 4F6-H5 were measured and compared.【Result】The Tn5 mutants of 4F6-G9 and 4F6-H5 were constructed after curing pKD46 plasmid at 42 ℃.No significant differences among Tn5 mutants (3G7-G9,3G7-H5,4F6-G9,and 4F6-H5) were observed from meta-cleavage activity assays.4F6 had higher meta cleavage activity than 3G7 during the ibuprofen and non ibuprofen treatments.Since there was only one C23O gene,the meta-cleavage activity differences between strains 3G7 and 4F6 may due to ibuprofen degradation regulators.【Conclusion】This research provides a simple and efficient method to target and knockout C23O gene. |
| Key words: ibuprofen degradable fosmid Tn5 mutants gene knockout |
