Screening And Identification Of Edwardsiella Tarda SRNA And Primary Function Of SRNA_EsR240

Edwardsiella tarda?Et?is often found in aquatic environment or aquatic animals,which infecting a wide range hosts.It is a facultative intracellular bacterium,So Et has to quickly adjust its own virulence gene's expression to adapting the various organisms and harsh environments.Therefore,there may be many factors regulating gene expression in the Et genome,such as non-coding RNA,also known as small RNA?sRNA?.We performed the analysis of sRNAs of ET13 strain according to the results of the RNA-Sequencing?RNA-Seq?and bioinformatics methods on the first Chapter.Firstly,the non-coding regions satisfied with certain conditions between the genes were extracted and 5838novel transcripts were obtained.Then,310 candidate sRNAs were obtained,which could not be compared with the annotated genes of protein library.16 annotated sRNAs were found,which were aligned with the sRNA library?sRNAMap?sRNATarBase and SIPHT?.Then 13new sRNAs with promoters and p-independent terminators were found by Software Promoter2.0 and RNAMotif,and named as EsR77,EsR128,EsR139,EsR150,EsR169,EsR190,EsR213,EsR214,EsR240,EsR255,EsR261,EsR285,and EsR289.ET13 strain of log phase continued to culture in pH 6.3 LB for 2h.8 of the 13 sRNAs were shown transcriptional levels by RT-PCR.In the LB of the different pH?4,5?,nutrition deficiency medium?GEM?,the LB of the different concentrations of H₂O₂?50 mM,100 mM,200 mM,300 mM,400 mM?,and the LB of the different concentrations of NaCl?100 mM,200 mM,300 mM,400 mM,500 mM?,the transcriptional levels of 8 sRNAs were examined by RT-PCR.On the stress conditions,the results demonstrated that their transcriptional levels were different,and all of the levels of the 3 sRNAs of EsR128,EsR139 and EsR240 were high at various conditions,and EsR240 was verified at log and stationary periods by Northern blot,and its size was 596bp.Then the transcriptional initiation and termination sites of EsR240 were verified by rapid amplification cDNA end?RACE?experiment.The analysis of the conservation of EsR240 was performed using BLAST.The results showed that as EsR240sequence was very high homology with the sequences of Et,it was consistent with the characteristics of sRNA.The target genes of EsR240 were predicted by IntaRNA software,According to their low free energies to high ones,the fronting eight target genes were selected.These target genes included ABC transporter binding protein,membrane protein,permease,sodium:proton reverse transcriptase and glycoside hydrolase,etc.Hydrolases and transporters were involved in the metabolic activities of bacteria,and EsR240 may participate in the regulation of bacterial metabolism and growth.On the Chapter 2,the biological function of EsR240 was further studied.A deleted strain of EsR240 in ET13 was constructed using the homologous recombination of suicide plasmid.Firstly,the ambilateral fragments of EsR240 were synthesized.Secondly,the suicide plasmid pHM5 and the fragments were connected to construct recombinational suicide plasmid pHM5-F1-F2.Lastly,the plasmid was transformed into SM10.After the SM10 containing the plasmid pHM5-F1-F2 and ET13 were conjugated in 1:4 ratio,the conjugants were screened out on two kinds of antibiotics plates by PCR.The colonies grown on 20%sucrose plates were inoculated both on two kinds of antibiotics plates and on one kind of antibiotics plate.The colonies only grown on one kind of antibiotics plates were selected and the EsR240deleted strains were screened by PCR and were identified by sequencing.Then construct the recombinant plasmid by connecting pACYC184 plasmid with EsR240 fragment.The recombinant plasmid was transformed into the EsR240 deleted strains by transformation.The complementary strain of EsR240 was got and identify by PCR.On Chapter 3,we first compared the survival rates of the wild type and EsR240 deleted strain under different stress conditions.Secondly,we compared the survival rates of the wild type,EsR240 deleted strain and the complementary strain within macrophages in vitro and mice in vivo.These results showed that the deletion of EsR240 affected Et growth in acid LB?pH 4.0?,or oxidation LB?including 0.4M H₂O₂?,or high osmosis LB?including 0.5M NaCl?,and affected Et growth in macrophages and survival ability in mouse liver and spleen in vivo.We further performed LD50 experiments with the EsR240 deleted strains and the wild strains.The results demonstrated that the virulence of the deleted strain of EsR240 was6.79 times weaker than the wild strain.In this study,we performed the sRNA prediction of ET13 strains with the results of the bacterial RNA-Seq and bioinformatics methods.The expressions of candidate sRNAs were done by experiments and the transcriptive level of EsR240 was high under various stresse of conditions.In addition,the promoter and?-independent terminator of EsR240 were looked for,and the target genes of EsR240 were searched.The EsR240 deleted strains and complementary strain were constructed.Our results showed that sRNA_EsR240 played the important roles in regulating Et growth in vitro,survival within macrophages and virulence.