The Inhibitory Effect And Mechanism Of Bacillus Megaterium On Aflatoxin Biosynthesis

Aspergillus flavus is one of the major moulds that colonize crops in the field and during storage. This mold produces the most potent known toxic aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. Countries in the world put a lot of manpower and material resources dedicated to the research of reducing the contamination of aflatoxins. The biological control methods, which are highly efficient and little to environmental pollution, are more popular. In this paper, a strain of marine Bacillus megaterium isolated from the sea was evaluated for its inhibitory effect on Aspergillus flavus growth and aflatoxin formation. Microarray analysis was used to detect aflatoxin pathway genes expression in Aspergillus flavus. It has important significance in the research of reducing aflatoxin.(1) The effect of B. megaterium on aflatoxin biosynthesis and genes expression in A. flavus was tested in two types of peanut kernels, UF 715133-1 and Jinhua 1012, respectively. Aflatoxin was determined by HPLC analysis,then KEGG and qRT-PCR were used for gene classification and analysis. The results showed that more than 85% of aflatoxin B1 was inhibited by the treatment of B. megaterium in UF715133-1 and Jinhua 1012. There are some coding protease genes highly downexpressed, such as short-chain dehydrogenase, non-ribosomal peptide synthetase. So, this strain of marine Bacillus megaterium isolated from the Yellow Sea of East China could significantly inhibit the aflatoxin biosynthesis in peanuts through inhibiting genes expression in A. flavus. While the role of most highly downregulated genes in aflatoxin biosynthesis hasn't been clarified, the mechanism remains to be further studied.(2) The effect of B. megaterium on aflatoxin biosynthesis and genes expression in A. flavus was also tested in two types of medium, potato dextrose broth liquid medium and liquid minimal medium, respectively. Aflatoxin was determined by HPLC analysis, then KEGG and qRT-PCR were used for gene classification and analysis. The results showed that aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost (reduced by 99.75% and 99.80%, respectively) inhibited by co-cultivation with B. megaterium. Compared with the control groups, biomass were also reduced by 81.15% and 74.67%, respectively. Microarray data showed that CPA biosynthesis related genes expression were downregulated in the experimental groups of PDB and MM media.(3) In this study, we selected three around 400 bp segments in Aspergillus flavus DNA as probes, and the 5' end were biotinylated. B. megaterium was incubated in beef extract peptone broth for 24 hours at 37? with shaking. Then the supernatant was harvested by centrifugation and concentrated by evaporation on a rotary evaporator, then dried with freeze dryer. Biotin-labeled DNA probes which was amplified by PCR, was used to interact with crude protein from B. megaterium. After the mixture of DNA and protein were electrophoresed on non-denaturing polyacrylamide gel, then the gel was transferred to nylon membrane. The complex of biotin-labeled DNA-protein was detected by chemiluminescence EMSA kit to determine the binding site. The results showed that the probe 3 combined with the metabolite of Bacillus megaterium and migration hysteresis occurred. The probe 3 was the DNA sequence which inhibitory protein of B. megaterium could bind to. It is evident that the metabolite of marine B. megaterium binds to the promoter position of aflS gene, so the aflS gene couldn't be transcribed and the biosynthesis of aflatoxin was interrupted.