The Screening And Identification Of A Non-aflatoxigenic Mutant Strain Induced By5-azacytidine

AFB1is a kind of hepatotoxin, the biosynthesis mechanism of AFB1is verycomplex. Methylation is an important chemical modification process which is closelyrelated with gene expression.5-azacytidine, an inactivator of DNA methyltransferase,was reported recently that5-AC could suppress the synthesis of AFB1obviouslyunder a relatively low concentration. In order to clarify the suppression mechanism, anon-aflatoxigenic mutant strain was obtained after treated with the DNA methylationinhibitor5-AC.Colony phenotypes of the A. flavus mutant (NT) were markedly different fromthose of the wild-type (WT) strain when grown on Czapek-Dox Agar medium andPDA. The mutant (NT) displayed a slightly flavescent conidial pigmentationcompared to the yellow of the wild-strain grown on Czapek-Dox Agar medium, whilethe mutants grown on PDA medium displayed no conidial pigmentation compared tothe dark green of the wild-type strain. In Czapek-Dox broth medium, the mutantshowed much bigger mycelial pellets than the wild-type. The kinetics of radial growthshowed that the mutant strain induced by5-AC exhibited vigorous growth on CzapekDox agar medium compared to the WT. Microscopic examination of the A. flavusmutant strains revealed markedly alterations in conidiophore morphology. Mycelia tiplength was longer for the mutant than the wild-type strain, and the average mycelia tiplengths for mutant and the wild-type strain were331.9±131.2μm and141.7±31.7μm, respectively. The distance of mycelium septa of the mutant was also longer thanthat of the wild-type strain, and the average length of mycelium septa distance for themutant and the wild-type strain were66.7±19.9μm and49.1±14.1μm, respectively.In addition to these, the AFB1production of A. flavus mutant (NT) is lower thanthat in wild-type, and no aflatoxin was even detected by TLC analysis after the mutantstrain was cultured in Martin Broth for10d. The result of the comparison betweenNT strain and wild-type strain, detected by qPCR, showed significant reduction bothin production and expression of genes of aflatoxin and conidia.Moreover, NT were more sensitive to strong oxidant, and the effect of oxidative stress to NT was verified by free radical accumulation and lipid peroxidation tests,which was associated with the loss of aflatoxin production, compared to the wild-type.These data indicate that5-AC, as an inactivator of DNA methyltransferase, plays avery important role on aflatoxin B1(AFB1) metabolism and the epigenetics ofAspergillus flavus. This work would make some contributions to the inhibitionmechanism of DNA methylation inhibitor on the synthesis of AFB1in A. flavus.