Transcriptome Study Of Aspergillus Oryzae RIB40 Based On RNA-Seq Technology

Aspergillus oryzae, an excellent strain used in Chinese traditional brewing industry, owns powerful capacity of protein expression and secretion, and has important applications in the field of grocery, beverage, cosmetic and medicine. And A. oryzae becomes an attractive expression system for heterologous recombinant protein genes, as the development of its genetic transformation system. However, the transcriptome research of A. oryzae progressed slowly. The research by EST and microarray technology, due to their limited depth of coverage and sensitivity, could not elucidate the functional complexity of A. oryzae transcriptome. Furthermore, it was still unknown why there were remarkable differences in growth phenotype and protein expression and secretion between solid-state culture (SC) and liquid culture (LC) in A. oryzae. And it was difficult to unpuzzle the fact that the expression level of heterologous genes in A. oryzae is lower when compared with homologous gene expression. Herein, in order to address the above fundamental issues in A. oryzae, the high-throughput RNA sequencing technology (RNS-Seq) was utilized to interrogate A. oryzae transcriptome. We hope to provide clue to the above problems on the transcriptome level.RNA samples of A. oryzae cultured under different conditions were sequenced by RNA-Seq technology. The obtained sequence fragments (reads) represent 145-fold A. oryzae genome lengths. Uniquely mapped reads accounts for >90% of all reads. The A. oryzae genome-wide transcriptome map was depicted according to the reads match in A. oryzae genome and genes. 11263 (93.28%) of the total 12074 A. oryzae protein-coding genes had transcriptional activity based on the RPKM value.The transcript structures of A. oryzae genes were analyzed on the genome scale based on RNA-Seq data. The results defined or extended 5′UTRs for 4198 transcribed genes and 3′UTRs for 4357 transcribed genes in A. oryzae, and the length distribution of UTRs and their functional categories were studied. 1345 upstream open reading frames (uORF) and 272 upstream initiation codons (uATG) were detected in the identified 5′UTRs. The amount of genes with uORF accounted for 11.14% of the total number of A. oryzae protein-coding genes, twice of that in yeast. The results also identified 1166 novel transcripts and 800 new exons in 513 annotated genes. 700 of the 1166 novel transcripts were likely to be protein-coding genes, based on the prediction by Augustus software.Alternative splicing (AS) of A. oryzae gene transcripts were analyzed on the genome scale based on RNA-Seq data. 1375 AS events were detected in 1032 A. oryzae genes (8.55% of all A. oryzae genes). This is the highest amount reported among fungi up to now. Retained intron (RI) accounted for the largest number of AS events (91.56%) in A. oryzae, while RI is a rare AS event in mammals. RI event is a feature of A. oryzae AS events, which was different from high eucaryotes. Based on the comparison of the amount and the length distribution between RIs and cassette exons (CEs), it is suggested that A. oryzae might perform splice site recognition predominantly by the intron definition (ID) mechanism.The differentially expressed genes between A. oryzae cultured in solid-state culture condition (SC) and liquid culture condition (LC), were analyzed based on RNA-Seq data. There were 4628 differentially expressed genes (p-value<0.001) between the two basic culture conditions (SC and LC), with 2355 up-regulated in SC and 2273 up-regulated in LC. GO functional enrichment analysis and KEGG pathway analysis of these genes, illustrated that the capacities of protein translation and modification and energy metabolism were much more powerful in SC than in LC. By comparison of the differentially expressed genes between SC and LC under ER stress (DTT treatment), the protein secretion model of A. oryzae under ER stress was depicted. The regulation of protein secretion in A. oryzae under ER stress was also analyzed, and the results illustrated that the regulation capacity for A. oryzae protein secretion was much more powerful in SC than in LC.The A. oryzae transcriptome was interrogated by RNA-Seq technology in this research. The results provided much information about gene structures, novel transcripts, metabolic pathways, alternative splicing events, protein expression and secretion and so on, which were valuable for such studies in A. oryzae as fundamental biological research, gene engineering and industrial applications.