| Vibrio parahaemolylicus is a Gram-negative halophilic bacterium that is mainly distributed in the seafood such as fish,shrimps and shellfish throughout the world.V.parahaemolyticus can cause diseases in marine aquaculture,leading to huge economic losses to the aquaculture industry.More importantly,it is also the leading cause of seafood-borne diarrheal disease in humans worldwide.In the case of infection,Vparahaemolyticus uses many different virulence factors to cause disease in animals or humans.The analysis of the pathogenic mechanism of V.parahaemolyticus is conducive to the development of new therapeutic strategies and prevention and control measures.The function of bacterial virulence factors plays an important role in the regulation of bacterial transcriptional regulators.GntR family transcriptional regulators,which are widely present in the bacterial genome,are often adjacent to the front end of the regulated gene,directly bind to their recognized DNA sequence(located in the promoter region of the target gene),and activate or inhibit transcription of the target gene.It is reported that GntR family transcriptional regulators play an important role in bacterial pathogenicity and participate in various biological processes of bacteria such as metabolism,biofilm formation,motility,stress,drug resistance and virulence.In this study,we investigated the molecular mechanism of GntR gene regulation of pathogenicity of Vparahaemolyticus by analyzing the biological characteristics and pathogenicity of GntR gene deletion strains.In addition,a method for quantitative detection of Vparahaemolyticus based on TaqMan probe method was established,which provided a reliable technical method for strengthening the prevention and control of Vparahaemolyticus.Experiment Ⅰ Establishment and application of triple Real-time PCR for the Detection of Vibrio parahaemolyticusTo establish a rapid,accurate and efficient detection method for V.parahaemolyticus,three pairs of specific primers and corresponding TaqMan probes were designed according to species-specific gene toxR and virulence genes tdh and trh of V.parahaemolyticus.The 5rahaemolyticusneic prtoxR,tdh and trh were individually labeled with FAM,HEX and CY5;The 3individually labeled with Fand correspondi;The concentration and reaction parameters of each reaction were optimized to build a triple Real-time PCR based on TaqMan probe method for the quantitative detection of V.parahaemolyticus.The results showed that the specificity of the assay has no cross-reaction with other pathogens,the minimum detection limit is 10 CFU/mL,the variation coefficient of repeated experiments is less than 1%.Triple Real-time PCR detected shrimp and shellfish samples contaminated by V.parahaemolyticus,the minimum detection reached 10 CFU/mL.The whole detection procedure time is approximately 1 h.A conclusion could be drawn that a newly triple real-time PCR method established in this study possesses strong specificity,high sensitivity,good repeatability and convenient.It’s an effective method for high-throughput detection of pathogenic V.parahaemolyticus.Experiment Ⅱ Construction of GntR gene deletion strain and complement of Vibrio parahaemolyticus and expression of GntR proteinThe GntR(VP 1277)gene deletion strain and the complementary strain of Vparahaemolyticus SH112 strain were constructed,and prokaryotic expression of the GntR recombinant protein was achieved.The transcription level of the deleted gene was detected by a real-time PCR assay,and the protein was correctly expressed by Western-blot.The results indicate that the gene was normally expressed in the wild strain SH112,but not in the deletion strain Δ gntR,and the expression level of the target gene in the complement strain C Δ gntR was comparable to that in the wild strain,which proved that the construction of the deletion strain and the complementary strain was correct.The deletion strain and the complementary strain showed no gene recovery after 20 passages,indicating that the deletion strain and the complementary strain have stable heritability.Western-blot showed that the recombinant protein GntR was expressed in prokaryotic expression in a highly soluble form with a molecular weight of 32 kDa.These results laid a foundation for further study of the functional studies of the GntR gene deletion strain in the biological characteristics and pathogenicity of Vparahaemolyticus,and the subsequent further study of GntR protein function.Experiment Ⅲ Biological characteristics and pathogenicity analysis of the GntR gene deletion strain of Vibrio parahaemolyticusIn order to explore the biological characteristics and pathogenicity of the gntR deletion strain of Vparahaemolyticus,many characteristical was tested,including the biofilm formation ability,the growth rate of different pH and detergent(TritonX-100),salinity resistance,lethal rate in ICR mice and the effect on toxicity level.The results indicated that the biofilm formation ability of the deletion strain Δ gntR was significantly decreased compared with the wild strain SH112 and the complementary strain C Δ gntR(P<0.05).Under different pressure factors,the pH and salinity had little effect on the strain,and the difference was not significant difference(P>0.05),but there was a difference in different concentrations of detergent(TritonX-100)(P<0.05).The toxicity of ΔgntR to Caco-2 cells was significantly decreased(P<0.05).The mortality of ICR mice was also significantly decreased(P<0.05).The above results indicate that the GntR gene of Vparahaemolyticus not only participates in the pathogenic process of cytotoxicity and inflammation of the host,but also plays a significant role in the mortality rate of mice.It fully demonstrates the GntR gene in V.parahaemolyticus plays an important role in the infection and the pathogenesis.Experiment Ⅳ Comparative analysis of transcriptome deep sequencing technology in wild-type SH112 and GntR gene deletion strainsThe Vparahaemolyticus strains of SH112 and GntR gene deletion strains were extracted in the logarithmic growth phase,and total RNA was extracted by Trizol method to construct a bacterial cDNA library.Entrusted companies to use the Illumina Hiseq TM2500 platform for sequence deep sequencing(RNA-Seq)to analyze wild and deleted strain transcription differential genes.Combined with significant differential expression gene screening,KEGG pathway metabolic pathway analysis and GO functional analysis,the differentially expressed genes were systematically classified to elucidate the regulatory gene network of GntR gene to Vparahaemolyticus.The results showed that 19 differential genes were found,including 4 up-regulated genes and 15 down-regulated genes.According to the analysis results of KEGG pathway and GO,the nearby gene groups VP 1273,VP1274,VP1275,and VP1276 have a large amount of data showing their functions.Identification was performed using real-time PCR.The results showed that the expression levels of VP1273,VP1274,VP1275 and VP1276 were up-regulated,and the difference was significant(P<0.001).There was significant difference in the expression of the down-regulated gene(P<0.001).Therefore,it was confirmed that VP1273,VP1274,VP1275 and VP 1276 are target gene groups regulated by the GntR gene,and subsequent experiments will further confirm whether or not it regulates the metabolism of histidine. |
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