| Siderophores have the high affinity to iron,then they are widely applied in medicine,agriculture industries and soil remediation,especially in the biological control of pathogenic fungi.Comparing with synthetic chelating agent,siderophores are environment friendly compounds that makes them have more widely and importance application prospects in environmental remediation.In the thesis,the Bacillus amyloliquefaciens SYBC H47 separated from honey has ability to secrete siderophore.It was then mutagenized by the atmospheric and room temperature plasma method?ARTP?to screen strains with remarkable increase in siderophore production.After that was the research of optimization of fermentation conditions;the cell proteins of mutant strains were analyzed by SDS-PAGE and protein mass spectrometry;genes that might lead to the increase of siderophore production of the mutant strain T7-14 were analysied and checked.The main results were as follows:1)In order to improve the yield of siderophore,the strain B.amyloliquefaciens SYBC H47 was induced by ARTP two times.Then 15 mutants with high-yield of siderophore were screened through the CAS double-layer plate and 96-well plate,followed by further screening with shake flask,and the siderophore production of the mutant T7-14 was the highest,reaching 5.98 mmol·g-1,which was increased by 155.0% compared with the original strain.2)The fermenation medium was optimized through single factor experiment and the optimum medium recipe was as follows: sucrose 15 g·L-1,glutamine 1.8 g·L-1 and pH 7.2.The yield of the optimized siderophore production was 14.4 mmol·g-1,which was 140.8% higher than that of the strain grown in the medium before optimization.3)The whole cell proteins of original strain and the mutant T7-14 in different culture periods were analyzed by SDS-PAGE.Seven proteins significantly increased in expression,were analyzed by protein spectroscopy.And Dhb A as one of them was involved in the synthesis of the catechol-type siderophore precursor DHB.4)The gene sequences and amino acid sequences of B.amyloliquefaciens SYBC H47 were analyzed by Blast.The results showed that there were thirty-four genes involved in siderophore production.And two of the thirty-four genes were regulation genes,twenty-five were associated with the transportation of siderophore or the complex of Fe-siderophore,seven of them were synthetic genes,included the gene cluster dhbACEBF.And it was predicted that the siderophore might be bacillibactin because of the gene cluster of dhb ACEBF.Moreover dhb A,dhbB and dhb C were involved in the synthesis of siderophore precursor DHB.The length of the three genes are 786 bp,927 bp and 1197 bp,respectively.The molecular weight of Dhb A,DhbB and DhbC were predicted to be 27.1 kDa,34.2 kDa and 42.8 k Da.Then dhb A,dhb B and dhb C were cloned to the p Cold? vector and transformed into E.coli BL21?DE3?,which were induced by IPTG and 15 °C to express the target genes.The siderophore yield was improved at different degrees.Comparing with the empty vector E.coli BL21?DE3?,the siderophore yield of the three recombinant strains were increased by 90.1%,39.6% and 209.2%,respectively.It was speculated that the overexpression of Dhb A could increase the yield of siderophore to some extent. |
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