Comparative transcriptomic and proteomic analyses provide insights into functional genes for hypoxic adaptation in embryos of Tibetan chickens

Background/Aims: Tibetan chickens, a unique plateau breed, have good performances to adapt to high-altitude hypoxic environments. A number of positively selected genes have been reported in Tibetan chickens; however, the mechanisms of gene expression for hypoxia adaptation are not fully understood. Methods: Eggs from Tibetan (TC) and Chahua (CH) chickens were incubated under hypoxic and normoxic conditions, and vessel density index (VDI) in the chorioallantoic membrane (CAM) of embryos was measured. Meanwhile, Transcriptomic and proteomic analyses of CAM tissues were performed in TC and CH embryos under hypoxic incubation using RNA-seq and iTRAQ. Results: We found that the vessel density index (VDI) in CAM of TCs was lower than in CHs under hypoxia incubation. In the transcriptomic and proteomic analyses, 160 differentially expressed genes (DEGs) and 387 differentially expressed proteins (DEPs) 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 adaption of chicken embryos. Conclusion: Combination of transcriptomic and proteomic data revealed several key candidate regulators and pathways that might play high-priority roles in the hypoxic adaptation of Tibetan chickens by regulating angiogenesis and promoting blood circulation, thus explaining the blunt responses to hypoxic conditions on CAM angiogenesis in Tibetan chicken embryos. This research provided insights into the molecular mechanism of hypoxia adaptation in Tibetan chickens. Chorioallantoic membrane (CAM) of embryos mRNA profiles of 11-day old Tibetan chickens (TCs) and Chahua chickens (CHs) were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000 platform.

背景/目的：藏鸡（Tibetan chickens）是我国特有的高原禽种，具备优异的高海拔低氧环境适应能力。目前已有多项研究在藏鸡中报道了正向选择基因，但针对其低氧适应的基因表达调控机制仍未完全明晰。
方法：本研究将藏鸡（Tibetan chickens，TC）与茶花鸡（Chahua chickens，CH）的种蛋分别置于低氧（hypoxic）与常氧（normoxic）环境下孵化，并检测胚胎尿囊绒膜（chorioallantoic membrane，CAM）的血管密度指数（vessel density index，VDI）。同时，采用RNA测序（RNA-seq）与同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation, iTRAQ）技术，对低氧孵化条件下藏鸡与茶花鸡胚胎的CAM组织开展转录组学与蛋白质组学分析。
结果：本研究发现，低氧孵化环境下，藏鸡胚胎CAM的血管密度指数显著低于茶花鸡。转录组与蛋白质组学分析共筛选得到160个差异表达基因（differentially expressed genes, DEGs）与387个差异表达蛋白（differentially expressed proteins, DEPs），这些分子主要富集于血管生成（angiogenesis）、血管发育（vasculature development）、血管形态发生（blood vessel morphogenesis）、血液循环（blood circulation）、肾素-血管紧张素系统（renin-angiotensin system）以及缺氧诱导因子-1（HIF-1）与血管内皮生长因子（VEGF）信号通路。此外，本研究鉴定出26个参与血管生成与血液循环的基因、2个离子转运相关基因以及6个调控能量代谢的基因，上述分子均可作为调控鸡胚胎低氧适应的候选功能基因。
结论：转录组与蛋白质组联合分析揭示了多个关键候选调控因子与通路，它们可能通过调控血管生成、促进血液循环，在藏鸡低氧适应过程中发挥核心调控作用，由此阐明了藏鸡胚胎CAM血管生成对低氧环境的钝性响应机制。本研究为阐明藏鸡低氧适应的分子机制提供了新的理论视角。
本研究采用Illumina HiSeq 4000测序平台，对11日龄藏鸡与茶花鸡胚胎的CAM组织开展深度测序，设置3次生物学重复，获取了胚胎CAM的mRNA表达谱数据。