Transcriptome Study Of Aspergillus Flavus CA43Based On Strand-specific RNA Sequencing Technology

Aspergillus flavus is a kind of conditional pathogenic bacteira due to its aflatoxinbiosynthesis, which could be infected with both crops and human. The fundmental researchin A. flavus could effectively prevent and cure the harm which was brought about to humanand agriculture by A. flavus. Sclerotia development in A. flavus is an importantphysiological process. Sclerotia development research is very important for further studyingmicrobial reproduction development and evolution. Furthermore, sclerotia development inA. flavus was closely related with a serious of secondary metabolic pathways such asaflatoxin biosynthesis. Advances in sclerotia development could contirbute to understandingaflatoxin biosynthesis and the bio-control of aflatoxin-producing strains. Recently, with theadvances in high-throughput genome sequencing technologies and the accomplishment of A.flavus genome sequencing, it is possible to study the regulation of A. lfavus physiologicalprecess and gene function using sequencing technologies.In this study, A. lfavus transcriptome under different physiological conditions werestudied using strand-specific RNA-seq technology. The high-resolution transcriptionalprofile was obtained by RNA-seq technology under basic cultivating condition, includinggene expression level, transcript structure and so on. Bioinformatic analysis of RNA-seqdata of A. flavus under different condition (aflatoxin-producing condition/aflatoxin-non-producing condition/mycelia condition/sclerotia condition) was done by GOfunction annotation, differential expression analysis and metabolic pathway analysis，toidentify the related genes and regulation network of sclerotia development and to constructthe biosynthetic model of aflatoxin by analyzing alfatoxin-related genes. The mainconclusions of this study are as follows.i) Strand-specific RNA-seq deeply profiles the genome-scale transcriptome of A. flavusThe genome-scale transcriptional profile oiA. flavus was obtained by mapping reads toits genome sequence. According to the RPKM value based on RNA-seq reads，9871geneswere detected on the transcirptional level among the13487protein-coding genes of A.lfavus，which accounts for73.19%of all A. lfavus genes. Gene structure of A. lfavustranscirpts were annotated based on RNA-seq data, of which UTR is the important annotation. In total，5’ UTR of5994genes and3‘UTR of6407genes were obtained，andUTR length distirbution was also analyzed. Abundant new transcirpts were identified andtheir function was analyzed. Furthermore, alternative splicing (AS) events was detected in A.lfavus transcriptome. The obtained1220AS events contained four types: skipping exon(SE)，retained intron (RI)，alternative5‘splicing site (A5SS) and;alternative3splicingsite (A3SS). The genes with AS events accounts for12.78%of all A. flavus multi-exongenes. A, flavus owned much more AS events than many other fungal strains. The ASmechanism was also analyzed.ii)Antisense transcripts in A. flavus transcirptomeAntisense transcripts (NAT) were detected in A. flavus transcriptome.1123and839NATs were detected in mycelia culture and sclerotia culture respectively, which were muchmore than the amount of A. flavus NATs based on EST data (352，2.8%). At the meantime,the role of NAT in gene regulation was analyzed. The protein expression, secretion andenergy production in A. flavus may be subjected to NAT regulation. This was the firstcomprehensive analysis of NAT in fungi.iii)Prediction of genes and network related with sclerotia development, and themolecular regulation mechanism of sclerotia developmentDifferentially expressed genes and their WEGO annotation identified14up-regulatedgenes and37down-regulated genes related with sclerotia development. It illustrated thatsclerotia development and secondary metabolism was inhibited with each other. Sclerotiawas closely linked with A. flavus reproduction, instead of a simple sexual vestige. Sclerotiadevelopment was regulated by complicated signal network, including antisense transcript.Furthermore, asexual sporulation was repressed under sclerotia condition. In total, RNA-seqdata provided rich information on sclerotia development. At the same time，sexual matingphenomena and sclerotia development in A. flavus demonstrated that A. flavus mightconduct sexual reproduction or it once owned sexual period, which could contribute tounderstanding the sexual reproduction, asexual reproduction and the genetic study of A.flavus strains.iv)Identification of aflatoxin related gene and construction of aflatoxin biosyntheticpathway model in A. flavus Results illustrated that all of aflatoxin pathway genes transcribed under myceliacondition，while three of them didnot transcirbe under sclerotia condition，suggesting thataflatoxin biosynthesis was inhibited under sclerotia condition. All genes related withaflatoxin in references was collected and to analyze their expression under mycilia andsclerotia condition, with11down-regulated genes and two up-regulated genes. According tothe previous study, these genes also participated in sclerotia development. Therefore, thesegenes might become the target genes linking sclerotia development and secondarymetabolisms. Due to the heavy burden on farmers resulting from aflatoxin contamination,these findings in this study could be used as the bio-control targets to inhibitaflatoxin-producing A. flavus strains.This study conld contirbute to deeply understanding the aflatoxin biosynthesis andsclerotia development in A. flavus. This was important for better understanding of A. flavusdevelopment and the bio-control of aflatoxin-producing strains.