摘要
目的:克隆、表达恶臭假单胞菌生物降解甲苯的关键酶基因。方法:恶臭假单胞菌WZ-1在含甲苯的MS培养基中培养以鉴定其降解甲苯的能力。设计引物扩增甲苯生物降解的关键酶——甲苯双加氧酶和邻苯二酚1,2双加氧酶基因,PCR扩增目的片段后进行T-A克隆,筛选阳性克隆并鉴定。构建甲苯双加氧酶-pET28a进行体外诱导表达。结果:恶臭假单胞菌WZ-1在含甲苯的MS培养基的生长曲线证明其具有降解甲苯的能力;通过重组技术获得阳性克隆,并以酶切、DNA测序进行鉴定;IPTG诱导后获得重组甲苯双加氧酶的高表达。结论:成功获得了用于降解甲苯的甲苯双加氧酶基因和邻苯二酚1,2双加氧酶基因,并在体外诱导表达了目的蛋白,为后续利用模式生物对苯系物的生物降解建立了实验基础。
Objective： To construct biosensor for the degeneration of toluene, the enzymes genes from Pseudomonas putida were cloned and expressed. Methods： A strain of Pseudomonas putida isolated from the water previously was cultured in MS medium for 24 h to identify the ability of degeneration of toluene firstly. Primers were designed according to toluene dioxygenase and catechol 1, 2-dioxygenase gene sequence. PCR was performed as the template of RDNA from the Pseudomonas putlda, T-A cloning as followed. Positive colonies were identified by restriction enzyme reactions and sequencing. Toluene dioxygenase gene was inserted to pET28a vector and the recombinant protein was expressed by IPTG induction. Results： This strain of Pseudomonas putida was proved high ability of degeneration of toluene. From its gDNA, acquirement of the two important genes in degeneration of toluene were demonstrated, and toluene dioxygenase gene was induced high level of expression in vivo. Conclusion： In this study, we complete in obtaining toluene dioxygenase and catechol 1, 2-dioxygenase gene sequence from pseudomonas putida and inducing the expression of toluene dioxygenase, which is significant for further construction of biosensors.
出处
《温州医学院学报》
CAS
2012年第6期556-560,共5页
Journal of Wenzhou Medical College
基金
浙江省大学生科技创新活动计划(新苗人才计划)(2010R413013)
浙江省教育厅科研基金资助项目(Y200804470)
关键词
恶臭假单胞菌
甲苯
克隆
原核表达
Pseudomonas putida
toluene
clone
prokaryotic expression
