鲁氏耶尔森菌invF基因无痕缺失突变株的构建及生物学特性

鲁氏耶尔森菌是一种具有广泛致病性的条件致病肠杆菌。三型分泌系统(T3SS)是该菌的重要毒力系统,其中invF基因是T3SS功能表达的重要调控因子。为探讨invF和T3SS对Y.ruckeri致病作用的影响,本研究构建了Y.ruckeri SC09株invF基因的无痕缺失株,并对其生物学特性进行研究。通过融合PCR方法,将invF基因的上、下游片段A、C融合,构建同源臂AC;将获得的同源臂AC连接入自杀质粒pLP12,构建pLP12-invF同源重组载体;pLP12-invF电转化进入供体菌株大肠杆菌β2163,并利用接合转移方法转入受体菌株Y.ruckeri SC09,利用抗生素正向筛选和vmt反向筛选分别对插入突变株和缺失突变株进行筛选,并利用PCR技术和序列测定对Y.ruckeri invF缺失株进行鉴定;对突变株和野生株进行菌体菌落形态观察、生化特性鉴定和生长曲线测定。结果显示,融合PCR、AC片段经氯霉素抗性正向筛选和vmt反向筛选,PCR鉴定和测序鉴定后,成功获得了Y.ruckeri SC09 invF基因的无痕缺失突变株,突变株和野生株菌体菌落形态和生化特性基本一致,突变株菌落较野生株小,各个时期突变株的生长浓度较野生株低。研究表明,采用自杀质粒pLP12和大肠杆菌β2163接合转移系统,利用抗生素正向筛选和vmt反向筛选技术,在对其基本生物学特性无显著影响的情况下,可简捷高效地获得invF基因的无痕缺失突变株。

Construction and identification of invF gene deleted Yersinia ruckeri and its biological characteristics

Yersinia ruckeri is a conditional pathogenic bacterium which has widespread pathogenicity. The most important virulence regulatory system of Y. ruckeri is its type Ⅲ secretion system (T3SS), while invF gene is an important regulatory factor of the T3SS. In this study, we constructed an unmarked deletion mutant with invF gene missing of a Y. ruckeri SC09, a highly virulent strain isolated from Ictalurus punctatus, and studied its biological characteristics. In order to investigate the effects of invF and T3SS on the pathogenicity of Y. ruckeri, construction of homologous arm AC, upstream fragment A and downstream fragment C of invF gene, was combined in overlap extension PCR. Then the homologous arm AC was ligated with the pLP12 suicide vector to generate the plasmid pLP12-invF. The plasmid was transformed into E. coli DH5α λ pir cells and it was amplified. The pLP12-invF was extracted and transformed into E. coli β2163 by electroporation, designated pLP12-invF-β2163. Then the pLP12-invF was transferred into Y. ruckeri SC09 strain through conjugation. Using the chloramphenicol for screening of insertional mutants and the vmt gene with L-arabinose for counter selection of deletion mutants. The deletion mutants were confirmed by PCR and subsequent sequencing. Then we observed morphology of bacteria and colonies, identified biochemical characterization and measured growth curve of the deletion mutants and wild type.In this study, the Y. ruckeri SC09 strain invF gene has been successfully knockouted. The morphology of bacteria and colonies, biochemical characteristics of deletion mutants and wild type were similar, but the colony size of deletion mutants was smaller than wild type and the growth of deletion mutants was slower than that of wild type. The method of pLP12 suicide vector, a highly efficient conjugation system form E. coli β2163 and screening by the antibiotic coupled with counter selecting by vmt gene, can be a simple and efficient gene editing operation to deal with Y. ruckeri, in the absence of a significant effect on its basic biological characteristics. In conclusion, this research obtained the unmarked mutant genes with the missing invF successfully, which laid a foundation for the studies of the pathogenicity of Y. Ruckeri in the future.