| Riemerella anatipestifer(R.anatipestifer)is an important pathogen causing duck serositis.After the onset of infection,its main pathological features are cellulosic pericarditis,perihepatitis,and air sacs inflammation,which causes major economic losses for the global duck industry.R.anatipestifer has a large number of serotypes,and there is no cross-protection between the serotypes,which poses a great challenge for the prevention and treatment of the disease.The two-component system can assist bacteria in coping with various stimuli,such as osmotic pressure changes,antibiotics,temperature,oxygen pressure,etc.,regulating virulence and antibiotic resistance.This study focused on the two-component regulatory gene G148-1936 of R.anatipestifer.The following experiments were carried out.The results are as follows:1.Bioinformatics analysis of two-component regulatory systemUsing the two-component regulatory system prediction website and the two-component regulatory system database,predictive analysis of 31 published R.anatipestifers from NCBI revealed that the two-component regulatory systems were widely present in R.anatipestifer.It contains the OmpR conserved domain and belonged to the YesN superfamily through conserved domain analysis for the protein encoded by G148-1936 gene.It is a DNA binding response regulator with a signal receptor domain.2.Construction of G148-1936 gene mutant strain and complement strain The target gene was successfully replaced by homologous recombination to obtain the mutant strain RA-CH-2?G148-1936.The recombinant plasmid was constructed by using Escherichia coli-Riemerella anatipestifer shuttle plasmid pLMF02 and introduced into E.coli DH5?.After identification,the recombinant plasmid was extracted and introduced into the RA-CH-2?G148-1936,and the complement strain RA-CH-2C?G148-1936 was successfully constructed.3.Functional study of G148-1936 geneThrough the growth curve measurement,biochemical reaction,serum bactericidal test,sedimentation curve measurement,median lethal dose measurement,and histological change observation,a series of phenotypic related tests showed no significant difference in phenotype between the mutant strain and the wild strain.In the drought tolerance test,the wild strain and the mutant strain were not tolerant to the dry environment,and the ability for against dry environment of the mutant strain was weaker than the wild strain;the minimum inhibitory concentration test found that the mutant strain was more sensitive to ?-lactamase antibiotics.4.Transcriptome sequencing analysis of RA-CH-2?G148-1936Transcriptome sequencing analysis and the differentially expressed genes analysis were performed for RA-CH-2?G148-1936 and RA-CH-2.The 177 genes were significantly up-regulated,and 252 genes were significantly down-regulated in the mutant strain.The total number of significant differentially expressed genes were 429,more than 1/5 of the total number of genes of the RA-CH-2 strain was significantly differentially expressed.GO enrichment analysis showed that differentially expressed genes were enriched in three major categories: biological processes,molecular functions,and cell components.The enrichment of KEGG pathways showed differential genes enriched in 56 pathways.Analysis showed that the G148-1936 gene has a regulatory effect on the transcription of R.anatipestifer.Combined with the dry tolerance test and antibiotic susceptibility test,the results of transcriptome sequencing showed that the gene G148-1936 can cope with the dry environment by controlling the changes of bacterial membrane components and its ability to respond to stimuli and antioxidant activities.meanwhile,take part in regulation of lactamase antibiotic resistance. |
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