Construction And Sequencing Of Hfq MRNA Library Of Burkholderia Pseudomallei

Objective Molecular chaperone protein Hfq is a key protein that promotes the paired binding of s RNA and target m RNA.Most s RNAs exert their regulatory effects by binding to target m RNA,and searching for relevant target m RNA is the main method to study its regulatory function.This paper aims to find Burkholderia pseudomallei Hfq-related m RNA lays the foundation for the further study of m RNA biological function and pathogenic mechanism.Methods 1)For this purpose,the recombinant vector p ET30a(+)-Hfq carrying Hfq gene was constructed and verified by PCR and restriction enzyme digestion analysis.Then the p ET30a(+)-Hfq was transferred in Escherichia coli BL21(DE3).After IPTG induction,the recombinant protein was expressed.The expressed product was detected by SDS-PAGE,identified in Western blot assay,then further purified using Ni2+ chelate column.2)The recombinant expression plasmid p ET30a(+)-Hfq was transformed into E.coli S17,positive monoclonal was picked for PCR verification,and the recombinant plasmid S17/p ET30a(+)-Hfq was ligated into Bp to induce the expression of the target protein Hfq.The bacteria were disrupted and the supernatant was extracted.The Hfq and its associated RNA were isolated by His-tag monoclonal antibody by co-immunoprecipitation,and the RNA in the RISC complex was extracted using TRIzol Universal.Then RNA concentration measured by Nano drop was used to takepart of the RNA for subsequent experiments.The rest of the reverse transcription was followed by rapid freezing with c DNA and stored for subsequent high-throughput sequencing and construction of m RNA sub-libraries.Results 1)Hfq gene fragment was amplified.Agarose gel electrophoresis showed that the size was 237 bp,which was consistent with the expected value.2)Subsequently,T-A subclones were successfully constructed.After double enzyme digestion and sequencing validation,the recombinant plasmid p ET30a(+)-Hfq was constructed correctly.3)The induced protein was detected by SDS-PAGE and identified by Western blot with Mouse Anti-His m Ab.The results showed that the molecular weight was about 9.4ku.4)The fusion protein mainly existed in the tcell filtrate.The target protein was purified via Ni2+-chelating chromatography.5)The recombinant plasmid S17/p ET30a(+)-Hfq was ligated into Bp and verified by PCR.6)The His-tag monoclonal antibody was isolated by immunoco-precipitation to obtain Hfq and its associated RNA.7)Hfq-related m RNA sub-libraries were constructed by high-throughput sequencing.A total of4185 genes were detected,of which 4155 were detected and 30 new genes were detected.The use of KEGG Pathway analysis and prediction in the new gene revealed that 5 genes can be found in the KEGG-A-class and KEGG-B-class signaling pathways,predicting that they may have metabolic,environmental information processing,membrane transport,amino acid metabolism and other Secondary metabolic biosynthesis.8)Additional 9 Hfq-related s RNAs were found.Conclusion: 1)The prokaryotic expression plasmid was successfully constructed.2)Hfq was successfully expressed in E.coli,and the s RNA chaperone protein Hfq was purified.3)Hfq-related m RNA libraries were successfully constructed and subjected to transcriptome analysis,polymorphisms,insertional mutations,RNA gene editing,and gene structure optimization,which provided the basis for further study of the functions of these m RNAs.The study of Burkholderia pseudomallei m RNA functionsmay reveal The pathogenic mechanism of Burkholderia pseudomallei in a certain area so as to find a corresponding therapeutic target.4)An additional discovery of 9Hfq-associated s RNAs provided the basis for the screening of s RNAs associated with this protein.