鸭疫里默氏杆菌TonB依赖性受体TbdR2基因缺失株的构建  

Construction of Gene Deletion Mutant TonB-dependent Receptor TbdR2 of Riemerella anatipestifer

作　　者：卢凤英[1,2] 苗双[2] 倪欣涛[1,2] 屠晶[2] 俞慧[2] 姜盼[2] 潘玲[1] 胡青海[2] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国家禽》2013年第16期21-24,共4页China Poultry

基　　金：国家自然科学基金(31072156和31272590);上海市自然科学基金(09ZR1438600)

摘　　要：用PCR方法扩增tbdR2基因的左右臂和壮观霉素抗性基因表达盒,然后将以上3个片段组成的同源重组同源臂LSR连接到自杀性质粒pDS132上,构建得到重组自杀质粒pDS-TbdR2-LSR。再将此质粒通过接合转导的方法导入到鸭疫里默氏杆菌CH3株。用含卡那霉素和壮观霉素的TSA筛选可能的基因缺失株,并对其进行PCR鉴定,获得基因缺失株CH3△tbdR2。CH3△tbdR2稳定性检测结果表明该缺失株能够稳定存在。另外,CH3△tbdR2突变株在电镜下的细菌形态以及在TSA培养基上的菌落形态与野生株CH3相似。表明通过自杀质粒同源重组的方法获得了鸭疫里默氏杆菌CH3株TonB依赖受体TbdR2的基因缺失株CH3△tbdR2,为下一步研究TbdR2的功能奠定了基础。The left and right flanking sequences of Riemerella anatipestifer tbdR2 gene,and a spectinomycin resistance cassette were amplified by PCR,respeetively. These PCR-derived fragments were digested with the restriction enzymes and ligated into the suicide vector pDS132 to produce the plasmid pDS-TbdR2-LSR. Then,the plasmid was introduced into R. anatipestifer strain CH3 by conjugative transduction. Transconjugants were selected by growth on TSA agar in the presence of kanamycin and speetinomyein. The deletion of the tbdR2 gene was confirmed by PCR amplification of tbdR2 DNA fragment and 16S rRNA sequence analysis. The mutant strain was designated CH3△tbdR2. Stability assaysshowed that CH3 A tbdR2 was stable in TSB medium. In addition, CH3 A tbdR2 exhibited similar bacteria and colony morphologies as the wild type CH3 observed on TSA agar or under transmission electron microscopy. Taken together, CH3 △tbdR2 mutant were eonstrueted successfully and would be useful to study the functions of TonB-dependent receptor TbdR2 further.

关 键 词：鸭疫里默氏杆菌 TonB依赖的受体 缺失突变株 自杀质粒 

分 类 号：S858.32[农业科学—临床兽医学]