| Vibrio alginolyticus is one of the Gram-negative marine bacteria and widespread in the seawater all over the world.It's an important pathogen to kinds of commercial fish,shrimp,and shellfish.The disease caused by this pathogen brings about serious economic loss to the aquaculture.It's also a kind of zoonotis microorganism and can cause tympanitis and wound infection to threat to human health.Hfq,the widespread and conserved chaperon protein of sRNA in bacteria,takes part in various physiological processes as the pleiotropic post-transcriptional regulator.The previous study demonstrated that Hfq played different roles in the kinds of physiological processes.It's known that Hfq realizes its regulation mainly through the promotion of the interaction between sRNA and their target mRNA.In this paper,five sRNA molecules,Qrr1-5,were cloned and the mutant strains,?qrr1,?qrr2,?qrr3,?qrr4 and?qrr5 were obtaind with the suicide plasmid,pDM4 based on the homologous recombination.Meanwhile,the complement strains were also constructed with the plasmid,p BAD33.The roles of these five sRNAs in Vibrio alginolyticus were studied and the main results were as follows:Five genes encoded for sRNAs(Qrr1,Qrr2,Qrr3,Qrr4,and Qrr5)were amplified with the genome of Vibrio alginolyticus EPGS020401 as template and the sequence sizes were 121 bp,133 bp,134 bp,133 bp,135 bp respectively.The sequences were analyzed by informatics methods and the results showed that the five qrr genes were highly homologous to those genes in other vibrio species and also they shared high similarity on the base constitution.They all contained the ?54 binding sites located between-12 and-24 region upstream from the transcription start site and the Rho-independent terminator in the 3'end.The secondary structures of these five sRNAs all formed four stemloops and four complement regions.The results of prediction on the target genes illustrated that Qrr1-5 can participate in different activities through kinds of target genes in Vibrio alginolyticus.The mutant strains,?qrr1(inframe deletion of 115 bp),?qrr2(120 bp),?qrr3(139 bp),?qrr4(128 bp),and ?qrr5(129 bp),were successfully constructed and so did the complement strains.The regulation of Qrr1-5 on the functions of Vibrio alginolyticus were preliminarily identified by comparison of the changes of the phenotypes in different strains.In ?qrr1-5 mutant strain,the growth rate and the maximum cell density were both reduced severely,that meaned Qrr1-5 affected the whole growth stage and the exponential stage most.For motility assay,all five mutant strains lost their swarming ability and the swimming ability was just absent in ?qrr1 strain,impired to some extent in?qrr2,?qrr3,?qrr4 strains,and least in ?qrr5 strain.Qrr3 and Qrr2 obviously repressed the biofilm formation,while Qrr1,Qrr4,and Qrr5 did nothing to the biofilm formation in Vibrio alginolyticus.Qrr5 played important roles in elevating the production of extracellular protease.For stress responces detection,Qrr1-5 had slight impact on the stress adaptation under different temperature(15°C and 43°C),30% sucrose,and the iron-limited conditions.Moreover,Qrr1-4 had little but Qrr5 did have some effect on the growth under 10%ethonal stress condition.SDS inhibited the growth of Vibrio alginolyticus and Qrr2 made some negative modulation for this stress adaptation ability.The results in this paper illustrated the roles of Qrr1-5 in regulation of virulence factor and other physiological processes in Vibrio alginolyticus briefly,riched the understanding of the virulence regulation mechanisms and layed some theoritical evidence for the diaganosis and development of protection method for vibriosis. |
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