| Bacillus is an typical plant growth promoting rhizobacteria(PGPR),which has important application value in biological control of field diseases.PGPR could colonize on the plant root or surface to compete with pathogens for nutrients,secret antibacterial substances to inhibit the growth of pathogens,and induce plant defense systems to resist the invasion of the pathogens during the process of its interation with plants..Numerous studies have shown that PGPR plays an effective role only if it can colonize on the plant roots,the process of colonization includes biofilm formation and swarming.In addition,Bacillus is able to generate spores to resist adverse environmental damages.SRNA is an important regulator in bacteria,which requires the assistance of Hfq protein to adjust physiological and biochemical functions.It is currently studied in Gram-negative bacteria,but little is known about that of Gram-positive bacteria.86 sRNAs have been previously identified in B.amyloliquefaciens FZB42 using RNA-Seq sequencing technology and verified by Northern blot,while the function of 73 sRNAs was not annotated yet.Therefore,it is worthwhile to explore the specific biological effects of sRNA with unknown function of Bacillus,and it has far-reaching guiding significance for the study of high-efficiency and stable microbial agents.In the study,we constructed 17 sRNA mutants and △hfq mutants.Meanwhile,we did related phenotypic tests on these mutants.The results showed that the growth of mutants were not affected after knockouting related genes.Compared with wild type,most mutants had no significant difference on biofilm formation and swarming except △Bas03 and△hfq.Additionaly,the sporulation efficiency of △Bas32 was significantly reduced.By detecting the ability of FZB42 sRNA mutant to resist environmental stress,we found that they can grow normally at 10℃ except △Bas03.The great majority of mutants did not change their ability to resist salt stress significantly except that △Bas12,△Bas15,△Bas23 have been more vulnerable.We also tested the tolerance of FZB42 wild type and sRNA mutants under acidic conditions(pH 3.0)and peroxide.The results showed that acidic conditions didn’t effect on their growth,while △Bas03,△Bas12,△Bas28,△ Bas32 and△hfq have weaker resistance to peroxides.Moreover,we examined the effect of FZB42 sRNA mutants on the seed germination of rice.The results showed that the growth promoting effects of △Bas13,△Bas20,△Bas23,△Bas32,△Bas37 and △Bas45 were reduced obviously compared with the wild type.In order to study the molecular mechanism of sRNA Bas03 and Hfq protein regulating biofilm and swarming,we detected the expression level of Bas03 in △hfq firstly.The results showed that the related-gene expression of Hfq and Bas03 were significantly down-regulated,which means the gene expression of Bas03 depends on Hfq.Then,we used the TargetRNA and real-time PCR to confirme preliminary that the target gene of Bas03 was srfAC that in charge of synthesizing surfactin.At the same time,the decreased yield of surfactin in △Bas03 further demonstrated the above conclusion.After that,the detection ofβ-galactosidase activity indicated that the srfAC promoter is not active,which means Bas03 may not act on the promoter region of srfAC,nor on the gene cluster that synthesize surfactin.Furthermore,the detection of srfAC mRNA’s half-period may confirm that Bas03 could affects the stability of srfAC mRNA,and EMSA results proved that sRNA Bas03 could combine with targeted gene srfAC in the presence of Hfq protein sRNA Bas03.In the early stage,we have found that compared with the wild type,the sporulation efficiency of the sRNA mutant △Bas32 have promoted.Therefore,We transferred the reported gene sspB with GFP into sRNA mutant.The results showed that the fluorescence density of FZB42-sspB-GFP△Bas32 weaker than FZB42-sspB-GFP,which explains that Bas32 related with sporulation.We compared the expression level relatively of Bas32 in wild type FZB42 with △hfq by Real-time PCR,the results indicated that the related-gene expression of Bas32 in △hfq were significantly down-regulated,which means the gene expression of Bas32 depends on Hfq.After that,we detected the spoVAE1 gene in △Bas32,the results shows there was no significant difference compared with the wild type.Our study have illustrated that Bas32 have palys a little regulation role in the process of sporulation efficiency.The results of this study will fill in the shortcomings of Bacillus in the research of sRNA biocontrol function,and provide new ideas for the research of biocontrol mechanism. |
