| Abuse of antibiotics has led to a global problem of bacterial resistance,with more and more drawbacks showed up,people hoping to develop an antibiotic that does not cause bacterial resistance,this is why the study of antimicrobial peptides was active in recent years.Antibacterial peptide has been considered an alternative to antibiotics in the future,and Rana for its long-term natural evolution,a lot of skin glands secrete antimicrobial peptides are formed,become a treasure trove of natural antimicrobial peptides resources.Moreover,the antimicrobial peptides from frog have many advantages such as small molecular weight,high antibacterial activity and low hemolytic.But a growing number of experiments indicate that after long-term co-evolution,pathogens can evolve an effective mechanism to combat antimicrobial peptide,it is necessary to explore these antimicrobial peptides generating process for solving the resistance problem.Brevinin-2CE is an antimicrobial peptide gene cloned from China Rana by our laboratory previously,it encodes an antimicrobial peptide Brevinin-2CE,which is a typical cationic antimicrobial peptide and has an excellent broad-spectrum antimicrobial activity.Brevinin-2CE could combined with pathogen membranes and lead to ruptured,bacterial lysates spilled outside and cell death.In this experiment,synthetic Brevinin-2CE was used as research material.To study the changes in transcriptome level of E.coli treated by antimicrobial peptides in a short time,and predict which kind of bacterial stress response would occurred,E.coli were short-term treated by Brevinin-2CE in a non-lethal concentration,then suppression subtractive hybridization,RNA-Seq and quantitative Real-time PCR have been performed respectively to detect genes expression differences between the experimental groups and the control groups.Subsequently,two genes showed different expression level in experimental groups and the control groups--cspB(experimental group raised)gene,srlE(experimental group down)gene were cloned into pET-28a plasmid to construct prokaryotic expression vector,then transformed the constructs into E.coli,detect transformation sensitivity to various antibacterial substances so that genes function can be predicted.In this study,10 genes expressed differentially were screened by subtractive hybridization,37 were screened by RNA-Seq,according to the function.20 of them were chosed by gene function and significance of differential expression,validate again by real-time quantitative PCR,got 11 genes eventually.Summarized below,upregulated genes covering several areas,cold stress,multi-drug efflux pump,cell wall synthesis,ribosome structure and other aspects,the two downregulated genes turned out both associated with stress.Most changes in expression of these genes are associated with Brevinin-2CE bactericidal mechanisms.Cell wall synthesis genes mraY,murE;frdD,uhpA,uhpB genes involved in metabolism,these two kind of genes increase may be considered as stimulated reaction after the cell membranes rupture.The over expression of multidrug efflux pump gene sugE can help the antimicrobial peptides in the cell discharged extracellular.CspB and cspE have been proven to help resist bacterial peptide when overexpressing.Gene srlE encoding sorbitol-specific enzyme IIB component,pressure response gene uspA come down regulation should be triggered by extra and intracellular osmotic pressure imbalance.During the experiments,we also found that the 16S rRNA significantly upregulated after Brevinin-2CE treatment,but it was reported that 16S rRNA always remained stable expression under all kinds of stimuli.Similarly,in RNA-Seq results,multiple genes encoding ribosomal subunit up-regulated,means that E.coli ribosome synthesis substantial increase in the initial treatment of Brevinin-2CE levels.In protein function test experiment,prokaryotic expression vectors were constructed successfully by upregulated gene cspB and down-regulated gene srlE,and were transformed into E.coli A70,formed two strains named as A70-pET-28a[cspB],A70-pET-28a[srlE].In the inhibition experiments with two strain A70 and A70-pET-28a as a control,use different concentrations of Brevinin-2CE,ampicillin and rifampicin to perform inhibition experiments,found the MIC values of A70-pET-28a[cspB]for Brevinin-2CE,ampicillin,rifampin did not change,and the inhibition rate at each concentration was also no significant change,sensitivity of A70-pET-28a[srlE]for Brevinin-2CE,rifampicin did not differ either,but its resistance to low concentrations of ampicillin decreased significantly. |
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