Pseudomonas putida ND6中萘降解调控基因nahR突变株的构建及萘降解调控机制初探  

Construction of a nahR Gene Knock-out Mutant of Pseudomonas putida ND6 and Exploration of the Regulation Mechanism

作　　者：朱琳[1,2] 李珊珊[3] 胡骁[2] 杨建佳[2] 王振英[1] 赵化冰[2] 

机构地区：[1]天津师范大学生命科学学院,天津300387 [2]武警后勤学院天津市职业与环境危害防治重点实验室,天津300162 [3]西安交通大学环境工程学院,陕西西安710049

出　　处：《武警后勤学院学报（医学版）》2013年第7期577-582,590,共7页Journal of Logistics University of PAP（Medical Sciences）

基　　金：国家自然科学基金(21077132)

摘　　要：【目的】构建Pseudomonas putida ND6中双拷贝萘降解调控基因nahR突变株,初步探讨ND6菌株的萘降解调控模式。【方法】扩增nahR基因的上下游同源片段及卡那霉素抗性基因Km;利用重叠延伸PCR技术将这三个片段连接起来,然后将其克隆到自杀性载体pEX18Tc,得到同源重组载体pEX18Tc-RKm;采用热激法将同源重组载体pEX18Tc-RKm转化到供体菌Escherichia coli S17-1,再利用接合转移的方法将供体菌中的载体转到受体细菌ND6菌株,通过同源重组敲除nahR基因,对突变菌株进行nahR基因调控分析。【结果】成功获得了nahR基因突变株。以萘为唯一碳源的无机盐培养基中,突变株生长明显比野生型延迟;以水杨酸钠为唯一碳源的无机盐培养基中,突变株与野生株生长曲线并无明显差异。以葡萄糖为唯一碳源培养时,野生型ND6中的nahR基因有一定量的本底表达量,加入诱导物水杨酸钠后,其表达水平没有明显的变化。【结论】本研究为深入研究ND6菌株萘降解操纵子调控机制奠定了基础。通过对突变株和野生型的比较研究,我们发现ND6菌株中的萘降解上游操纵子受到NahR蛋白的调控,而下游操纵子不受NahR蛋白的调控,且nahR基因为组成型表达。[ Objective ] To To analyze the regulation model for the mineralization of naphthalene by Pseudomonas putida ND6 via the construction of regulatory gene nahR-deficient strain. [Methods]Firstly, the up and downstream homologous recombinant fragments of nahR gene and kanamycin resistance gene were amplified. Secondly, the three fragments were assembled together and cloned into suicide vector pEX18To. Finally, the homologous recombinant vector was introduced into Escherichia cell S17-1 by thermal stimulation method and then transformed into Pseudomonas putida ND6 by conjugation. After knocking out nahR gene by homologous recombination, we can analyze the regulation model of nahR gene. [Results] The nahR-mutated strain was successfully constructed. Mutant strain ND6- △ △ R exhibited an obvious growth delay comparing with the wild-type strain, when cultured in naphthalene-supplemented mineral media. The growth curve of mutant strain NDr- △ △ R is quite similar with the wild-type when cultured in salicylate-supplemented mineral media. The uahR gene expressed constitutively when cultured in glucose-supplemented mineral media, and the expression level showed almost no increase when the salicylate was added. [Conclusion]The study laid a good foundation for further research on operon regulation model for the mineralization of naphthalene by ND6 strain. It could be deduced that the NahR protein only regulates naphthalene degradation upstream operon of ND6 strain by comparison of the growth curve and nahR gene expression level between NDr-△ △R mutant and wild-type. The nahR transcription was constitutive.

关 键 词：PSEUDOMONAS PUTIDA nahR基因 突变株 重叠延伸PCR技术 

分 类 号：R459.6[医药卫生—治疗学]