| Aspergillus flavus is an opportunistic pathogen,which is one of the major causes of food contamination around the world.Development and the formation of secondary metabolites of Aspergillus flavus are affected by many environmental external factors,in which the osmotic pressure is one of the most crucial factors.Although the mechanism of osmotic pressure has been studied more thoroughly in S.cerevisiae,how to respond to changes in osmotic pressure and how toxigenic regulatory mechanism is changed in osmotic pressure are not yet known in A.flavus.Therefore,this research pays attention to mitogen-activated protein kinase kinase(MAPKK)AflPbsB in the high osmotic glycerol(HOG)response pathway of A.flavus.In this study,A flavus pbsB gene knockout mutant(?pbsB)was constructed using the principle of homologous recombination.The results showed that the growth of mutants was suppressed compared to wild-type strain.The results also shown that the number of sclerotium in ApbsB was significantly decreased compared to wild-type strain.The number of sclerotium in wild-type strain was even as twice as that of the mutants.The results of QPCR showed that expression of sclerotia related gene nsdC in ApbsB was decresed at the transcription level.And the results demonstrated that aflatoxin producing capacity of ApbsB mutant decreased significantly.As far as the concentration of the toxin,the wild-type strain was more than twice of mutant's.But knockout strains didn't lose toxigenic ability completely.It was still able to produce a small amount of toxin.At the transcription level,the relatively expression of two toxin synthesis structure genes(aflD and aflQ)were down regulated.In terms of number of spores,there was also a significant difference between ApbsB mutant and the wild-type strain.The number of spores of wild-type strain was over one time than mutant's.Comparing the morphology of microscopic conidiophores,it was found that conidiophores of the wild-type strain developed more completely and dense compared to ApbsB mutant.And ApbsB mutant's number of conidiophores was less.Besides,the length of spore stem of ApbsB mutant was shorter and the number of spores at the top of stems was significantly less than wild-type strain.In response to osmotic stress,OpbsB mutants became more sensitive to osmotic pressure due to the lack of MAPKK in the HOG pathway,so that it influenced by hypertonic stress a lot.In the medium added sodium chloride,inhibition rate to growth of the ApbsB was higher than that of wild type.The inhibition rate of WT was 33.96%,and the inhibitory rate of knockout strains were 37.50%and 40.98%,respectively.In the medium added sorbitol,the inhibition rate of ?pbsB strains was also higher than the wild type.Inhibitory rate of WT was 10.93%,whereas those of knockout strains were 41.62%and 30.15%,respectively.Moreover,the impact on wild-type by sorbitol was less than impact by the sodium chloride suppression.In this study,the ability of the ApbsB strains to infect peanut seeds and maize kernel were examined.The results shown that the infection ability of A.flavus strains decreased significantly for lacking of pbsB gene.The wild-type strain tended to produce more mycelia and spores.Its toxin biosynthesis ability was also stronger than ?pbsB strains.All in all,MAPKK AflPbsB protein in HOG pathway is critical for A.flavus.Lacking of pbsB gene,Aspergillus flavus strain's ability to grow,to produce sclerotium,to produce aflatoxin,to produce spores,to adapt to hypertonic stress and to infect will be decreased.The results provide a basis for elucidating the role of HOG pathway in the growth and aflatoxin producing of Aspergillus flavus,and it would lay a theoretical foundation for the prevention and control of A.flavus in the production and life. |
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