荧光假单胞菌高效广谱载体的构建及分析

为促进高GC含量基因在荧光假单胞菌(Pseudomonas fluorescens)中表达效果更加理想、操作更加简便,本研究首先采用不依赖基因序列和连接反应的克隆(Sequence and ligation independent cloning,SLIC)方法将载体pCIBhis上与复制相关的序列和标记基因片段构建成克隆载体pCIBS1.然后优化荧光假单胞菌转化方法,用电转化法将pCIBS1导入荧光假单胞菌BL915中,随后又将T7和tac基因启动子分别插入pCIBS1中,成功构建了表达载体pCIBS3和pCIBS2.研究发现载体pCIBS1在大肠杆菌和荧光假单胞菌中均较为稳定,并且将绿色荧光蛋白基因插入表达载体中,在大肠杆菌BL21(DE3)中获得表达,验证了表达载体功能.本研究构建的表达载体和建立的荧光假单胞菌BL915电转化方法,为高GC含量基因在荧光假单胞菌中的表达奠定了基础.

Construction and Analyses of High Efficient and Broad Host Range Vectors of Pseudomonas fluorescens

Pseudomonas fluorescens is a useful common bacterium and has GC content as high as above 60%.Because the genes with high GC content are not easy to be expressed in conventional expression systems,constructing a novel expression system is important in P.fluorescens.The plasmid pCIBhis is commonly used to express protein in P.fluorescens.Since it is above 20 kb in length conjugation is usually used to transfer this plasmid vector into P.fluorescens which results in a more complicated operation.In this study,the replication-related segment and genetic marker gene were amplified from pCIBhis by using PCR and then recombined together with the method called "sequence and ligation independent cloning(SLIC)" to construct the vector pCIBS1.The transformation method of P.fluorescens was optimized and the plasmid pCIBS1 was electroporated into P.fluorescens.Then T7 and tac promoters were inserted into pCIBS1,respectively,and the expression vectors pCIBS3 and pCIBS2 were constructed.It was found that pCIBS1 could be stable either in P.fluorescens or in Escherichia coli.The green fluorescent protein gene was finally cloned into the two expression vectors and the transformants harboring the recombinant plasmid could express the functional protein in E.coli BL21(DE3).The results lay a foundation for the expression of those genes with high GC content in P.fluorescens.