Project description:Objective: Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of GO analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. This study aimed to investigate the integrative function of the aflR gene in A. flavus. Design: In this study, we used Aspergillus flavus NRRL3357 as a wild-type strain (WT) and constructed a knockout strain of A. flavus ΔaflR by homologous recombination. Based on the transcriptomics technology, we investigated the metabolic effects of aflR gene on growth, development and toxin synthesis of A. flavus, and discussed the overall regulation mechanism of aflR gene on A. flavus at the transcriptional level. Results: The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. In addition, disrupted strains of the aflR gene produced relatively sparse conidia and a very small number of sclerotia. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly down-regulated. Meanwhile, the ΔaflR strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT strain. Conclusions: The results showed that the A. flavus aflR gene also played a positive role in the growth and development of fungi.
Project description:RNA-seq was used to compare differential gene expressions for Aspergillus flavus wild type strain and ASPES transcription factor deletion strains.The goals of this study are to explore the aflatoxin regulation pathway in A. flavus.
Project description:Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs. Background The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis. Results Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs. Conclusions This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies. Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available.
Project description:Aspergillus flavus and A. parasiticus are two of the most important aflatoxin-producing species that contaminate agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here, we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four out of forty-five attempted interspecific crosses between compatible mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were isolated and were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of putative allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important species.