鼠伤寒沙门菌ydiU基因敲除株的构建及其在压力培养条件下的生长特性研究  

Construction of a ydiU gene deletion mutant of Salmonella typhimurium and its growth characteristics under pressure

作　　者：宋楠楠 杜忠君[2] 岳盈盈[3] 于功昌[2] 贾强[2] 李冰清 邵华[1,2] SONG Nan-nan;DU Zhong-jun;YUE Ying ying;YU Gong chang;JIA Qiang;LI Bing-qing;SHAO Hua(Shandong University of Traditional Chinese Medicine,Jinan,China 250355;不详)

机构地区：[1]山东中医药大学,山东济南250355 [2]山东省职业卫生与职业病防治研究院 [3]山东省医学科学院基础医学研究所

出　　处：《中国病原生物学杂志》2019年第12期1415-1419,1428,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金青年基金项目(No.31800054);山东省重点研发计划(No.2019GSF107055,2019GSF107026)。

摘　　要：目的构建鼠伤寒沙门菌ydiU基因敲除株,并比较敲除株与野生株在不同生存压力条件培养基中的生长曲线,为进一步研究ydiU基因及其编码蛋白YdiU的功能奠定基础。方法以鼠伤寒沙门菌ATCC14028株作为母本PCR扩增含有ydiU基因上下游同源臂和氯霉素抗性片段的线性打靶基因,通过同源重组操作得到ydiU基因敲除株ATCC14028AydiUo分别测定敲除株与与野生株在营养限制、氧化应激、缺铁、高盐,高糖、低PH和阿嗥等压力培养基中的生长曲线,并对两株细菌的生长特性进行比较。结果经PCR内、外侧鉴定及测序鉴定表明ydiU基因敲除株构建成功。敲除株和野生株在LB培养基中培养6 h即对数生长期后期时平均A600值分别为0.805和0.894,而在培养时间9h即平台期前期时检测平均值分别为1.022和1.220。两个生长关键期的细菌生长速度均表现为敲除株略为缓慢,但两株菌最终在平台期后期达到相同A值。在限制营养培养基(M9)中,敲除株增殖速度落后于野生株细菌,敲除株培养时间6 h、9 h和22 h的平均Ago。值分别为0.511.0.590和0.693,相同培养时间野生株平均值为0.595.0.719和0.8450以上数据表明在营养匮乏的生存压力下,ydiU基因对于细菌的生存和增殖具有关键作用。敲除株与野生株在培养基中活性氧增高、高糖、低PH、高眄|喙浓度等压力下生长速度和状态也有不同,提示ydiU基因在细菌的各种压力应激中可能发挥重要作用。结论成功构建了ydiU基因敲除株ATCC14028AydiU,其在多种压力培养基中显示出与野生株不同的生长特性,为进一步探究YdiU蛋白的功能提供了良好的基础和思路。Objective To construct a ydiU knockout strain of Salmonella typhimurium and to compare the growth curve of the knockout strain and a wild-type strain in media with different survival pressures in order to lay a foundation for further study of the function of the ydiU gene and the YdiU protein that it codes for.Methods A linear target gene containing the upstream and downstream homologous arms of the ydiU gene and a fragment conferring chloramphenicol resistance was amplified using the Salmonella typhimurium ATCC14028 strain as a female parent,and the ydiU gene knockout strain ATCC14028 O ydiU was obtained via homologous recombination.The knockout strain and wild-type strain were subjected to nutrient restriction,oxidative stress,an iron deficiency,a high salt concentration,a high sugar concentration,a low PH,and indole,and growth curves were plotted.The growth characteristics of the two strains were compared.Results The ydiU knockout strain was successfully constructed according to PCR using inside and outside primers and sequencing.The average OD600 value was 0.805 for the knockout strain and 0.894 for the wild-type strain cultured in LB medium for 6 h,when the bacterium was in the late logarithmic growth phase.The average OD600 value was 1.022 for the knockout strain and 1.220 for the wild-type strain during the early plateau phase.During the two key growth phases,the knockout strain grew at a slightly slower rate,but the two strains ultimately had similar A values in the late plateau phase.In nutrient restricted medium(M9),the growth rate of the knockout strain lagged behind that of the wild-type strain.The average 0D600 value for the knockout strain was 0.511 at6 h,0.590 at9 h,and 0.693 at 22 h.The average OD600 value for the wild-type strain was 0.595 at6h,0.719 at9 h,and 0.845 at 22 h.The growth rate of the knockout strain was significantly slower than that of the wild-type strain,indicating that ydiU gene plays a key role in the survival and proliferation of the bacterium under the pressure of a lack of nu

关 键 词：鼠伤寒沙门菌 ydiU基因 敲除株 压力应激 生长曲线 

分 类 号：R376.1[医药卫生—病原生物学]