| Background:Vibrio parahaemolyticus is a gram-negative halophilic bacterium found in Oceans and other salt water and almost marine product throughout the world.People who ingestion uncooked or unmerited cooking food can cause enterogastrtis or foodborne illnesses. Its popular has seasonality and outbreak common be in warmer monthes(May-October). This organism is firstly isolated from seafood food poisoning in Osaka, Japan in1950, Vibrio parahaemolyticus poisoning accounted for40-60%in this event. Inrecent years, caused by V. parahaemolyticus of acute gastroenteritis accounted for in the world,and the cases of food poisoning are more than all of other bacterias beyond Salmonella in our country, especially in some littoral cities. The most common syndrome is gastroenteritis, the symptoms of which includes diarrhea with abdominal cramps, regurgitation, vomiting, even septicemia and death if not treated in time. For the control of outbreak of food poisoning, we must pay much attention to Vibrio parahaemolyticus,and it is necessary t to study and research the pathogenic mechanism of it.Vibrio parahaemolyticus can form biofilms in life cycle which is the one key factor for environmental survival and transmission, as well as Vibrio cholerae, Vibrio harveyi and Vibrio vulnificus. Biofilm formation and disperse are a dynamic process, this process is controlled by Quorum sensing(QS) system. The homology of Quorum sensing regulator protein between V. parahaemolyticus OpaR and Vibrio harveyi LuxR is96%, so the Quorum sensing both of them should have a similar mechanism of action. Although phenotype experiments and expression profiling data has indicated that OpaR can regulate capsular polysaccharide gene locus, extracellular polysaccharide gene locus, c-di-GMP molecule metabolism and flagellar gene and so on genes expression related with biofilm formation, the global regulater OpaR how to transcriptional control these genes has not been clarified, so it is necessary to do further research.Objective:The study is going to construct the biofilm phenotype and the LacZ fusion assay platform in Vibrio parahaemolyticus RIMD2210633. so there will be a solid and mature experimental platform for further investigations of transcriptional regulatory mechanisms, not only for OpaR but also the other regulaters.Methods:(1) Phenotype experiment project of biomembrane:biofilm formation of V. parahaemolyticus should be affected by many factors,including temperature, pH, salinity, culture method, fosters time nutrition and so on, by comparison and analysis of these factors, three different biofilm phenotype analysis methods have been established(rugose colony morphology, congo red staining assays, crystal violet staining assays).and then measure and assay the biofilm of mutant strains (AopaR, AtoxR)and wild type strain (J5421)for validating the stability of these phenotype experiments.(2) The LacZ fusion assay experimental technique:The entire promoter regions of the target genes were amplified by PCR, and then cloned into the vector of pHRP309. The recombinant plasmid of VPA1513was transformed into the wild type strain (J5421) and OpaR mutant strain (AopaR) to determine the β-Galactosidae activity (Miller units) in cellular extracts, through which the transcriptional regulation of VPA1513by OpaR regulator can be determined.Results:Compared to37℃,30℃is more fit for the growth of V. parahaemolyticus and its biofilm formation, and no matter what the temperature, it is fully in the growth plateau after12h’shaking culture with the speed of200rmp. V. parahaemolyticus can manifest more rugose in HI which is a nutrient-rich medium compared to LB, M and APW#3after complete habituated culture, so does in low salt concentration(0.5%) medium;Biofilm formation in neutral and slightly alkaline environment is not very different;Even though static culture is more fit for phenotype of wrinkle, shaking with the speed of100rmp is necessary to crystal violet staining assays which is used to quantitative determination for biofilm. Incubation time is less influential to congo red staining assays compared with crystal violet staining assays, but both of their phenotypes tend to be more obvious with time; These trials have identified OpaR negative and ToxR positive regulate biofilm formation. The nine recombinant plasmids for LacZ fusion assay were successfully constructed. The expression of VPA1513was negatively controlled by regulator OpaR.Conclusion:Each stabilised platform of rugose colony morphology, congo red staining assays, crystal violet staining assays and the LacZ fusion assay platformshave been successfully constructed. What’s more, discovering temperature, salinity, culture method, fosters time and nutrition effect biofilm formation, but Slightly alkaline environment do not effect.greatly. These could be used for V. parahaemolyticus biofilm phenotype and the transcriptional regulation mechanism studies in future. |
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