| Objective:The Salmonella enterica serovar Typhi(S.Typhi)is an important intestinal pathogen,and also an important model bacterium for the study of prokaryotic gene expression regulation.The biofilm formed by S.Typhi can resist the host’s immune effect.The formation of biofilms is controlled by a complex regulatory network,and non-coding RNA plays an important role in the process of biofilm formation.In the early stage of the research group,RNA-Seq technology was used to analyze the transcriptome of S.Typhi in the biofilm state and the planktonic state,and a number of differentially expressed nc RNAs were found in the two states.The expression abundance of nc RNA617 in the biofilm bacteria is higher than planktonic bacteria.In this study,we focus on the molecular characteristics and expression characteristics about the newly discovered nc RNA617 and study its role and potential functions and mechanisms in the formation of biofilm.Methods:1.Molecular identification of nc RNA617(1)Design specific reverse transcription primers and probe primers in the known sequence region of nc RNA617,verify the existence of nc RNA617 in S.Typhi by RT-PCR and northern blot.(2)5’end and 3’end of nc RNA617 was detected by 5’RACE and 3’RT-PCR,and analyze the full length of nc RNA617 molecule with the northern blot result and determine its gene location.2.Expression analysis of nc RNA617(1)The total RNA of S.Typhi was extracted in different growth periods and the expression of nc RNA617 was detected by northern blot.(2)The total RNA of S.Typhi under different stress conditions was extracted and the expression of nc RNA617 was detected by q PCR.3.Construction of strains(1)Δ617: According to the full-length identification result of nc RNA617,design specific primers to amplify the upstream and downstream homologous arm fragments of nc RNA617,use suicide plasmid to construct nc RNA617-deficient strain(Δ617).(2)Δ617-c617: Design nc RNA617 specific primers,amplify specific fragments,use plasmid p BAD33 to construct nc RNA617 high expression vector,electro transform positive recombinant vector into Δ617 to construct complement strain(Δ617-c617).(3)007-p BAD33 and Δ617-p BAD33: The empty plasmid was introduced into wild strain(007)and defective strain(Δ617)to construct wild control strain(007-p BAD33)and defective control strain(Δ617-p BAD33).4.Analysis of the function of nc RNA617(1)Growth curve determination: the wild strain and defective strain were cultured at37°C,record the OD600 value every 1 hour,and draw the growth curve of the wild strain and defective strain,compare the effect of nc RNA617 on bacterial growth.(2)Motility experiment: The wild strain and defective strain were cultured in LB medium to logarithmic phase,transfer the bacterial liquid to semi-solid medium,and place it in 37℃ incubator for culture,and determine the diameter of the motility circle of bacteria,analyze the influence of nc RNA617 on the motility of bacteria.(3)Antimicrobial susceptibility test: The sensitivity of the wild strain and defective strain to seven different antibiotics was detected by the K-B method,determine the size of the inhibition zone,and analyze the effect of nc RNA617 on the impact of resistance.(4)Biofilm formation experiment: Strains about 007-p BAD33,Δ617-p BAD33 and the complemented strain Δ617-c617 were cultured in TSB medium to the logarithmic phase,and inoculate the bacterial into 96-well plate at 30°C for 96 hours,after staining,fixing,dissolving,and measuring,the effect of nc RNA617 on the formation of biofilm was analyzed.5.Study on mechanism of action(1)The m RNA level of genes related to biofilm formation was detected q PCR.(2)Predict the interaction between nc RNA617 and genes related to biofilm formation:The interaction between nc RNA617 and the m RNA of regulated genes was predicted and analyzed by RNA Structure,Inta RNA and other websites.(3)The potential target gene of nc RNA617 was predicted by bioinformatics methods and the m RNA level of nc RNA617 potential target gene was detected q PCR.Results:1.Molecular identification of nc RNA617(1)RT-PCR and northern blot results indicate that nc RNA617 is indeed expressed in S.Typhi,with a length of about 300 nt.(2)5’RACE results show that the transcription start site of nc RNA617 is located967 nt downstream of the mig-14 stop codon;3’RT-PCR results show that the transcription termination site of nc RNA617 is located between 2378 and 2560 nt upstream of the start codon of t2681 gene.2.Expression analysis of nc RNA617(1)The result of q PCR experiment showed that compared with the mid-log phase the expression level of nc RNA617 was lower in the lag phase,late logarithmic and stationary phase.(2)The result of northern blot experiment showed that compared with the untreated group,the expression level of nc RNA617 was lower under acid,oxygen and hypertonic stress.3.Construction of strainsStrains about Δ617,Δ617-c617,007-p BAD33 and Δ617-p BAD33 were successfully constructed.4.Analysis of the function of nc RNA617(1)The growth curve determination showed that there was no significant difference between the growth of the wild strain and the defective strain in 37℃ LB medium.(2)Motility experiment results show that compared with the wild strain,the diameter circle of the defective strain has no obvious change.(3)The results of the drug susceptibility test showed that there was no significant difference in the inhibition zone size of the 7 antibiotics between the wild strain and defective strain.(4)The biofilm formation experiment showed that compared with the wild control strain,the biofilm formation ability of the defective control strain was increased,and the formation ability of the complemented strain was restored.5.Study on mechanism of action(1)The results of q PCR analysis of the m RNA levels of biofilm-related genes showed that the expression of csg D,fim A,flh D,flj B,bap A,and yih P m RNA in the defective strain were up-regulated.(2)Predictive analysis showed that there were different binding regions between nc RNA617 and the m RNA of regulated biofilm-related genes.(3)Predictive analysis found that there was a series of target gene about nc RNA617 in S.Typhi,and the m RNA level of related genes analyzed by q PCR showed that the expression of cit E,ubi F,mur G,kgt P m RNA were up-regulated.Conclusion:This study found that nc RNA617 is a non-coding RNA located in the intergenic region between mig-14 and t2681(hypothetical gene).Its full length is between 270 and 452 nt.The expression level of nc RNA617 is the highest in the mid-log phase.The expression is reduced under acid oxygen and hypertonic stress.nc RNA617 can negatively regulate biofilm formation;nc RNA617 can negatively regulate biofilm formation by affecting the m RNA level of regulated biofilm-related genes.At the same time,nc RNA617 can negatively regulate the he expression of cit E,ubi F,mur G,and kgt P. |
PDF Full Text Request