解淀粉芽孢杆菌ptsGHI基因的敲除及缺陷株生长特性  

Knockout of ptsGHI Gene of Bacillus amyloliquefaciens and Growth Characteristics of Corresponding Deficient Strain

作　　者：杨慧林[1] 王坤[1] 廖瑜玲[1] 王斌[1] 林影[1] 潘力[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《华南理工大学学报（自然科学版）》2012年第8期95-100,共6页Journal of South China University of Technology(Natural Science Edition)

基　　金：国家"863"计划项目(2011AA100905)

摘　　要：在解淀粉芽孢杆菌磷酸转移酶系统中,葡萄糖主要是由ptsGHI操纵子编码的酶EI、HPr、EIIGlc转运入细胞.文中运用PCR技术,扩增出ptsG和ptsHI基因上下游的DNA片段,共约1kbp,用融合PCR连接上下游片段后,再连接到温敏型质粒pKS2上,然后电转化到解淀粉芽孢杆菌XH7中,最终构建了ptsG和ptsHI基因缺陷株.实验结果显示:在LB培养基中,ptsG及ptsHI基因缺陷株的生长状况与野生型菌株无明显差异;在含葡萄糖的LB培养基中,ptsG基因缺陷株的最高菌密度比野生型菌株提高了28%,且平稳期比野生型菌株要长,而ptsHI基因缺陷株的最高菌密度比野生型菌株降低了约32%,与其在LB中的生长状况基本一致;ptsG和ptsHI缺陷株及野生型菌株经鸟苷发酵实验后,ptsG基因缺陷株的鸟苷产量比野生型菌株提高了约24%,ptsHI基因缺陷株的鸟苷产量则比野生型菌株降低了约82%.以上结果表明:解淀粉芽孢杆菌ptsG基因缺陷株具有良好的生长能力和产物合成能力.In the phosphotransferase system of Bacillus amyloliquefaciens, glucose is transported into the cell mainly through enzymes EI, HPr and EIIGlc encoded by the ptsGHI operon. In this investigation, first, about 1 kbp up- stream and downstream DNA fragments ofptsG and ptsHI genes were amplifed and were ligated into one fragment by using fusion PCR. Then, the fused fragment was inserted into the temperature-sensitive plasmid pKS2 and trans- formed by electroporation into Bacillus amyloliquefaciens XH7, thus constructing ptsG- and ptsHl-deficient strains. Experimental results show that （1） in LB medium, there is no obvious difference in the growth characteristics be- tween the deficient strains and the wild-type strains; （2） in LB medium supplemented with 2% glucose, the ptsG gene-defcient strain shows a maximal cell density that is 28% higher than the wild-type one as well as a longer steady period than the control, while the ptsHI gene-defcient strain shows a maximal cell density that is about 32% lower than the wild-type one, and its growth accords well with that in LB medium; and （3） in guanosine fermenta- tion experiments, the guanosine production yield of the ptsG gene-deficient strain is about 24% higher than that of the wild-type one, while that of the ptsHI gene-deficient strain is about 82% lower These results demonstrate that ptsG gene-defcient strains are of good ability in growth and guanosine biosynthesis.

关 键 词：解淀粉芽孢杆菌 磷酸转运系统 基因敲除 代谢工程 

分 类 号：Q812[生物学—生物工程]