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Expression of target genes in itaconic acid cluster and electron transport chain in Aspergillus terreus NRRL1960 cultivated under low dissolved oxygen

ABSTRACT: Aim: We aimed to study the differential gene expressions of the target genes encoding H+-ATPase, the mitochondrial respiratory chain complex, and the alternative oxidase (AOX) in Aspergillus terreus NRRL1960 cultivated under low dissolved oxygen Methods:The fermentation samples were collected for RNA extraction.The barcoded RNA libraries were prepared using Lexogen’s Quant-Seq 3’ mRNA seq kit (Ion Torrent, Lexogen, Vienna, Austria). DNA templates for sequencing were prepared from 200 bp v3 OT2 kit and the Ion One Touch 2 platform. Sequencing was performed on the Ion Proton by Ion Torrent Suite v5.0.4. Raw sequences form each sample were uploaded and the dapter sequences were trimmed. The reads were aligned to A. terreus NIH2624 genome references downloaded from http://fungi.ensembl.org/info/website/ftp/index.html using Star 2.4.1d. The bam index files were generated. The quantification of gene expression was performed by Htseq v0.6.0 with -f and -s options indicating bam inputs and un-stranded reads, respectively. Cuffdiff v.2.1.1 was used to estimate the transcript abundance with the -no-diff and default options to generate the differential analysis for each described comparison. Results:From the gene expression (FPKM) results, genes in Cytochrome C complex and aoxA had the high expression level compared to pma encoding H+-ATPase. However, the different gene expression analysis results shown that the target genes in the mitochondrial respiratory chain complex were predominantly down regulated as observed from the log2(fold_change) less than -1 and and the gene encoding AOX (aoxA) were down regulated. Conclusions: In this study, the transcriptomic analysis were performed for better understanding the biosynthesis pathway and the regulation of target genes in itaconic acid cluster and the ATP regeneration pathway.

ORGANISM(S): Aspergillus terreus

PROVIDER: GSE135329 | GEO | 2019/08/05

REPOSITORIES: GEO

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Project description:Purpose: to reveal the small RNA profile of postnatal retina in Wistar WU albino rat by Ion-Torrent PGM sequencing technology. Methods : Wistar WU rats ranging in age from P1 to P21 were anesthetized by inhalation using Forane prior to sacrifice at the same hour to avoid circadian variation. Total RNA was extracted from the retinal tissues of various developmental stages using NucleoSpin miRNA kit (Macherey–Nagel, Düren, Germany) following manufacturer's instructions. The miRNA concentration was assessed using Qubit microRNA Assay Kit (ThermoFisher Scientific, MA, USA). The RNA integrity number (RIN), an algorithm for judging the integrity of RNA samples, were evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) following the manufacturing instruction of the RNA 6000 Nano kit and RIN>7 was considered acceptable. MicroRNAs was also determined using the Agilent Small RNA Kit to have deeper view in the 10 to 40-nucleotide size range. Small RNA library construction from pooled retinal samples (N=3, in each age-group) were carried out according to the Ion Total RNA-Seq Kit v2 protocol (Revision E) with slight modification. Enzymatic reaction was performed in half reaction volume, while cleaning procedures were executed with the accurate final volume according to the protocol. The Ion 316 or 318 ™ Chip v2 was used for sequencing on the Ion Torrent PGM™ instrument according to the protocol of Ion PGM™ Hi-Q View Sequencing Kit. To assess reliability of miRNA workflow solution in 316 v2 chip, two independent P7 samples were assayed (biological replicates) and P21 sample were sequenced as technical replicates in every sequencing procedure. Some samples were also sequenced in 318 v2 chip (P3, P5, P10, P15 and P21) as biological replicate. Ion Torrent Suite Platform was used to trim the raw sequence data and remove any residual sequencing adapter fragments that remained on the 5′ or 3′ ends. Reads were mapped to the non-coding RNAs from ENSEMBL [Rnor_6.0 (GCA_000001895.4)] using TMAP algorithm. These aligned BAM (Binary Alignment Map) files were further processed in Galaxy Web-based platform (Afgan, 2018) via Cufflinks, Cuffmerge and Cuffdiff (Version 2.2.1.3) application (Trapnell, 2010). Further analysis and visualising of the datasets was carried out in R Studio Software environment. qRT–PCR validation was performed using TaqMan assays.

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Expression of target genes in itaconic acid cluster and electron transport chain in Aspergillus terreus NRRL1960 cultivated under low dissolved oxygen

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