Abstract
Aflatoxins are extremely toxic and carcinogenic substances produced by mainly Aspergillus flavus and Aspergillus parasiticus.Contamination of agricultural commodities with aflatoxins is very serious problem all over the world including China.Elucidation of the biosynthetic mechanism of aflatoxins is very important as for how to control and prevent aflatoxin contamination of feed and food. Disruption of the genes involved in aflatoxin biosynthesis.Recently, two novel and short putative genes, hypB1 and hypB2, were discovered in the aflatoxin gene cluster in Aspergillus parasiticus.To investigate their function, I disrupted either hypB1 or hypB2 gene by gene replacement strategy.The resulting mutants were then compared with the wild type strain.The hypB1 as well as hypB2 gene was expressed in aflatoxin-conductive (YES) medium, but not in non-conductive (YEP) one, indicating their expressions were regulated by AflR.Aflatoxin productivity of the hypB1-disrupted mutant was about 10% of that of the wild strain, SYS4.Norsolorinic acid (NA) was also accumulated in the mycelia of the mutant.RT-PCR analysis showed that expression of nor-1 gene, a down-stream gene of the hypB1, slightly decreased in the mutant.Since the nor-1 gene is known to be involved in the reaction from NA to averantin, HYPB1 may be necessary for maximum transcription of the nor-1gene.In contrast, the hypB2-deleted mutant produced almost same amounts of total aflatoxins as those of the wild strain.However, ratio of the amounts of 2-group aflatoxins, aflatoxins B2 and G2, to 1-group aflatoxins, aflatoxin B1 and G1, were partly increased.Since the neighbor verB gene is known to be involved in the branching step from 2-group aflatoxins to 1-group aflatoxins, it suggested that deletion of the hypB2 gene likely affects expression of the verB gene.In fact, RT-PCR showed that expression of verB decreased in this disruptant.The hxtA and glcA gene are located within the sugar utilization gene cluster which is adjacent to the aflatoxin gene cluster.Aflatoxin productivity of the hxtA as well as glcA-disrupted mutant remarkably increased in the medium containing glucose as a carbon source, but not in other media containing xylose or glycerol.These results indicated that the hxtA and glcA gene are directly involved in the carbon source dependency of aflatoxin production. Purification of the pigment accumulated in mycelia of the norB gene-deleted mutant.The norB-deleted mutant accumulated a yellow pigment in the mycelia.I purified the pigment using thin layer chromatography and high performance liquid chromatography.Finally, about 0.2 mg of the purified substance was obtained.Analysis of the pigment by Nuclear Magnetic Resonance (NMR) demonstrated that the pigment has very similar structure to aflatoxin G1.
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