Abstract
The Tibetan chicken is a unique breed that has adapted to the high-altitude hypoxic conditions of the Tibetan plateau. A number of positively selected genes have been reported in these chickens; however, the mechanisms of gene expression for hypoxia adaptation are not fully understood. In the present study, eggs from Tibetan and Chahua chickens were incubated under hypoxic and normoxic conditions, and vascularization in the chorioallantoic membrane (CAM) of embryos was observed. We found that the vessel density index in the CAM of Tibetan chickens was lower than in Chahua chickens under hypoxia conditions. Transcriptomic and proteomic analyses of CAM tissues were performed in Tibetan and Chahua chicken embryos under hypoxic incubation using RNA-Seq and iTRAQ. We obtained 160 differentially expressed genes and 387 differentially expressed proteins that were mainly enriched in angiogenesis, vasculature development, blood vessel morphogenesis, blood circulation, renin-angiotensin system, and HIF-1 and VEGF signaling pathways. Twenty-six genes involved in angiogenesis and blood circulation, two genes involved in ion transport, and six genes that regulated energy metabolism were identified as candidate functional genes in regulating hypoxic adaptation of chicken embryos. This research provided insights into the molecular mechanism of hypoxia adaptation in Tibetan chickens.
Highlights
Identification and functional analysis of differentially expressed genes (DEGs)
The 160 DEGs were classified into two categories for the six samples, and the heat tones of fragments per kilobase of transcript per million (FPKM) values showed the expressed quantity of the DEGs have good repeatability within groups (Fig. S4)
The results indicated that six proteins were markedly up-regulated and 14 proteins down-regulated in the TC compared with CH, and the expression changes of all 20 proteins were consistent with fold changes in iTRAQ (Fig. S7, Table 2)
Summary
Animals and samples preparation.We collected eggs of TC and CH from the Experimental ChickenFarm of China Agricultural University (CAU). Eighteen CAM samples of Tibetan (n = 9) and Chahua chickens (n = 9) under hypoxic conditions were collected from embryos at day 11 of incubation, immediately frozen in liquid nitrogen, and stored at − 80 °C for sequencing. Tibetan and Chahua chickens incubated in the normoxic and hypoxic conditions. On days 6, 10, 14, and 18 of incubation, eggs were removed from the incubator and opened with a needle at the blunt end where the air sac is located. The VDI (intersections/mm) was determined from the number of intersections between all discrete blood vessels and three concentric circles having diameters of 2.05, 2.55, and 3.01 mm (total circumference = 23.9 mm)[28]. Eight embryos were measured for CAM images and VDI in each group at each time point
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