Transcriptome sequencing of fat-1 transgenic bovine fetal fibroblasts and wild-type

Purpose:The fatty acid dehydrogenase fat-1 gene derived from Caenorhabditis elegans encodes n-3 polyunsaturated fatty acid dehydrogenase and catalyzes the 18-20 carbon n-6 polyunsaturated fatty acids to generate corresponding n-3 PUFA balance ratio between n-6 PUFA and n- 3 PUFA in fat-1 transgenic cells. Changes in fatty acid composition will affect the entire metabolic system. We used transcriptome sequencing to analyze the expression of which genes are altered by fat-1 transgenic cells, and which metabolic pathways these genes mainly aggregate in. Methods:Total RNA of bovine fetal fibroblast cells was extracted witth Trizol reagent (Invitrogen, CA, USA). Poly (A) RNA was purified from total RNA (5μg) by poly-T oligo-attached magnetic beads and broken into small fragments. The spliced RNA fragments were reverse-transcribed to construct the final cDNA library and then the paired-end sequencing on IlluminaHiseq4000 (LC Sceiences, USA). The read values of the samples were aligned with the UCSC (http://genome.ucsc.edu/) reference genome using HISAT package, which initially removed a portion of the reads based on the quality information accompanying each read value, and then mapped the reads to the reference genome. All the transcripts in the samples were combined and a comprehensive transcriptome was reconstructed using Perl Scripts, and StringTie and edgeR to estimate expression levels of all transcripts. The differentially expressed mRNAs and genes with log2 (fold change) >1 or log2 (fold change) < -1 and with statistical significance (P value < 0.05) were screened by R package. Results:Total of 27,366 differential genes were obtained, among which 1157 genes significantly up-regulated and 1,200 genes significantly down-regulated . KEGG pathway enrichment showed that ECM-receptor interaction, axon guidance, and MAPK signaling pathway were significantly upregulated . GO enrichment revealed the differential genes were associated with cell migration, positive regulation of cell migration and substrate adhesion-dependent cell spreading. Genes related to oxidative phosphorylation, thermogenesis, and TCA were down-regulated, and these genes were involved in oxidation-reduction process, single-stranded DNA binding. Interestingly, we found many items related to ATP synthesis, oxidation-reduction process, proton-transporting ATP synthase complex, coupling factor F(o), ATP synthesis coupled proton transport pathways were significantly changed between fat-1 transgenic cells and wild-type cells . Conclusions:These results suggest that the transgenic fat-1genes not only change the fatty acid composition but also regulate and influence energy metabolism, possibly the ATP synthesis pathway. Transcriptome sequencing of two cell types and three samples of each type.