λRed技术构建肺炎克雷伯氏菌sRNA-sgrS基因缺失突变株  

Construction and verification of Klebsiella pneumoniae sRNA-sgrS gene deletion mutant strain byλRed homologous recombination technique

作　　者：王雪萌 修宝林 吕雨泽 葛菁萍[1,2] WANG Xue-Meng;XIU Bao-Lin;LV Yu-Ze;GE Jing-Ping(Key Laboratory of Microbiology of Heilongjiang University,School of Life Science,Heilongjiang University,Harbin 150080;Agricultural Microorganisms Technology Education Engineering Research Center,Heilongjiang University,Harbin 150500,China)

机构地区：[1]黑龙江大学生命科学学院微生物黑龙江省高校重点实验室,哈尔滨150080 [2]黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨150500

出　　处：《黑龙江大学工程学报》2020年第4期81-88,共8页Journal of Engineering of Heilongjiang University

基　　金：国家自然科学基金面上项目(31570492);黑龙江省教育厅重点项目(HDJCCX-2016Z05)。

摘　　要：肺炎克雷伯氏菌(Klebsiella pneumoniae)以葡萄糖为底物进行发酵生产2,3-丁二醇(2,3-butanetriol,2,3-BD)的过程中,糖磷酸胁迫非编码小RNA(sugar-phosphate stress sRNA,sRNA-sgrS)通过调控菌体内葡萄糖浓度来解除菌体的应激效应。通过λRed同源重组技术,将构建的同源重组片段sgrS A1-EGFP-sgrS A2,经过酶切、纯化后,通过电转化将重组片段转入到含有pKD46质粒的野生型菌株K.pneumoniae HD79及突变型菌株K.pneumoniae HD79-02(△ldh,△ack)的感受态细胞中。经过荧光显微镜观察筛选阳性转化子,经过PCR验证K.pneumoniae HD79及K.pneumoniae HD79-02 sRNA-sgrS基因缺失重组菌株构建成功。利用λRed技术构建肺炎克雷伯氏菌sRNA-sgrS基因缺失突变株,可为后续探究菌体内外葡萄糖浓度对菌株产量2,3-BD的影响奠定了基础。During Klebsiella pneumoniae fermentation of glucose to produce 2,3-butanediol(2,3-BD),sugar-phosphate stress sRNA(sRNA)relieved the stress effect by regulating the glucose concentration in the bacteria.TheλRed homologous recombination technique was used to construct the recombinant fragment sgrS A1-EGFP-sgrS A2.Then after enzymatic digestion and purification,the fragment was electrically transformed into competent cells of K.pneumoniae HD79 and K.pneumoniae HD79-02 containing pKD46 plasmid.The positive transformants were screened by fluorescence microscopy.The successful construction of sRNA-sgrS gene-deletion recombinant strains were verified by polymerase chain reaction.This research showed that theλRed homologous recombination technique can be used to construct a sRNA-sgrS gene-deletion mutant strain.The constructed mutant K.pneumoniae HD79 can lay a foundation for further research on the effect of glucose concentration in vivo and in vitro on 2,3-BD production.

关 键 词：sRNA-sgrS基因 λRed同源重组技术 基因突变 肺炎克雷伯氏菌 

分 类 号：Q78[生物学—分子生物学]