Zhang Xiaojing, Cai Xuwang, Qi Yi, Liu Yunbao, Cao Qi, Wang Xiangru, Chen Huanchun, Xu Xiaojuan
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University,Wuhan, Hubei 430070,China.
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University,Wuhan, Hubei 430070,China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei 430070, China.
Plasmid. 2018 Jun;98:8-14. doi: 10.1016/j.plasmid.2018.07.001. Epub 2018 Jul 9.
Some Haemophilus parasuis strains display resistance to transformation with Escherichia.coli-derived plasmids. This property limits the application of genetic approaches previously developed for H. parasuis. The present study showed that natural transformation with the shuttle plasmid pS2UK led to allelic exchange in H. parasuis strains SH0165 and CF7066. Furthermore, natural transformation with pS2UK yielded allelic exchange mutants in 10 of 17 H. parasuis strains, similar to results using the suicide plasmid pK2UK. Subsequently, 17 H. parasuis strains were transformed with pS2UK by electroporation and 13 obtained the transformants harboring the complete plasmid molecules. As a result, natural transformation of homologous blank strains with the H. parasui-derived plasmids significantly improved the transformation efficiency targeted at obtaining allelic exchange mutants. In addition, shuttle plasmids pS1UG and pSHUK that carried the different homologous arm sequences also displayed the increased transformation efficiency after they were replicated in homologous H. parasuis cells. The approach described here not only improved the efficiency of natural transformation of H. parasuis, but also enlarged the range of transformable H. parasuis strains, thereby enabling application of H. parasuis-specific genetic manipulation techniques in a wider range of isolates.
一些副猪嗜血杆菌菌株对源自大肠杆菌的质粒转化表现出抗性。这一特性限制了先前为副猪嗜血杆菌开发的遗传方法的应用。本研究表明，穿梭质粒pS2UK的自然转化导致了副猪嗜血杆菌菌株SH0165和CF7066中的等位基因交换。此外，用pS2UK进行自然转化在17株副猪嗜血杆菌菌株中的10株中产生了等位基因交换突变体，这与使用自杀质粒pK2UK的结果相似。随后，通过电穿孔用pS2UK转化了17株副猪嗜血杆菌菌株，其中13株获得了携带完整质粒分子的转化体。结果，用副猪嗜血杆菌衍生的质粒对同源空白菌株进行自然转化显著提高了获得等位基因交换突变体的转化效率。此外，携带不同同源臂序列的穿梭质粒pS1UG和pSHUK在同源副猪嗜血杆菌细胞中复制后也显示出转化效率的提高。这里描述的方法不仅提高了副猪嗜血杆菌自然转化的效率，而且扩大了可转化的副猪嗜血杆菌菌株的范围，从而使副猪嗜血杆菌特异性遗传操作技术能够应用于更广泛的分离株。
