Study On The Relationship Between CRISPR/Cas And Drug Resistance In E.coli.

In recent years,bacterial resistance has become a serious threat to human health and a major challenge in the field of global public health.At present,the relationship between CRISPR/Cas system and drug resistance is not clear.Therefore,this study uses the chicken-derived Escherichia coli CRISPR/Cas system as the research object.Firstly,the positive rate of CRISPR in E.coli was detected and the difference of drug resistance between CRISPR positive and negative strains was compared.Secondly,bioinformatics analysis of CRISPR sequence was carried out,and the expression levels of cas gene and CAS protein were analyzed.which laid the foundation for further study of CRISPR/Cas function and drug resistance mechanism.The main research contents and results are as follows:1.The relationship between CRISPR and drug resistance.Among the 130 strains of E.coli,39 strains of E.coli detected CRISPR,and the positive rate of CRISPR was 30%.The CRISPR-positive strains were multi-drug resistant strains,and the resistance rate(100%)was significantly higher than that of CRISPR-negative strains(87%).It is suggested that the presence of CRISPR is associated with the phenotype of multidrug resistance.2.Bioinformatics analysis of CRISPR.Bioinformatics analysis of CRISPR by BLAST,RNA secondary structure prediction,CRISPRTarget and other methods.Among the 12 strains of E.coli detected,there are 5 repeat sequences,15 of which are very conservative,and repeat sequences in different strains.Some bases are mutated;168 shared spacer sequences,of which 77 are newly discovered sequences,and homology alignments are found.Among the 112 spacer sequences with alignment results,72.3%of the spacer sequences and the plasmid genome The sequence has homology,25%has homology withphage,2.7%has homology with bacteria and virus,indicating that partial base mutation of repeat sequence and variability of spacer sequence are related to multi-drug resistance of strain.3.Fluorescence quantitative PCR analysis.RT-q PCR was used to detect the relative expression of 8 cas genes in ATCC 25922 and multi-drug resistant strain.Compared with the ATCC 25922,there was no significant difference in the expression of 8 cas genes in multi-drug resistant strain,suggesting that the expression of cas genes may not be related to drug resistance.4.Proteomics analysis.Proteomic analysis of ATCC standard strain and multi-drug resistant strain by tandem mass spectrometry tagging and parallel reaction monitoring targeting protein validation techniques,a total of 300 differentially expressed proteins were screened in multi-drug resistant bacteria,of which 167 were up-regulated and 133 were down-regulated,differentially expressed protein function enriched in bacterial chemotaxis,motor response,metabolism and other pathways.Nine proteins related to drug resistance:Tsr,Tar,CheA,CheW,HisJ,ProV,OppF,Hfq,YbhC,PutA,AdhP,Prr,CysJ,mainly enriched in bacterial chemotaxis and ABC transporter.There was no significant difference in the expression of CAS protein,suggesting that the expression of CAS protein may not be related to drug resistance.In this study,130 strains of Escherichia coli were detected by CRISPR,and their relationship with drug resistance phenotype was analyzed.The bioinformatics analysis of CRISPR repeats and spacer sequences was carried out to investigate the relationship between CRISPR sequences and drug resistance.Furthermore,the relationship between cas gene expression level and CAS protein expression level and drug resistance was further studied to lay a foundation for further study of drug resistance mechanism of E.coli.