利用绿色荧光蛋白报告基因筛选植物乳杆菌强启动子

Screening of Strong Promoter in Lactobacillus plantarum with Green Fluorescent Protein Reporter Gene

[目的]结合已知植物乳杆菌启动子和绿色荧光蛋白报告基因,筛选能够进行稳定强表达的启动子,为构建植物乳杆菌高效特异表达载体提供基因元件。[方法]分别合成已知植物乳杆菌启动子P11、Pefp、Ptuf33和Ptuf34及其下游绿色荧光蛋白报告基因,并使用上述合成序列分别替代pMG36e乳酸菌表达载体原有多克隆位点及其上游启动子部分,形成启动子筛选载体。利用电转方法将成功构建的启动子筛选载体转化至植物乳杆菌,通过检测重组子绿色荧光信号强弱对比分析启动子强弱特性。[结果]4个启动子中转录延伸因子TU基因启动子Ptuf33和Ptuf34在受体植物乳杆菌中表达目的基因水平显著(P<0.05)高于其他启动子。[结论]结合该研究结果及相关报道可知,启动子Ptuf33和Ptuf34在植物乳杆菌中普遍高表达,可以为后续高表达植物乳杆菌载体构建提供元件。

[Objective]To screen an enhanced expressed promoter with high stability using the known Lactobacillus plantarum promoter and the green fluorescent protein reporter gene,and to provide genetic element for construction of the highly efficient and specific expression vectors in Lactobacillus plantarum.[Method]The known Lactobacillus plantarum promoters of P11,Pefp,Ptuf33 and Ptuf34 as well as the corresponding downstream green fluorescent protein reporter genes were synthesized,respectively.To construct the promoter-screening vectors,the above synthesized sequences were used to replace the original multiple cloning sites and the corresponding upstream promoters of the pMG36e,a lactic acid bacteria expression vector,respectively.The successfully constructed promoter-screening vectors were transformed into Lactobacillus plantarum by electroporation,and the promoters were screened by detecting the strength of green fluorescent signal of recombinants.[Result]Among the four constructed promoters,Ptuf33 and Ptuf34,the promoters of transcription elongation factor TU gene,had significantly(P<0.05)higher expression levels in the target genes compared with the other two promoters.[Conclusion]In combination with the results obtained in this study and related reports,it is known that the promoters of Ptuf33 and Ptuf34 are generally highly expressed in Lactobacillus plantarum,and they can serve as the candidates for subsequent construction of highly expressed vectors in Lactobacillus plantarum.