利用蛋白质组和磷酸化组学技术研究肉鸡热应激调控机制

发布时间：2018-10-13 09:48

【摘要】：在集约化养殖环境下,热应激已成为现代家禽生产中普遍关注的全球性问题。热应激可导致肉鸡的采食量下降、生长速度变慢、饲料转化率低,肉品质变差等不利影响,并且严重影响肉鸡免疫力甚至导致肉鸡死亡率,给肉鸡生产带了巨大经济损失。为了阐明高温环境下肉鸡热应激调控机制,本项目使用高通量蛋白质组和磷酸化蛋白质组学技术,分析了热处理前期和后期肉鸡肝脏中蛋白质和磷酸化修饰的变化。首先使用SWATH非标蛋白质组技术研究热处理中期(72小时)肉鸡肝脏中蛋白质变化,一次鉴定到4271个蛋白及对应的36073条多肽,并发现257个蛋白与热应激调控密切相关。GO注释和通路分析表明,这些蛋白主要涉及机体氧化还原过程、蛋白质折叠、信号传导及负凋亡调控等生物功能,参与能量代谢通路、氨基酸代谢通路、脂类代谢通路和免疫调节相关的代谢通路。通过生物信息学深度挖掘蛋白功能发现,热应激下肉鸡主要通过抑制细胞外信号调节激酶(ERK)通路影响肝细胞凋亡；通过增强甲硫氨酸的体内合成增强机体抗氧化能力；通过增加儿茶酚胺类化合物加快呼吸频率以达到降温的目的。蛋白质磷酸化和去磷酸化是调节生命活动的一个重要蛋白质翻译后修饰过程,对体内信号传导、蛋白功能和新陈代谢具有至关重要的调控作用。我们在72小时样品的蛋白质组分析过程中发现ERK信号通路受到抑制,且与ERK信号通路有密切关系的蛋白质磷酸化多数发生在处理早期。为了调查热处理早期肉鸡肝脏中磷酸化蛋白质的变化,我们使用Ti02富集热处理24小时后样品中的磷酸化蛋白,并使用iTRAQ同位素标记实现对磷酸化肽段的定量分析。实验最终鉴定到6556个磷酸化修饰多肽(FDR0.5%),对应1 686个磷酸化修饰蛋白,寻找到46个差异表达磷酸化蛋白。通过生物信息学分析,证实肉鸡热应激调控过程中,调控信号传导是通过SRC→PBK→AKT→RAS→C-RAF→MAPK/ERK信号途径实现的下游信号传递。通过以上两个方面研究发现与热应激调控相关的相关蛋白和磷酸化蛋白,并确定磷酸化蛋白的修饰位点的变化,并使用生物信息学系统地分析磷酸化蛋白之间的联系,进一步分析这些蛋白参与机体调控机理和途径,最终在蛋白质水平阐明肉鸡响应热应激调控的分子机制,为今后通过遗传育种和营养调节等措施提高肉鸡的自我适应能力提供理论基础。
[Abstract]:In intensive farming environment, heat stress has become a global problem in modern poultry production. Heat stress could lead to the decrease of feed intake, slow growth rate, low feed conversion rate, poor meat quality and other adverse effects. It also seriously affected the immunity of broilers and even led to the mortality of broilers, which caused a huge economic loss to broiler production. In order to elucidate the regulation mechanism of heat stress in broilers under high temperature, the changes of protein and phosphorylation modification in the liver of broilers were analyzed by using high-throughput proteomics and phosphorylation proteomics techniques in the early and late stages of heat treatment. SWATH nonstandard proteome technique was used to study the changes of protein in the liver of broiler chickens at the middle stage of heat treatment (72 hours). 4271 proteins and 36073 corresponding polypeptides were identified at one time. It was found that 257 proteins were closely related to heat stress regulation. GO annotation and pathway analysis showed that these proteins were mainly involved in biological functions such as redox process, protein folding, signal transduction and negative apoptosis regulation, and participated in energy metabolism pathway. Amino acid metabolic pathway, lipid metabolic pathway and immune regulation related metabolic pathway. Through deep bioinformatics, it was found that heat stress mainly affected hepatocyte apoptosis by inhibiting extracellular signal-regulated kinase (ERK) pathway, and enhanced the antioxidant capacity by enhancing the synthesis of methionine in vivo. By increasing catecholamines to speed up the respiratory rate to achieve the purpose of cooling. Protein phosphorylation and dephosphorylation is an important process of protein post-translational modification, which plays an important role in regulating signal transduction, protein function and metabolism in vivo. We found that the ERK signaling pathway was inhibited during the 72-hour proteome analysis, and that the phosphorylation of proteins closely related to the ERK signaling pathway occurred in the early stage of treatment. In order to investigate the changes of phosphorylated proteins in the liver of broilers at the early stage of heat treatment, we used Ti02 to enrich the phosphorylated proteins in the samples after 24 hours heat treatment, and to quantitatively analyze the phosphorylated peptides using iTRAQ isotopic markers. 6556 phosphorylated polypeptides (FDR0.5%) were identified, corresponding to 1 686 phosphorylated modified proteins, and 46 differentially expressed phosphorylated proteins were found. Through bioinformatics analysis, it was proved that during the process of heat stress regulation in broilers, the regulated signal transduction was downstream signal transduction through SRC PBK AKT RAS C-RAF MAPK/ERK signal pathway. Through the above two studies, we found the related proteins and phosphorylated proteins related to heat stress regulation, and determined the changes of phosphorylated protein modification sites, and used bioinformatics to systematically analyze the relationship between phosphorylated proteins. Further analysis of these proteins involved in the organism regulation mechanism and pathway, finally at the protein level to elucidate the regulation of the response to heat stress in broilers molecular mechanism, It provides a theoretical basis for improving the self-adaptive ability of broilers by means of genetic breeding and nutrition regulation in the future.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：S858.31

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