降解布洛芬克隆菌株转座子插入突变体的构建  

Construction of transposon insertion mutants for ibuprofen degradable cloning strains from Fosmid Library

作　　者：卫亚红[1,2,3] 段敏[1] 向照举[4] 曲东[5] 王瑶[1] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学旱区作物逆境生物学国家重点实验室,陕西杨凌712100 [3]西北农林科技大学旱区生物质能研究中心,陕西杨凌712100 [4]陕西师范大学生命科学学院,陕西西安710062 [5]西北农林科技大学资源环境学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2016年第4期135-141,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：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)

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3G7Tn5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。[objective] The C230 gene was knocked out from ibuprofen clone strain 4F6 and its Tn5 transposon insertion mutants were constructed to provide basis for understanding factors affecting ibupro- fen degradation. [Method] Tn5 transposon was transferred from clone 3G7 to 4F6 through electroporation mediated with ）,-red homologous recombinase of pKD46 plasmid. After curing pKD46 plasmid and knocking out C230 gene from 4F6, the meta-cleavage activities of Tn5 mutants of 3GT-G9,3GT-H5,4F6-G9 and 4F6- H5 were measured and compared. [Result] The Tn5 mutants of 4F6-G9 and 4F6-H5 were constructed af- ter curing pKD46 plasmid at 42 ℃. No significant differences among Tn5 mutants （3GT-Gg, 3GT-HS,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 C230 gene,the me- ta-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 C230 gene.

关 键 词：可降解布洛芬福斯质粒 Tn5突变体 基因敲除 

分 类 号：Q933[生物学—微生物学]