Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生相关联

为探讨黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生的关系,收集黄曲霉菌、米曲霉菌和寄生曲霉菌若干株。在有利于黄曲霉毒素产生的条件下培养后,提取各菌株的总RNA,RT-PCR法检测aflR基因的mRNA表达水平;并应用ELISA法检测各菌株产生黄曲霉毒素B1的情况。提取各菌株的基因组DNA,PCR扩增aflR基因启动子序列并测序。应用基因分析软件将不产毒素的黄曲霉菌与产毒黄曲霉菌的aflR基因启动子序列进行比较,找出不产毒菌株aflR基因启动子序列的变异位点。ELISA法和RT-PCR法结果表明,产毒的黄曲霉菌菌株均有明显的aflR基因转录,而在2株不产毒的黄曲霉菌菌株中,一株aflR基因无转录,另一株仅有较低水平的转录。序列比较结果表明,不产毒黄曲霉菌菌株的aflR基因启动子序列存在如下共同变异位点:-90、-236、-253、-262、-282位。米曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均为阴性,并且其aflR基因启动子序列中存在与上述不产毒黄曲霉菌菌株相同的变异位点。寄生曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均呈阳性,并且其aflR基因启动子序列的上述5个位点与产毒黄曲霉菌完全一致。在不产毒素的黄曲霉菌aflR基因启动子序列中发现了5个共同变异位点,实验结果提示这些变异位点可能与黄曲霉毒素的产生有关。     

黄曲霉毒素生物合成径调节基因aflR

黄曲霉毒素生物合成途径调节基因在黄曲霉毒素产生过程中发挥十分重要的作用,它为绝大多数黄曲霉毒素合成相关基因的表达所必需。黄曲霉毒素生物合成途径调节基因的启动子中,含有若干真菌转录因子同源物的假定结合位点。AflR蛋白是黄曲霉毒素生物合成途径中的主要正性转录因子,它调节大多数黄曲霉毒素合成相关基因,也包括其自身基因的表达。     

Detection of Aflatoxin in Aspergillus Species Isolated from Pistachio in Iran

To estimate the incidence contamination of fresh pistachio nuts by aflatoxigenic fungi in Iran, nut samples were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan regions. Out of the 200 Aspergillus isolates obtained, 11 species were identified as A. alliaceous, A. candidus, A. flavus, A. niger, A. niveus, A. ochraceus, A. parasiticus, A. tamari, A. terreus, A. unguis and A. wentii. For detection of aflatoxin production ability of the isolates, three target genes, namely aflR, aflJ, and omtB, used in PCR amplification. In all the examined cases, the degenerate primer designed for amplification of omtB gene, named omtBII, was able to amplify an expected 611 bp fragment in aflatoxigenic isolates in this study and yielded the same result as those obtained from TLC analysis and fluorescence ability by application of methylated β‐cyclodextrin in culture media. Using this procedure the significant incidence of aflatoxin‐producing aspergilli was confirmed in pistachio nuts produced in different regions of Iran. The results indicated that PCR method described here, in combination with fluorescence assay, is a reliable and simple confirmatory test for monitoring pistachio nuts contaminated with aflatoxinogenic aspergilli.