Bacillus Amyloliquefaciens FZB42 SRNA Igr3927 Transcription Analysis

The use of chemical fertilizer in agricuLture and forestry gradually were restricted due to its serious harm caused to the environment,then comes to a more secure,low pollution and efficient biological fertilizer.Let the biological fertilizer to better play its advantages,we must understand the mechanisms of biological fertilizer.Bacillus amyloliquefaciens FZB42 is a growth promoting rhizobacteria which has great development and application prospect for plant,but at present it’s plant growth promoting mechanism is not clear.Our laboratory found a special non-coding sRNA of this strain during the previous study and named it Igr3927.Interestingly,the expression of sRNA was significantly inhibited by root exudates,so we specuLated that the sRNA may play a key role in the relationship between plant and microorganism.Therefore,this study hope to make a further exploration on the influence factor of the sRNA transcription,the effects of sRNA Igr3927 expression on the bacterial phenotype and the possible target of this sRNA.We hope to discuss the key role of this sRNA when it response to the stimuLator of plant root exudates and in the process of the reguLation of target gene expression.The main resuLts of this study are as follows: 1 The test of FZB42 sRNA Igr3927 transcription by adding Phosphate and asparagineUse the different length of sRNA Igr3927 promoters which were amplified by PCR method from Bacillus amyloliquefaciens FZB42 to generate the report gene GFP to contrust the recombinant plasmid pFB26,pFB27.The recombinant plasmids were transformed into Escherichia coli DH5 a competent cells with heat shock method.Detect the effects on the expression of report gene GFP by adding different concentrations of phosphate solution.ResuLts showed that the expression of GFP was increased with the increasing of phosphate concentration in the range of tested phosphate concentration.We presume that phosphate directly or indirectly promote the transcription of FZB42 sRNA Igr3927 in Escherichia coli.Transfer the plasmid pFB26,pFB27 into Bacillus FZB42,through homologous recombination system to construct the mutant strains FB26,FB27.Add phosphate solution with different concentrations and 0.5mg/mL asparagine for test on mutant strain.The resuLts revealed that the addition of phosphate solution causes excessive interference for the phenotype of the tested strains,which effect the detection of the expression of report gene GFP.2 The exploration of BS168 sporuLation and biofilm formation via sRNA Igr3927 over expressionLink the Bacillus amyloliquefaciens FZB42 s RNA Igr3927 which is amplified by PCR method with the vector pDG148 stored in our laboratory to construst the sRNA induced plasmid p JCL03,with the empty vector as control.Transfer the recombinant plasmids into Bacillus subtilis BS168 seperately.Observe the effect on BS168 sporuLation and biofilm formation when s RNA is induced expressed in different humidity and temperature.The resuLts show that,the over expression of s RNA Igr3927 had a significant influence on BS168 spores,but under different humidity conditions,the over expression of sRNA did not increase the amount of spores produced by BS168.In addition,the over expression of sRNA has significant effect on biofilm formation under different temperatures.In the range of the tested temperature,the biofilm formation increases more significantly with the test temperature increasing.By killing the nutrient cells through high temperature of 85 degree,we analyse the relationship between BS168 and the over expression of sRNA.The experimental resuLts show that the spores quantity of sRNA over expression strain was 625 times higher than the control group.3 Certify the target gene of sRNA Igr3927Amplify the target gene spolVA with PCR method.The target gene is detected in our laboratory before with a list of methods-knockdown of sRNA,total protein SDS-PAGE and bioinformatics analysis.Then,construct the recombinant plasmid pFB36,pFB37 with the two region predicted the binding region of Igr3927 on the target gene spolVA and report gene GFP for fusion expression.Transform into Bacillus BS168.Obtain the mutant strain of Bacillus through homologous recombination.In addition,the inducible expression plasmid pJCL03 and empty vector were also transformed into BS168,and construct the target gene detection system.Through the induction of IPTG to observe the expression of GFP.The resuLts showed that,over expression of sRNA Igr3927 significantly enhanced the expression of report gene GFP.We argued that spolVA is the target gene of FZB42 sRNA Igr3927.