Screening, Identifying And Function Primary Researching Of Yersinia Pestis Exogenous Obtained Plasmid Coded Small RNA

BackgroundPestis is a kind of disease of natural focus and its pathogenic bacteria is Yersinia pestis (Y. pestis). Y. pestis has three palsmids, thus pCD1, pMT1 and pPCP1 in which pPCD1 can also be found in Y. pseudotuberculosis but pMTl and pPCP1 were exogenous obtained through evolution, so Y. pestis is specific of these two plasmids and it is closely related to its pathogenicity.There are many other regulatory factors besides protein and also much non-coding RNA that have regulatory effects which also called small RNA in bacterias. As a regulatory factor, small RNA regulate iron metabolism, glycometabolism, quorum sensing, OMPs synthesis and virulence, and it plays an important role of regulation in prokaryote physiology and virulence.This research used high-throughput sequencing technology to extract total RNA to proceed sequencing of transcriptome in Y. pestis under different growth conditions,104 small RNA were found which including many small RNA in plasmid pMTl and pPCPl, and these two plasmid were obtained though Y. pestis evolution, so verifying the function of small RNA in plasmid pMT1 and pPCPl is an important part of Y. pestis pathogenic mechanism research. This project is to use Northern blot to identify newly found small RNA and structure small RNA gene deleted strains, and through a serious of phenotype screening experiments to primarily study the influence of survival and virulence of small RNA to Y. pestis.Result and discussion(1) We identified the existence of 10 small RNA:sR18, sR3425, sR3457, sR3461, sR3446, sR3411, sR4338, sR4340, sR6142 and sR 6143, and test the length and dependencies to Hfq of small RNA that have been identified in this project and partial of previously identified small RNA. The length that have been identified in this project of small RNA are sR16, sR271, sR82, sR138, sR524, sR503, sR3446, sR3457, sR3461, sR4338, sR4340, sR6142, sR6143, sR18 and sR3411, together fifteen of them. The length of these small RNA are basically the same as the result of prediction. The expression quantity of sR16, sR4338, sR82, sR138 and sR510 in wild strain is more than that in Hfq mutant strain, so that the steady expression of these small RNA are rely on Hfq protein; the expression quantity of sR271, sR363, sR524, sR3461, sR18 and sR3411 in wild strain is basically the same as that in Hfq mutant strain, and that the expression quantity of sR503 and sR147 is less than that in Hfq mutant strain, so that the steady expression of these small RNA are not rely on Hfq protein.(2) Using the method of λ Red reconstruct system basic on the mutagenesis technique, we successfully constructed 16 strains of small RNA deleted strains, which are ΔsR4338, ΔsR4339, ΔsR4340, ΔsR3457, ΔsR3446, ΔsR6143, ΔsR378, ΔsR3383, ΔsR3440, ΔsR18, ΔsR6094, ΔsR3411, ΔsR6106, ΔsR6097, ΔsR3425 and ΔsR3438. And we successfully constructed an over-expression strain pBAD-sR3383 based on the deleted strain.(3) In this project, we used phenotype and virulence experiment to identify the regulatory relationship of small RNA to the physiology and virulence of Y. pestis. In the aspect of biofilm formation, the surface of Y. pestis bacterial colony was smoother than that of wild strain after deleted of sR3446, so that sR3446 may positively regulate the biofilm formation of Y. pestis; the surface of Y. pestis bacterial colony was rougher than that of wild strain after deleted of sR3425, sR3383, sR3457 and sR6143, so that these small RNA may negatively regulate the biofilm formation of Y. pestis. We proceeded crystal violet staining experiment based on this result to make the quantitative analysis of biofilm formation, it showed that the result of the regulation of sR3446, sR3425, sR3383, sR3457 and sR6143 to biofilm formation of Y. pestis is the same as the result of bacterial-colony-surface-corrugation experiment. We cultured Y. pestis under normal condition, high salt condition and acidic condition and test the change of growth rate of wild strain and small RNA mutant strain, and indentified the influence of small RNA to the survival ability of Y. pestis. The result showed that only the growth rate of ΔsR3425 is less than wild strain on these three conditions, so thatΔsR3425 can enhance the ability of coping with environmental changes of Y. pestis. We identified the influence of small RNA to the virulence of Y. pestis through mice challenge test, and analyzed the experimental result and calculated the growth curve P value difference of wild strain and small RNA mutant strain using software GraphPad Prism 5. When P≤0.05, it’s been considered to have statistical significance. The result showed that:the P value of Δ sR3440, Δ sR3383, Δ sR378, Δ sR6094, Δ sR3411, Δ sR4339 and Δ sR6097 is respectively 0.0258,0.0075,0.0004,0.0003,0.0267,0.0156 and 0.0143, so that we considered these small RNA can influent the virulence of Y.pestis.