Transcriptomic Difference Analysis In Tibetan Chicken Embryo At Hypoxia

Tibetan chicken have lived at Qinghai Tibetan plateau for thousands of years, and the hatchability of the Tibetan chicken in hypoxia is higher than low land chicken. They have a distinctive suite of traits are clearly the result of adaptation to this environmental hypoxia. These physiological traits are often complex traits with a polygenic basis. Although hypothesis-driven studies of candidate genes have made important contributions to our understanding of the mechanisms of high-altitude adaptation of Tibetan chicken, but the genetic basis of the coordinated work of many interacting genes remains unknown. Here we profiled the heart and liver transcriptomes of embryos of the Tibetan Chicken and low land chicken which were incubated in hypoxia with the13%oxygen concentration by RNA-Seq, to uncover the novel gene which relate to the adaptations to the Tibetan plateau.Our RNA-Seq results,26171genes termed as expressed genes were identified in all heart samples of chicken embryos, in total. Genes accorded with the following criteria were termed as differentially expressed genes(DEGs):padj<0.05.652DEGs between Tibetan Chicken(T) and White Leghorn(W), and167DEGs between Tibetan Chicken(T) and Silky Chicken(S). Compared with T, W and S are both low land breeds.6DEGs were randomly selected for real-time RT PCR to validate the gene expression data generated by RNA-seq. Correlation analysis revealed that the RNA-seq data was greatly consistent with the real-time PCR results. We compared the gene ontology annotation and KEGG pathway enrichment of DEGs among the three breeds pairwise. The results show that these DEGs trend enrich in HIF signaling pathways, ECM-receptor interaction, fatty acid metabolism, linoleic acid metabolic pathways, etc. The DEGs enriched in response to biotic stimulus, transferase activity, nucleic acid metabolic process, etc.The results of chicken embryo liver transcriptome of three breeds show that a total of14580common transcripts were expressed in all three breeds. According to the employed cut off(Pvalue<0.05and FDR<0.1),222transcripts were identified as differentially expressed genes between T and W,171DEGs between T and S. The pathway analysis revealed that these genes belong to Metabolic pathways, Metabolism of xenobiotics by cytochrome P450, Alanine, aspartate and glutamate metabolism, etc. and gene ontology annotation related to response to stimulus, glutamine family amino acid metabolic process, long-chain fatty acid transport etc.In our RNA-Seq results, glutathione peroxidase8(GPx8), glutathione S transferase(GST) were termed as DEGs between T and W in the heart, liver tissuses, respectively. Activities of GPx, GST, and GR and levels of glutathione were determined spectrophotometrically. Under normoxia conditions and hypoxia(13%oxygen concentration) conditions, the activities of glutathione peroxidase and glutathione S-transferase in Tibetan Chicken were higher than those of the lowland chicken. The activities of glutathione peroxidase and glutathione S-transferase between the Tibetan chicken and the low land chicken under hypoxia(13%oxygen concentration) conditions were dramatically higher than those under normoxia conditions.Some DEGs screened from heart RNA-Seq were enriched in AKT-mTOR pathway and Ca2+signaling which involved in the pathway regulated the expression of hypoxia-inducible factor (HIF-1). Recently, researches revealed that the gene prolyl hydroxylase2(EGLN1/PHD2) from the hypoxia-inducible factor (HIF) pathway involved with hypoxia adaptation of Andeas and Tibetans. To analysis gene expression of EGLN1gene of Tibetan Chicken and White Leghorns and its effect on hypoxia adaptation of Tibetan Chicken, the chickens were incubated to days16separately in hypoxia and normoxia. Real-time PCR, western blotting techniques were directed to determinate the expression of EGLN1gene of embryo liver. The expression of livers with White Leghorn of EGLN1gene in the normoxia condition was significantly higher in value than that of hypoxia condition(P<0.01). Oxygen concentration of15%had a significant different effect on the expression of livers between the White Leghorn and Tibetan Chicken(P<0.05). The results showed that the protein expression of EGLN1gene in the liver was declined with the decreasing of oxygen concentration. In hypoxia, expression of EGLN1gene in white leghorn is lower than that of Tibetan chicken, and the trend was similar with the expression of the mRNA in liver.In the present study, differential gene expression analysis showed the potential genes which affect metabolic pathways, HIF signaling pathways, response to stimulus. The results indicate that the Tibet chicken responded better than the Silky chicken to oxidative stress in extreme hypoxia through glutathione enzymes of detoxification. Accordingly, to response the stress of hypoxia, the chicken decreased the expression of EGLN1gene to change the expression of HIF-1. This work provides reference for future studies on the association of genetic basis with the Tibetan Chicken adaptation to hypoxia. This work provides reference for future studies on the association of the EGLN1gene expression with the adaptation to hypoxia.