| Vibrio alginolyticus is a major cause of Vibriosis in farmed marine aquatic animals and has caused large economic losses to the Asian aquaculture industry in recent years.Therefore,it is necessary to control V.alginolyticus effectively.The virulence mechanism of V.alginolyticus,the Type III secretion system(T3SS),is closely related to its pathogenicity.In this study,a regulatory gene tyeA on T3 SS was taken as the target.Firstly,we designed specific primers with the help of the whole genome sequence of V.alginolyticus on Gen Bank,performed PCR cloning,bioinformatics analysis.The sequence of HY9901 tyeA gene from V.alginolyticus was found to be 285 bp in length with a theoretical molecular weight of 10.98 k D.The transcriptional expression results showed that the expression of tyeA in Dulbecco’s Modified Eagle Medium(DMEM)and bile salt medium was significantly lower than that in normal TSB medium.The expression vector p GEX-4T-tyeA was constructed,it was used to prepare mouse antibody,and WB test showed that it had immunogenicity.The ΔtyeA mutant strain without antibiotic markers was obtained by using Overlap PCR and two homologous recombination techniques to knock out tyeA on HY9901 of V.alginolyticus,combined with forward and reverse screening methods.And the biological phenotypic analysis was carried out.The results showed that V.alginolyticus was able to grow normally with good genetic stability in the absence of tyeA gene,its growth curve and extracellular enzyme activity did not change significantly compared with the wild strain,its biofilm generation capacity at 24 h was higher than that of the wild strain(p<0.01),and its swamming ability was significantly weaker compared with the wild strain(p<0.01).The deletion of tyeA was found to significantly reduce the virulence of HY9901 of V.alginolyticus by median lethal dose on zebrafish(p<0.01).Drug sensitivity tests showed that the wild and mutant strains were highly sensitive to amikacin,minocycline,gentamicin,and cefoperazone,and resistant to benzocillin,clindamycin,and ceftazidime.RNA-Seq was used to compare the transcripts of HY9901 and ΔtyeA in DMEM culture,and a total of 194 differentially expressed genes were found in both strains,of which 82 genes were up-regulated and 112 genes were down-regulated.The differentially expressed genes were annotated,and the results showed that the up-regulated genes were mainly proteins related to the type III secretion system.qPCR verified the transcription level of genes.It was found that the m RNA level of the ΔtyeA was consistent with the expression level of RNA-Seq,indicating that the results of RNA-Seq were credible.qPCR was used to analyze the effect of tyeA deletion on the m RNA expression of T3 SS effector proteins Vop N,Vop S,Vcr V and Hop.These effector proteins were found to be significantly up-regulated at all growth periods of ΔtyeA.It was also found that the expression of effector proteins decreased at 48 h.The regulatory relationship of tyeA on effector protein hop,vop S,vop N and vcr V genes was investigated using Lac Z reporter gene fusion assay,and it was found that the activity of β-galactosidase detected in HY9901 was significantly lower than that of ΔtyeA(p<0.05),indicating that tyeA could inhibit the expression of hop,vop S,vop N and vcr V genes.The results of the Electrophoretic mobility shift assay(EMSA)assay showed that no blocking bands appeared with the increase of TyeA protein concentration,indicating that there was no direct regulation of hop,vop S,vop N and vcr V by tyeA.The deletion strain ΔtyeA was used as a live attenuated vaccine to immunize zebrafish,and the relative immune protection against Vibrio alginolyticus was found to be 71.2%.Real-time PCR analysis showed that ΔtyeA immunized zebrafish could enhance the expression of immune genes such as Ig M and IL-1β in the fish.Histopathologic examination showed that the sections infected with HY9901 had the pathological features of congestion and lymphocyte,while the negative control group and the immune group had no such features. |
