Prediction And Partial Function Study Of SRNA In Brucella

Brucellosis is a zoonotic disease caused by Brucella that poses a huge threat to the public health and livestock.Brucella is an intracellular bacterium that face a harsh environment after invade host cells.In order to cope with the complex intracellular environment,Brucella need to regulate the expression of related genes to ensure its survival and replication.As sRNA is continuously discovered in bacteria,the function of sRNA is gradually known,and it plays an important role in the regulation of bacterial gene expression,which can affect biofilm formation,metabolism,quorum sensing,stress response and virulence.Hfq is an RNA-binding protein that mediates gene regulation of sRNA.sRNA is also present in Brucella,but the transcriptional regulation is not clear.In order to study the function of sRNA in Brucella,this study used second-generation sequencing technology and sRNA-Detect algorithm to predict the sRNA of B.abortus 2308,and select partial verification.The functional of correct sRNA were studied,including its effects on bacterial virulence and response under stress conditions,the targets of these sRNAs.1.Total RNA of the strain was extracted,and high-throughput sequencing of RNA-Seq was performed.Based on the sequencing results,the sRNA prediction was performed by sRNA-Detect algorithm,and verified by RT-PCR.The results showed that a total of 3,253 candidate sRNAs were predicted,and 14 sRNAs were randomLy selected and verified.In addition,we found that the transcription levels of 13 sRNAs decreased more than 2-fold in B.abortus 2308hfq mutant,indicating that Hfq protein is closely related to the stability of these sRNAs.2.The upstream and downstream homologous arms of sRNA were amplified by fusion PCR and fused,cloned into pBlue vector,and then the kanamycin resistance gene was inserted between the upstream and downstream homologous arms to serve as a marker for screening for deletion strains.The pBlue-sRNA homology arm-Km plasmid was electroporated into B.abortus 2308,cultured with Km-resistant TSA plates,and single colonies were picked for identification.The mutant strain was used to infect mouse macrophage RAW264.7 at 100MOI,and the number of bacteria surviving in the cells was counted at 1h and 48h respectively.The results showed that the survival ability of sRNA0304 mutant strain in mouse macrophages decreased significantly at 48h.3.After inoculation of B.abortus into TSB culture for 48h,each 1mL was centrifuged and discarded supernatant,and resuspended in1mL of TSB containing 500mM NaCl,pH 4.5 and 2.5mM H₂O₂,and placed at 37°C for 1h,extract bacterial total RNA.qPCR was used to determine the transcriptional changes of sRNA under stress conditions.The results showed that the transcriptional changes of sRNA0009,sRNA0283,sRNA0304and sRNA0344 were significantly up-regulated under hypertonic,acidic and strong oxidative conditions,even more than 4 times,the transcriptional changes of sRNA0365 up-regulated under acidic more than 2 times,it is indicated that these five sRNAs are all related to bacterial stress response.4.TargetRNA2 is used to predict sRNA targets and validated by a dual plasmid reporter system.The sRNA and its promoter were amplified by PCR and cloned into the vector pUT18C,and the predicted target and its promoter were amplified by PCR and cloned into the pMR-LacZ plasmid,and then the two plasmids were sequentially transferred to BTH101,and cultured on LB plates with X-gal,and determine the regulation of sRNA on its target according to the color change on the plate.The results showed that the targets of sRNA0009,sRNA0283 and sRNA0304 were BAB1₂057,BAB1₁813 and BAB1₁023,respectively,and both were negatively regulated.In this study,RNA-seq technology was used to screen out new sRNA,and research on environmental stress,virulence and target was provided to provide reference for studying the pathogenesis and treatment of brucellosis.