2010 Annual Report
1a.Objectives (from AD-416)
Sequence the whole genome of Aspergillus flavus and analyze the genome in comparison with closely related Aspergillus species such as A. oryzae in their genome structure, gene sets, and gene functions. Construct whole genome Aspergillus flavus microarray and whole genome Aspergillus flavus/peanut microarray. Perform high throughput gene profiling and functional genomics studies using these microarray resources.
1b.Approach (from AD-416)
Aspergillus flavus genome size is about 36 Mega basepairs. Genomic DNA will be isolated and size-fractionated. Different libraries with 40 kb, 10 kb and 2 kb DNA inserts will be constructed. Sequence will be determined by shotgun sequencing strategy and assembled by J. Craig Venter Institute (JCVI) assembler software. The sequence will be annotated and the putative coding sequences will be identified with the help of the A. flavus and A. oryzae Expressed Sequence Tag (EST) data as well as A. oryzae gene model. Comparative analysis of the A. flavus genome will be made in reference to the A. oryzae genome using the Sybil software developed by JCVI. The A. flavus whole genome oligo microarray will be designed according to the annotated putative coding sequences. Those unique genes in A. oryzae but absent in A. flavus will be included in addition to some of the identified corn genes that showed resistance to A. flavus infection in the whole genome microarray design. A total of over 20,000 peanut EST sequences that represents about 10,000 unique peanut ESTs will be cleaned and assembled at JCVI. 70mer oligoes will be designed from these unique ESTs for a comprehensive peanut/A. flavus microarray construction. This microarray will include all of the currently available peanut ESTs and identified peanut genes, all genes in the A. flavus whole genome, unique set of genes in the A. oryzae genome, and some corn genes of interest as well. Gene profiling experiments and related high throughput functional genomics studies will be performed at JCVI in the cooperator’s laboratory.
The major objective of this project is to sequence all the deoxyribonucleic acid (DNA) in the Aspergillus flavus genome and to prepare microarrays. These arrays are for use in further research in understanding the aflatoxin contamination process in crops for controlling aflatoxin contamination in all vulnerable crops including corn, cotton, peanut, and tree nuts.
For examining all the genes and the genetic mechinery involved in the production of the harmful (carcinogenic) compound aflatoxin by the fungus Aspergillus (A.)flavus on crops, the entire fungal deoxyribonucleic acid (DNA) was determined through whole genome sequencing at J. Craig Venter Institute (JCVI) in collaboration with North Carolina State University. Primary analysis of the DNA indicated that the Aspergillus flavus genome size is about 36.8 Mega Base pairs. Comparing the whole genome between the fungus Aspergillus flavus and Aspergillus oryzae (a non-toxigenic food grade industrial organism) demonstrated that the genome size, genome structure, gene categories, and gene homology are quite similar. However, each species contains a unique set of about 300 genes. The ones in A. flavus may contribute to aflatoxin production.
Using glass slides contaning all the genes of the fungus (microarray fabricated at JCVI), genome wide gene profiling experiments have been conducted under specific conditions that favor aflatoxin production in the fungus. Genes and gene clusters that are putatively involved in aflatoxin formation have been identified. Several research papers have been published and a few manuscripts are under preparation.
In parallel with microarray gene profiling experiments, we are currently shifting to Next Generation Sequencing technologies to identify genes potentially involved in the formation and regulation of aflatoxin production. Preliminary results by the technique ribonucleic acid (RNA)-Seq (Illumina) revealed the mechanism of aflatoxin production under specific temperature regimen. Under high temperature, the expression of specific regulatory genes, aflR and aflS, is significantly reduced. The change in ratio of aflR to aflS in high temperature is the main reason for shutting off aflatoxin production.
The DNA sequence data obtained A. flavus expressed sequence tags (EST) and whole genome sequence have been submitted to National Center for Biotechnology Information (NCBI) GenBank (genetic sequence) database. The microarray data are also submitted to the NCBI Gene Expression Omnibus (GEO) database. A database web server containing Aspergillus flavus EST and whole genome databases has been established at the Mid South Area Genomics Center for free access by United States Department of Agriculture/Agricultural Research Service scientists. Progress by cooperators was monitored through routine teleconferencing, meetings, and scientific presentations of recent findings to the project.