单核细胞增生李斯特菌非编码RNA rli87基因缺失株的构建、鉴定及其生长特性初步研究  

Construction,Identification of ncRNA rli87 Gene Deletion Strains Listeria monocytogenes and Its Growth Characteristics

作　　者：谢堃[1] 乔军[1] 孟庆玲[1] 彭叶龙[1] 赵海龙[1] 马玉[1] 陈诚[1] 才学鹏[2] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院动物疾病防控兵团重点实验室,石河子832003 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,兰州730046

出　　处：《畜牧兽医学报》2015年第6期998-1003,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家国际科技合作专项(2014DFR31310);国家自然科学基金(30960274;31360596)

摘　　要：拟构建单核细胞增生李斯特菌(LM)非编码RNArli87基因缺失株,对其进行分子鉴定,分析rli87基因缺失对LM生长的影响。用融合PCR方法构建LM-SB5 rli87基因缺失突变体,并构建pKSV7-Δrli87穿梭载体,将其转化入LM-SB5感受态细胞,利用温度(42℃)和氯霉素(10μg·mL-1)的抗性双重压力来实现同源重组,筛选同源重组菌进行分子鉴定,测定缺失株与母源野毒株37℃条件下生长曲线。结果显示:PCR和测序结果证实成功获得了LM-Δrli87重组菌。通过25代的连续传代,PCR鉴定结果显示,该缺失株具有良好的遗传稳定性。与野毒株进行对比,在37℃条件下缺失株与野毒株的生长差异不显著(P>0.05)。非编码RNArli87基因在37℃条件下对LM生长没有明显的调控作用。In this study,non-coding RNArli87 gene deletion strain of LM was constructed and identified,and growth characteristics of deletion strain was studied.The rli87 gene deletion mutant was constructed by fusion PCR method,and then pKSV7-Δrli87shuttle vector was constructed,which was transformed into competent cells LM-SB5.Homologous recombination was conducted using temperature(42 ℃)and chloramphenicol(10μg·mL-1)resistance to achieve the rli87 gene deletion strain,and growth curves of wild strain and deletion strain were assayed at 37℃.The PCR and sequencing results confirmed that LM-Δrli87 was successfully obtained.PCR identification results showed that the deletion strain has good genetic stability through 25 generations of continuous passage.Comparison with the wild strain,there was no significant difference(P>0.05)in growth between deletion mutant and wild strain at 37 ℃.The results suggest that rli87 gene did not play significant role in regulatory growth under the conditions of 37 ℃.

关 键 词：rli87基因 LM感受态细胞 缺失株 生长特性 同源重组 

分 类 号：S852.61[农业科学—基础兽医学]