基于TMT蛋白组学及生物信息学分析牦牛抗冻差异蛋白

【目的】从蛋白水平阐明牦牛抗寒性能机理，并进一步从营养学角度提高其代谢性能，为牦牛有效抵御外界恶劣气候条件提供科学依据。【方法】应用TMT蛋白组学技术对寒冷季节（1月）和温暖季节（8月）的牦牛抗冻蛋白进行挖掘，并对鉴定到的牦牛抗冻蛋白进行亚细胞定位、结构域、GO功能富集、KEGG信号通路注释、蛋白相互作用等生物信息学分析。【结果】从牦牛耳组织中共鉴定获得21856个肽段（Peptide），其中特有肽段（Unique peptide）序列为18452个，定量获得4519个蛋白，最终筛选出144个差异蛋白，其中上调蛋白89个、下调蛋白55个。144个牦牛抗冻差异蛋白亚细胞定位到7个条目上，分别是细胞核蛋白56个、细胞质蛋白51个、质膜蛋白24个、细胞外蛋白23个、线粒体蛋白18个、细胞骨架蛋白1个和溶酶体蛋白1个；共鉴定到194个结构域。GO功能富集分析结果显示，生物过程主要富集到细胞过程蛋白79个、代谢过程蛋白70个和生物调控蛋白42个等，分子功能主要富集到结合功能蛋白75个和催化活性蛋白64个等，细胞组分主要富集到细胞部分蛋白89个和细胞蛋白89个等。144个牦牛抗冻差异蛋白在KEGG数据库中注释到205条KEGG信号通路，主要涉及核糖体、氮代谢、胞质DNA感受、动物体内生热作用、氧化磷酸化、白细胞介素-17及钙离子信号等通路。牦牛抗冻差异蛋白相互作用网络分析发现L8IHE5的关联度最高，且冷诱导RNA结合蛋白（CIRP）和HSP70结合蛋白在蛋白相互作用网络中具有更多的相互作用关系。【结论】基于TMT蛋白组学对牦牛抗冻差异蛋白进行挖掘，结果鉴定获得144个抗冻差异蛋白（上调蛋白89个，下调蛋白55个），其中CIRP和HSP70在冷应激条件下呈上调趋势，能促使牦牛肌体适应低温环境，可作为牦牛抗冻性育种的候选分子标记。

TMT proteomics and bioinformatics to analyze differential proteins in cold resistance of yaks

【Objective】To elucidate the mechanism of cold resistance of yak from protein level，and to further improve its metabolic performance from the perspective of nutrition，so as to provide scientific basis for yak to effectively resist harsh climate conditions.【Method】The yak antifreeze proteins were extracted by TMT proteomics in cold season （January）and warm season（August），and the subcellular localization analysis，domain analysis，GO functional analysis， KEGG signaling pathway annotation analysis ，protein interaction were analyzed.【Result】A total of 21856 peptides were identified，of which 18452 were specific peptides and 4519 proteins were identified. There were 144 subcellular proteins， including 89 up-regulated proteins and 55 down-regulated proteins. 144 yak antifreeze differential proteins were subcellular mapped to 7 items，including 56 nuclear proteins，51 cytoplasmic proteins，24 plasma membrane proteins，23 extracellular proteins，18 mitochondrial proteins，1 cytoskeleton protein and 1 lysosomal protein. A total of 194 domains were identified. GO enrichment of function analysis indicated that，biological process mainly enriched to cell protein 79，70 metabolic process protein and 42 regulation protein，molecular function mainly enriched to combine functional protein 75 and 64 catalytic activity of the protein，cell component part mainly enriched to the 89 cell protein and 89 cell proteins. The 144 yak antifreeze differential proteins were annotated into 205 KEGG signaling pathways in KEGG database，which mainly involved in ribosomes，nitrogen metabolism，cytoplasmic DNA sensing，in vivo thermogenesis，oxidative phosphorylation，interleukin-17 and calcium ion signaling. It was found that L8IHE5 had the highest correlation，and cold-induced RNA-binding proteins（CIRP）and HSP70 binding protein had more interaction in the network.【Conclusion】According to TMT proteomics，144 differential antifreeze proteins（89 up-regulated proteins and 55 down-regulated proteins）are identified，among which CIRP and HSP70 are up-regulated under cold stress，which can promote yak body to adapt to low temperature environment，and can be used as candidate molecular markers for yak antifreeze breeding.