畜牧与兽医2018，Vol.50Issue(5)：80-83,4.
牛支原体08M株突变体文库的构建及鉴定
Construction and appraisal of a Mycoplasma bovis strain 08M mutant library
摘要
Abstract
This study was to construct a mutant library of Mycoplasma bovis (M.bovis) strain 08M by transposon insertion,which could provide a platform for identification of the virulent genes of the pathogen.Firstly,the color change units (CCU) and the gentamicin (Gm) minimum inhibitory concentration (MIC) of M.bovis strain 08M were determined.Secondly,the plasmid pISM2062 carrying transposon Tn4001mod was introduced into the cells of strain 08M by electroporation.Thirdly,the mutant library of strain 08M was constructed by mutant clones screening,culturing,and PCR identification.Finally,ten clones were selected,underwent 10 passages,had the stability of transposons in them detected by PCR;and two clones were selected to detect by Southern blot to see whether the transposons had been inserted many times.The results showed that the concentration of strain 08M was 1.13× 107 CCU/mL,and that of the gentamicin MIC was 16 ng/μL.A total of 445 colonies were picked up after electroporation with 380 of them being grown colonies,and 263 of the grown colonies were identified as positive Tn insertion clones by PCR.The positive rate of the mutant library was 69.21％ (263/380).PCR and Southern blot analyses showed that the transposons underwent stable passage,and no multiple insertion of the transposons were detected.A mutant library of M.bovis was successfully constructed.关键词
牛支原体/转座子/突变体文库Key words
Mycoplasma bovis/transposon/mutant library分类
农业科技引用本文复制引用
季文恒,陈胜利,郝华芳,李明霞,赵萍,王展慧,储岳峰..牛支原体08M株突变体文库的构建及鉴定[J].畜牧与兽医,2018,50(5):80-83,4.基金项目
国家自然科学基金(31402223、31602088) （31402223、31602088）
国家重点研发计划(2016YFD0500907、2017YFD0500905) （2016YFD0500907、2017YFD0500905）
国家科技支撑计划(2015BAD12B02) （2015BAD12B02）
甘肃省重点研发计划(17YF1NA070) （17YF1NA070）
农业部科研院所基金(1610312016027) （1610312016027）
